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Molecular Characterization and Developmental Expression
Patterns of the Zebrafish twist Gene Family
Yeo Gare Hoon
(B.Sci, University of Melbourne)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
YOOG LOO LIN SCHOOL OF MEDICINE
(DEPARTMENT OF PAEDIATRICS)
NATIONAL UNIVERSITY OF SINGAPORE
2009
Table of Contents
_____________________________________________________________
Acknowledgements
v
List of Tables
vi
List of Figures
vii
Abbreviation
ix
Summary
x
Abstract
xiii
________________________________________________________________________
Chapter 1:
Introduction
1
1.1
TWIST gene family
1
1.1.1
TWIST1 gene
1
1.1.2
TWIST2 gene
5
1.1.3
Twist3 gene
6
1.2
Why zebrafish is used as an animal model in this study?
7
1.3
Phylogenetics
8
1.3.1
DNA or protein sequences
9
1.4
Gene duplication
10
1.4.1
Evolutionary fates of duplicate genes
12
Chapter 2:
Materials and Methods
16
2.1
Animal stocks and maintenance
16
2.2
Isolation of genomic DNA and total RNA
16
2.2.1
Isolation of genomic DNA
16
2.2.2
Isolation of total RNA
17
2.3
Full-length cDNA sequence
17
2.3.1
Rapid amplification of complementary DNA ends
2.3.2
(RACE) of zebrafish twist1b
17
Assembly of zebrafish twist1b full-length cDNA
20
ii
2.4
Genomic sequence of zebrafish twist1b
21
2.5
RT-PCR
22
2.6
Synthesis of RNA probes for in situ hybridization
analysis
2.6.1
Identification of unique 3’UTR sequences of the zebrafish
twist gene family
2.6.2
23
23
Isolation of unique 3’UTR sequences of zebrafish twist
gene family
23
2.6.3
Linearization of plasmid DNA
28
2.6.4
RNA labeling with Digoxenin / Fluorescein RNA
labeling kits (SP6/T7/T3)(Roche)
29
2.6.5
Purification of RNA probe
29
2.7
Whole mount in situ hybridization
30
2.8
Cryosection
34
2.9
Image processing
35
2.10
Phylogenetic analysis
35
2.10.1
Alignment and phylogenetic tree
35
2.10.2
Calculation of genetic distances
36
2.10.3
Comparative synteny analysis
37
Chapter 3:
Results
38
3.1
Characterization of zebrafish full-length cDNA of
twist1b
38
3.2
Genomic organization of zebrafish twist1b
39
3.3
Alignment of TWIST family peptides
41
3.4
Comparison and alignment of zebrafish twist gene
family
3.5
41
Identification and confirmation of the true orthologs
of zebrafish twist genes
47
3.5.1
Comparison of zebrafish twist gene family with other species
47
3.5.2
Phylogenetic analysis
48
iii
3.5.3
Calculation of genetic distances
50
3.5.4
Comparative synteny analysis
51
3.6
Embryonic expression patterns of the zebrafish twist
gene family
58
3.6.1
RTPCR analysis
58
3.6.2
In situ hybridization analysis
60
Chapter 4:
Discussion
76
4.1
The zebrafish twist gene family
76
4.2
Phylogenetic relationships of the twist genes in fish
77
4.3
Genetic distance analysis of twist1a and twist1b among
the fishes
78
4.4
Comparative synteny analyses
79
4.5
Comparison of zebrafish twist family expression pattern
with other species.
83
4.5.1
Zebrafish twist1a and twist1b genes
83
4.5.2
Zebrafish twist2
85
4.5.3
Zebrafish twist3
86
4.6
Shared and unique expression sites of the zebrafish
twist genes
86
4.6.1
Importance of using unique 3’UTR sequences as riboprobes
86
4.6.2
Comparison of zebrafish twist genes expression sites
with other publications
86
4.7
Evolutionary fates of the zebrafish twist gene family
91
Chapter 5:
Conclusions
94
Chapter 6:
References
95
iv
Acknowledgement
________________________________________________________________________
Firstly, I thank God for the strength and perseverance that have sustained me through
this research project. Thank God for His wisdom and for directing my path.
My utmost gratitude goes to my supervisor, Assoc Prof Samuel Chong for giving me the
opportunity to further my studies in his lab. Thank you for your kind understanding,
encouragement and patient supervision to me.
Special thanks to Assoc Prof Christoph Winkler for your constructive advice, for sharing
with me your invaluable knowledge. Your help is very much appreciated.
Thank you Prof Byrappa Venkatesh for your enormous help in phylogenetic analyses, for
enlightening me on the topics of evolution and phylogeny, an area which I am very green.
To Assoc Prof Vladimir Korzh, thank you for your precious recommendation and time.
Your insightful advice has been most helpful. Thank you too, for the gifts of pax2.1 and
wt1 plasmids.
To Dr Karuna Sampath, I am very grateful for both your helpful technical advice and
your patient guidance.
Special thanks to Felicia and Ben Jin, for your words of encouragement and support and
for sharing with me your laboratory expertise and personal experiences on time
management as a part-time student.
Big thanks to Haibo, Shanta and Xiaoyu for helping with the care and maintenance of the
fish system. Without your meticulous care, I wouldn’t have healthy embryos and fish for
my project.
To Arnold, Wang Wen, Weijun, Chia Yee, Pooi Eng, Clara, Yvonne, Jack, Siew Hoon and
Victor thanks for the words of encouragement, your friendship and moral support along
the way.
Thanks to Monte Westerfield and Andrew D. Sharrocks for their gifts of dlx2a and fli1a
(pAS160) plasmids respectively.
And last but not least, thanks to my parents, my sisters and Patrick for giving me the love
and support to press on.
v
List of Tables
________________________________________________________________________
Table 1 :
The synthesis of RNA probes and in situ hybridization conditions
Table 2:
Nucleotide identity of the coding region, bHLH domain, and WR domain
of the zebrafish twist genes
Table 3:
Comparison of zebrafish twist gene sequences with TWIST sequences
from other species
Table 4:
Comparative synteny analysis of chromosomal regions around zebrafish
twist1a and twist1b and human TWIST1
Table 5:
Comparative synteny analysis of chromosomal regions around zebrafish
twist1a and medaka twist1a and twist1b
Table 6:
Comparative synteny analysis of chromosomal regions around zebrafish
twist1b and medaka twist1a and twist1b
Table 7:
Comparative synteny analysis of chromosomal regions around twist2 from
zebrafish, human and medaka
Table 8:
Comparative synteny analysis of chromosomal regions around zebrafish
twist3 and medaka twist3a and twist3b
Table 9:
Expression domains of the four zebrafish twist genes
Table 10 :
Twist expression sites in selected species
vi
List of Figures
________________________________________________________________________
Figure 1.1:
Terminologies used to classify homologs
Figure 1.2:
The model of synfunctionalization: a mechanism for gene loss or function
shuffling.
Figure 2.1:
The incomplete cDNA sequence of zebrafish twist1 gene
Figure 2.2:
Agarose gel electrophoresis of 5’ and 3’ RACE experiment
Figure 2.3:
5’ UTR sequences of the zebrafish twist1 (twist1b) gene obtained from
5’RACE experiment.
Figure 2.4:
3’ UTR sequences of the zebrafish twist1 (twist1b) gene obtained from
3’RACE experiment.
Figure 2.5:
The complete full-length cDNA sequence of the zebrafish twist1 gene.
Figure 2.6:
cDNA sequence of zebrafish twist1a
Figure 2.7:
cDNA sequence of zebrafish twist1b
Figure 2.8:
cDNA sequence of zebrafish twist2
Figure 2.9:
cDNA sequence of zebrafish twist3
Figure 3.1:
Full-length cDNA sequence of zebrafish twist1 (twist1b) and its
deduced amino acid sequence.
Figure 3.2:
Genomic DNA sequence of zebrafish twist1 (twist1b).
Figure 3.3:
Alignment of predicted Twist proteins
Figure 3.4:
Alignment of zebrafish full-length cDNAs
Figure 3.5:
Cladogram and unrooted radial tree of Twist proteins generated by the
neighbor-joining method
Figure 3.6:
Gene structure of twist1a, twist1b, twist2 and twist3
Figure 3.7:
RT-PCR of zebrafish twist genes
Figure 3.8:
Expression of zebrafish twist genesexpression during the cleavage period
vii
Figure 3.9:
Expression of zebrafish twist genesexpression during the gastrula period
Figure 3.10:
Expression of zebrafish twist genesexpression during the early
segmentation period
Figure 3.11:
Expression of zebrafish twist genes during mid-somitogenesis
Figure 3.12:
Zebrafish twist expression along the trunk
Figure 3.13:
Zebrafish twist1b and twist3 expression during somitogenesis
Figure 3.14:
Zebrafish twist1b expression in the somites
Figure 3.15:
Expression of zebrafish twist genesexpression during the prim-5 stage
Figure 3.16:
Expression of zebrafish twist genesexpression during the long-pec stage
Figure 3.17:
Expression of zebrafish twist genesexpression during the hatching period
Figure 4.1:
A model for the evolutionary history of twist genes.
viii
Abbreviations
________________________________________________________________________
cDNA
complementary DNA
dNTP
Deoxyribonucleotide triphosphate
UTR
Untranslated Region
PCR
Polymerase Chain Reaction
hpf
hours post fertilization
dpf
days post fertilization
DEPC
diethylpyrocarbonate
SDS
sodium dodecyl sulfate
RACE
Rapid amplification of complementary DNA ends
bHLH
basic Helix-Loop-Helix
WR
tryptophan-arginine
RT
Reverse Transcription
SCS
Saethre-Chotzen Syndrome
ORF
Open reading frame
LG
Linkage Group
ix
Summary
________________________________________________________________________
The Twist gene is essential for development and survival, and is present in
animals ranging from Drosophila to humans, either in single copy or as a gene family of
two to five members. In 2007, a paralog of twist1 was identified by Gitelman, who
renamed the genes according to their relationships with those of other species (Gitelman,
2007).
This study aims to characterize the zebrafish twist family of genes, their
phylogenetic and evolutionary relationships, and their developmental expression profiles.
I performed a comprehensive alignment, phylogenetic and comparative synteny analysis
to determine the relationship of these genes to each other and to those of other species.
Phylogenetic analysis showed that the Twist peptides were clustered into three
clades, with Twist1, Twist2 and Twist3 peptides in each clade. Interestingly, the Twist1b
peptides of the Acanthopterygii (medaka, fugu, spotted green pufferfish and stickleback)
were clustered together with the Twist3 peptides instead of Twist1 peptides whereas
zebrafish twist1a and twist1b peptides were clustered with the Twist1 peptides.
Comparative
nucleotide
substitution
analyses
revealed
a
faster
nucleotide
mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b,
thus explaining the anomalous clustering of the former group of Twist1b peptides.
x
Comparative synteny analysis of the chromosomal regions flanking the zebrafish,
medaka, and human twist genes showed that the zebrafish twist1a and twist1b are coparalogs and co-orthologs of human TWIST1. Furthermore, zebrafish twist1a and twist1b
are orthologous to medaka twist1a and twist1b, respectively, despite the different
phylogenetic clusterings of zebrafish and medaka twist1b. The orthology of zebrafish
twist2 to human and medaka TWIST2/twist2, was also confirmed. Finally, zebrafish
twist3 showed greater chromosomal synteny to medaka twist3b than to the medaka
twist3a. Based on these results, a model for the evolutionary history of the twist genes
has been reconstructed.
I also performed a comprehensive developmental expression analysis of all four
twist genes. All four genes were expressed in the pharyngeal arches. Zebrafish twist1a
and twist1b were expressed in the sclerotome and twist3 in the somite during the
segmentation period. Zebrafish twist1b and twist3 were found to be present as maternal
transcript. Many expression sites were unique. Transcripts of twist1a were detected
specifically in the premigratory neural crest cells during early somitogenesis and in the
heart valve at the hatching period. Zebrafish twist1b was expressed in the intermediate
mesoderm during segmentation period and in the olfactory placode at the hatching period.
Zebrafish twist2 expression was observed in the organizer at the shield stage,
presumptive vasculature during the segmentation period, and in the hypochord and dorsal
aorta during the prim-5 stage. Zebrafish twist1a and twist3 were expressed in the fin bud,
with twist3 expression concentrated in the endochondral disc and twist1a expression
xi
strongest in the actinotrichs. Minimal expression overlap was observed among the four
twist genes using unique 3’UTR sequences for riboprobes.
The contents of this thesis have been published in two paper, “Zebrafish twist1 is
expressed in craniofacial, vertebral, and renal precursors
(Yeo et al., 2007) and
“Phylogenetic and evolutionary relationships and developmental expression patterns of
the zebrafish twist gene family” (reference in press).
xii
Abstract
_____________________________________________________________
Four members of the twist gene family (twist1a, 1b, 2 and 3) are found in the
zebrafish, and they are thought to have arisen through three rounds of gene duplication,
two of which occurred prior to the tetrapod-fish split. Phylogenetic analysis groups most
of the vertebrate Twist1 peptides into clade I, except for the Twist1b proteins of the
acanthopterygian fish (medaka, pufferfish, stickleback), which clustered within clade III.
Paralogies and orthologies among the zebrafish, medaka, and human twist genes were
determined using comparative synteny analysis of the chromosomal regions flanking
these genes. Comparative nucleotide substitution analyses also revealed a faster rate of
nucleotide mutation/substitution in the acanthopterygian twist1b compared to the
zebrafish twist1b, thus accounting for their anomalous phylogenetic clustering. Based on
these analyses, a model for the evolutionary history of the twist genes has been
reconstructed. I observed minimal expression overlap among the four twist genes using
unique 3’UTR sequences for riboprobes, suggesting that despite their significant peptide
similarity, their regulatory controls have diverged considerably, with minimal functional
redundancy between them.
xiii
Chapter 1: Introduction
_____________________________________________________________
1.1
The TWIST gene family
The TWIST genes are a group of transcription factor genes whose peptides contain
two highly conserved domains, the basic helix-loop-helix (bHLH) domain and the
tryptophan-arginine (WR) domain (Atchley and Fitch, 1997; Spring et al., 2000). The
bHLH domain can be found in a number of other proteins and is involved in growth
regulation, myogenesis and neurogenesis (Jan and Jan, 1993). The function of the WR
motif is unclear although it has been suggested to be required either for TWIST activity,
for the stability of its mRNA or for normal protein folding (Gripp et al., 2000; Castanon
and Baylies, 2002).
Twist was first isolated in Drosophila as a zygotic gene involved in the
establishment of dorso-ventral patterning, mesoderm specification and myogenesis
(Thisse et al., 1987; Thisse et al., 1988; Baylies and Bate, 1996). At gastrulation,
homozygous twist mutant embryos were abnormal and failed to differentiate their
mesoderm (Simpson, 1983; Thisse et al., 1987).
Since this initial discovery, Twist
orthologs and paralogs have been identified in many other animal species.
1.1.1 TWIST1
The TWIST1 gene is located on human chromosome 7p21.2 and has been reported
to be the causative gene for Saethre-Chotzen Syndrome. Twist1 has been the most
intensively studied gene among the TWIST gene family and its expression profile has
1
been reported in many species including the mouse (Wolf et al., 1991; Fuchtbauer, 1995;
Stoetzel et al., 1995), rat (Bloch-Zupan et al., 2001), Xenopus (Hopwood et al., 1989;
Stoetzel et al., 1998), chick (Tavares et al., 2001), medaka (Yasutake et al., 2004) and
zebrafish (Rauch, 2003; Germanguz et al., 2007; Yeo et al., 2007).
In the mouse, maternal transcript of Twist1 was first detected in the extraembryonic tissue and embryonic ectodermal cells of the primitive streak (Stoetzel et al.,
1995). As the embryo develops, Twist1 is expressed in the head region, trunk and limbs.
In the head region, transcripts of Twist1 is found in the vicinity of the neural structures,
including the forebrain and area of the nasal placodes, the diencephalon and the optical
vesicles, the rhombencephalon and around the otic vesicles. Furthermore, a high level of
expression was observed in the branchial arches. In the trunk, Twist1 expression is
detected in the sclerotome and somatopleura. In addition, Twist1 expression is also found
in the posterior limb buds and tail, the mesenchyme cells forming the internal ear, face,
lingua and the skin (Wolf et al., 1991; Fuchtbauer, 1995; Stoetzel et al., 1995).
In Xenopus, twist1 is also present as a maternal transcript (Stoetzel et al., 1998).
Expression of twist1 is also detected in head, body and tail region. In the head, twist1
transcript accumulates in the internal mesoderm. In the trunk region, expression of twist1
is detected in the notochord, neural crest, lateral mesoderm and somites (Hopwood et al.,
1989).
2
Expression of Twist1 has also been described in other species including rat
(Bloch-Zupan et al., 2001), chick (Tavares et al., 2001), medaka (Yasutake et al., 2004)
and zebrafish (Tavares et al., 2001; Yasutake et al., 2004; Germanguz et al., 2007; Yeo et
al., 2007).
The functions of Twist1 have been reported in many species. In mouse, Twist1
protein is known to be involved in myogenesis. Mouse Twist1 proteins can interfere with
the activity of myogenic transcription factor MyoD (myogenic determination) and MEF2
(myocyte-enhancing factor 2) by preventing the formation of functional MyoD-E proteins
heterodimers and inhibiting MEF2-mediated transactivation process (Spicer et al., 1996).
In addition, a study done in a metastatic breast cancer mouse model showed that Twist1
is necessary for the onset of metastasis (Yang et al., 2004).
Twist1 is also known to participate in transcription regulation.
It has been
reported that TWIST1 functions as a prometastic oncogene. TWIST1 protein can interact
directly with two independent HAT (histone acetyltransferases) domains of p300 and
PCAF (p300/CBP-associated factor) acetyltransferases via its N-terminus. The binding
of Twist inhibits the acetyltransferase activities of p300 and PCAF, thereby preventing
subsequent histone acetylation process that is essential for unwinding the densely packed
chromatin to allow the access of transcriptional machinery during transcription process
(Hamamori et al., 1999; Massari and Murre, 2000).
3
TWIST1 also plays a role in human osteoblast metabolism. The level of TWIST1
protein can influence osteogenic gene expression and it may act as a master switch in
initiating bone cell differentiation by regulating the osteogenic cell lineages (Lee et al.,
1999).
Twist1 has also been reported to induce epithelial to mesenchymal transition
(EMT) by repression of E-cadherin and induction and regulation of N-cadherin (Yang et
al., 2004; Alexander et al., 2006). Additionally, overexpression of Twist1 has been
described to induce angiogenesis and chromosomal instability (Mironchik et al., 2005).
In knockout mice, the Twist1-/- null mice died at embryonic day 11.5, exhibiting a
failure of neural tube closure specifically in the cranial region. They also had defects in
head mesenchyme, branchial arches, somites, and limb buds, suggesting that Twist1 is
involved in regulating the cellular phenotype and behavior of head mesenchymal cells
that are essential for the morphogenesis of cranial neural tube (Chen and Behringer,
1995). Further studies show that absence of Twist activity in the cranial mesenchyme
region causes improper closure of the cephalic neural tube and this subsequently leads to
a malfunction of the branchial arches in Twist1-/- null mice. The authors later found that
Twist1 activity is required in both the cranial mesenchyme for directing neural crest cells
migration as well as the neural crest cells within the first branchial arch to ensure correct
localization of the progenitor cells. Furthermore, Twist1 is also required for the proper
differentiation of the first branchial arch tissues into bone, muscle, and teeth (Soo et al.,
2002).
4
In medaka twist knockdown morphants, the neural arches were absent.
Subsequent experiments performed suggest that twist is involved in the differentiation
process of sclerotomal cells into neural arch-forming osteoblasts (Yasutake et al., 2004).
1.1.2 TWIST2
Twist2 (previously known as Dermo1) is another family member that is found in
human (Lee et al., 2000), mouse (Li et al., 1995), rat (Maestro et al., 1999), chick (Scaal
et al., 2001), medaka (Gitelman, 2007), Fugu (Gitelman, 2007) and zebrafish
(NM_001005956). Its expression profile has been described in mouse (Li et al., 1995),
chick (Scaal et al., 2001) and zebrafish (Thisse and Thisse, 2004; Germanguz et al.,
2007).
In mouse, Twist2 is expressed in both the sclerotome and dermatome of the
somite, the cranial mesenchymal cells around the nose, pharyngeal arches and tongue,
whiskers, somites, limb and branchial arches (Li et al., 1995). In chick, Twist2 is
expressed in the somites, head mesenchyme, limbs, branchial arches and mesenchyme of
the feather buds (Scaal et al., 2001).
Twist 2 is involved in transcriptional regulation and is a transcriptional repressor
of p65 (an NF-kB subunit) and myocyte enhancer factor 2 (MEF2) (Gong and Li, 2002;
Sosic et al., 2003). A study showed that Twist2 protein bound the E-box consensus
sequence in the presence of E12. Furthermore, Twist2 act as a repressor in Myo-D
mediated transactivation via its C-terminal and HLH domains and has been suggested to
5
regulate gene expression in a subset of mesenchymal cell lineages including developing
dermis (Li et al., 1995; Gong and Li, 2002). Furthermore, Twist2 interacted directly with
MEF2 and selectively repressed MEF2 transactivation domain (Gong and Li, 2002; Sosic
et al., 2003). Additionally, Twist2 has been identified to be an interacting protein with
adipocyte determination and differentiation dependent factor 1 (ADD1)/sterol regulatory
element binding protein isoform (SREBP1c). ADD1/SREBP1c is a transcription factor in
fatty acid metabolism and insulin dependent gene expression. Overexpression of Twist2
specifically suppresses the transcriptional activity of ADD1/SREBP1c by interfering with
ADD1/SREBP1c binding to its target DNA and histone deacetylation (Lee et al., 2003).
Twist2 is also suggested to function as an oncoprotein, antagonizing the activation
of p53-dependent apoptosis in response to DNA damage (Maestro et al., 1999). It is
found that Twist2 is expressed in osteoblastic cells and it possibly act as a negative
regulator of the differentiation of osteoblast (Tamura and Noda, 1999).
1.1.3 Twist 3
A third family member Twist3 is absent in mammals but found in Xenopus, chick,
medaka, stickleback and zebrafish (Gitelman, 2007). In contrast to Twist1 and Twist2,
little is known about the role of Twist3.
6
1.2
Why zebrafish is used as an animal model in this study?
The laboratory mouse Mus musculus has become the predominant model
organism used to study human development, however, the zebrafish Danio rerio has
emerged
as
a
promising
complement
for
embryological,
genetic/genomic,
cellular/biochemical and other functional studies.
The zebrafish was first introduced by George Streisinger as a system for genetic
analysis of vertebrate development (Streisinger et al., 1981; Walker and Streisinger,
1983). Its increased use in research is attributed to the many advantages of the zebrafish.
Firstly, the zebrafish is small in size (up to 6 cm) and thus can be economically
maintained with relative ease in the laboratory compared to mouse and Xenopus.
Secondly, it has a short generation time of about 3 months. Thirdly, zebrafish eggs are
fertilized externally and each mating can generate approximately 100 eggs. In addition,
zebrafish embryos are transparent and develop rapidly. Rudimentary organs such as
eyes, ears, brain and heart can be observed one day after fertilization.
Moreover,
zebrafish form essentially all of the same skeletal and muscle tissue types as their higher
vertebrate counterparts, but in much more simple spatial patterns composed of smaller
numbers of cells and this is achieved within a short period of time (Schilling, 2002).
Furthermore, many of the features that govern craniofacial development in higher
vertebrates are conserved and zebrafish contain craniofacial elements similar to those of
higher vertebrates (Schilling, 2002; Yelick and Schilling, 2002).
7
1.3
Phylogenetics
Phylogenetics is the study of evolutionary history in which the nucleotide
characters in DNA or protein sequences are compared among different species. This is
based on the assumption that closely related organisms have sequences that are similar
and more distantly related organisms have sequences that differs greatly.
These
sequences are known as homologs and they are believed to be inherited from a common
ancestor.
Other terminologies are used to classify homologs. Homologs that are produced
by speciation are known as orthologs. They represent genes that were derived from a
common ancestor that diverged because of divergence of the organism. Orthologs may or
may not have the same functions. Homologs that are produced by gene duplication are
known as paralogs. They represent genes that were derived from a common ancestral
gene that duplicated within an organism and diverged. Paralogs are believed to have
different functions (Figure 1.1). Phylogenetics reconstructs the evolutionary relationship
between species and allows the estimation of the time of divergence between two
organisms since they last shared a common ancestor.
8
Early globin gene
GENE
DUPLICATION
α chain gene
Frog α
Chick α
β chain gene
Mouse β
Mouse α
PARALOG
Chick β
Frog β
PARALOGS
ORTHOLOG
ORTHOLOG
HOMOLOG
Figure 1.1:
Terminologies used to classify homologs. An example of the globin gene.
(Adapted from: http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/Orthology.html)
There are generally two classes of phylogenetic trees, namely, the rooted and
unrooted tree. A rooted tree has a particular node (root), representing a common ancestor
from which a unique path leads to any other nodes. An unrooted tree only specifies the
relationship among species, without identifying a common ancestor or evolutionary path.
1.3.1 DNA or protein sequences?
Both nucleotide characters in DNA and protein sequences are used for
constructing phylogenetic trees, in estimating phylogenetic relationships and times of
divergence among taxa. In general, DNA sequences are used for relatively recent events,
for example, in closely related species such as human and chimpanzee. This is because
the protein sequences between human and chimpanzee are too conserved to be useful
9
(Hedges, 2002). Both the coding and non-coding regions of the DNA sequence can be
used. The rate of mutation is assumed to be the same in both coding and non-coding
regions; however, there is a difference in the substitution rate. It is important to note that
non-coding DNA regions have more substitutions than coding regions.
Proteins are much more conserved since they “need” to conserve their function.
Hence, protein sequences are more useful for more ancient events – for example, in
human and fish – when DNA sequences are usually too divergent to make accurate
estimates on the basis of nucleotide substitutions of DNA (Hedges, 2002).
However, there is a limitation of using either nucleotide or protein sequences
because unequal base or amino acid composition among the genomes of different species
is common. In addition, sequence length is a limiting factor, in that the average gene
(coding) or protein sequence (~1,000 nucleotides, ~350 amino acids) is usually not long
enough to yield a robust phylogeny or time estimate, and therefore many genes and
proteins must be used (Hedges, 2002).
1.4
Gene Duplication
In 1936, Bridges observed gene duplication in a mutant of the fruit fly Drosophila
melanogaster, where the doubling of a chromosomal band results in extreme reduction in
eye size (Bridges, 1936; Zhang, 2003).
Gene duplication is a key mechanism in
evolution. Duplicated genes contribute genetic raw material for the emergence of new
functions through natural selection. Lynch and Conery (2000) reported that there are
10
around 15% of genes in the human genome there are found to be duplicated. The average
rate of duplication of a eukaryotic gene is estimated to be on the order of
0.01/gene/million years, which is of the same order of magnitude as the mutation rate per
nucleotide site (Lynch and Conery, 2003).
A number of mechanisms have been described to attribute gene duplication.
These are unequal crossing over, retroposition, gene conversion and chromosomal (or
genome) duplication (Ohta, 2000; Zhang, 2003; Hurles, 2004). However, depending on
the mode of duplication different outcomes are generated.
Unequal crossing over usually results in tandem gene duplication in which the
duplicated genes are linked in a chromosome. The duplicated region can contain a
portion of a gene, the entire gene or several genes, depending on the exact position of
crossing over (Zhang, 2003).
Retroposition is the integration of reverse transcribed mature RNAs at random
sites in a genome. The resultant duplicated genes, known as retrogenes, usually display
several molecular features such as lack of introns and regulatory sequences, the presence
of poly-A tails and presence of flanking short direct repeats (Zhang, 2003; Hurles, 2004).
In addition, a duplicated gene generated by retroposition is usually unlinked to the
original gene as the insertion of cDNA process is random (Zhang, 2003; Hurles, 2004).
11
Chromosomal (or genome) duplication occurs probably by a lack of disjunction
among daughter chromosomes after DNA replication (Zhang, 2003). Many of these
duplicated segments are located in regions that are hot spots of chromosomal and/or
evolutionary instability (Samonte and Eichler, 2002).
In the TWIST gene family, gene duplication is observed. TWIST1 and TWIST3
genes are found to be duplicated in some species. Duplication of this Twist1 gene
(twist1a and twist1b) has been observed specifically in Actinopterygii (ray-fined fishes)
(Gitelman, 2007). In stickleback and medaka, there are also two copies of the twist3 gene
(Gitelman, 2007).
1.4.1
Evolutionary fates of duplicate genes
The consequences of gene duplication play a key mechanism of evolution as it is
the survival and fitness of the organism harboring the newly duplicated gene/genome that
determine whether either copy of the gene persists or not. Different mechanisms/models
have been described to contribute to different evolutionary fates of duplicate genes.
The nonfunctionalization model explains how one copy of the duplicate genes is
assumed to be redundant and acquires degenerative mutations that eventually eliminate
its function (silenced). The non-functional copy is referred to as pseudogene (Woollard,
2005; Sjodin et al., 2007). Pseudogenes can be classified into processed and unprocessed
pseudogenes.
Processed pseudogenes are generated by the integration of reverse
transcription products of processed mRNA transcript into the genome whereas
12
unprocessed pseudogene are generated by the integration of non-processed RNA
transcript and thus has retained the original exon-intron structure of the functional gene
(Zhang et al., 2008).
The neofunctionalization model specifies that one member of a duplicate gene
acquires mutation that results in a novel, beneficial function (Conant and Wolfe, 2008).
This requires varying numbers of amino acid substitutions. Here, directional selection or
by genetic drift can contribute to the duplicate fixation (Conant and Wolfe, 2008).
The subfunctionalization model showed that both members of the duplicate gene
are stably maintained or persevered by purifying selection and they differ in some aspects
of their functions (Conant and Wolfe, 2008). Each daughter gene adopts part of the
function of their parental gene. The division of gene expression occurs after duplication.
The synfunctionalization model provides a mechanism for gene loss or function
shuffle (Figure 1.2). After gene duplication and prior to synfunctionalization, each copy
of the duplicate gene is retain in the genome as they possess unique function. In the
process of synfunctionalization, one copy of the duplicate gene acquires a unique
expression domain of the other and hence, all unique functions of this gene can be found
on one copy of the duplicates. Therefore, the other copy of the duplicate gene becomes
redundant and leads to gene loss (Figure 1.2) (Gitelman, 2007). If both copy of the
duplicate gene are retained due to their remaining unique functions, function shuffling
13
could result whereby a function found in both copies of the duplicate gene may now be
redundant in one copy and lost (Figure 1.2) (Gitelman, 2007).
Gene X
Gene
Duplication
Gene X1
α
Gene X2
β
β
ε
δ
synfunctionalization
α
β
α
gene loss
α
β
α
Gene X1
Figure 1.2:
β
ε
δ
Gene X2
β
ε
δ
function shuffling
α
β
α
Gene X1
ε
δ
Gene X2
The model of synfunctionalization: a mechanism for gene loss or function
shuffling. Boxes indicate expression domains. Adapted from (Gitelman, 2007).
Phylogenetic and gene expression studies are invaluable tools that aid in our
understanding of the regulation and function of conserved genes and gene families such
as the TWIST gene family. Together, they provide important clues to the evolutionary
events and functional changes that have occurred in these genes in different species.
14
1.5
Aims
In this study, I aimed to:
1. Determine the complete cDNA sequence, genomic structure, and map location of the
zebrafish twist genes
2. Sort out the confusion in evolutionary orthology of the zebrafish twist genes with
their mammalian counterparts through comparative gene structure and linkage
synteny analyses
3. Characterize and compare the developmental and tissue-specific expression profiles
of the zebrafish twist genes during embryogenesis and in adult zebrafish
4. Compare expression patterns in zebrafish with other species.
15
Chapter 2: Materials and Methods
_____________________________________________________________
2.1
Animal stocks and maintenance
Singapore wild-type zebrafish were maintained in an aquarium system at 28oC.
Embryos were obtained by natural spawning and kept at 28oC and staged according to
Kimmel et al. (1995) using standard morphological criteria. Older embryos were treated
with 0.003% (2nM) 1-phenyl-2-thiourea to inhibit pigment formation and facilitate whole
mount examination.
2.2
Isolation of genomic DNA and total RNA
2.2.1 Isolation of genomic DNA
A healthy adult male fish was transferred into a 2-ml tube and frozen in liquid
nitrogen. The frozen fish was immediately grinded into powder and homogenized in 10
ml genomic DNA extraction buffer (10 mM Tris pH8.0, 100 mM EDTA pH8, 0.5%
sodium dodecyl sulfate (SDS) and 200 μg/ml proteinase K (Sigma)). The mixture was
mixed well and incubated at 50oC for 4 hours with gently swirling. After incubation, the
mixture was cooled to room temperature and extracted twice with one volume of
equilibrated phenol and once with phenol:chloroform:isoamyl alcohol (25:24:1). The
mixture was mixed gently until an emulsion has formed and centrifuged at 3000-5000 x g
for 10 minutes. The aqueous phase was transferred into a fresh tube and precipitated with
16
200mM sodium chloride (NaCl2) and 2 volumes of ethanol. The DNA was washed with
70% ethanol, air-dried and dissolved in 5 ml of TE buffer (10 mM Tris pH8, 5 mM
EDTA) and treated with 100 μg/ml RNAse A (Sigma) at 37oC for 3 hours. DNA solution
was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) to remove RNAse A.
The DNA was precipitated with 0.1 volume of 7.5 M ammonium chloride and two
volumes of ethanol. The DNA was washed twice with 70% ethanol, air-dried, dissolved
in TE buffer and stored at -20oC. The concentration of the DNA was measured with a
spectra-photometer (SpectraMax M5).
2.2.2
Isolation of total RNA
Approximately 50 zebrafish embryos were harvested; chorion was removed and
embryos were collected in a 2 ml microfuge tube. Excess water was removed with a
pipette. The embryos were frozen in liquid nitrogen and homogenized in 1 ml of
TRIZOL® RNA isolation reagent (Invitrogen) with a plastic pestle.
The insoluble
material was removed by centrifugation at 12,000 x g for 10 minutes at 4oC. The
aqueous phase was transferred to a fresh tube and RNA was precipitated with 2 volumes
of isopropyl alcohol and centrifuged at 12,000 x g for 10 minute at 4oC. The RNA pellet
was washed with 70% ethanol, air-dried and dissolved in 20 μl of 0.1%
diethylpyrocarbonate (DEPC, Sigma) treated water. The RNA was stored at -80oC and
concentration was measured with a spectra-photometer (SpectaMax M5).
2.3
Full-length cDNA sequence
2.3.1
Rapid amplification of complementary DNA ends (RACE) of zebrafish twist1b
(formerly twist1)
17
Based on the then available 624 bp cDNA sequence of zebrafish twist1b
(GenBank accession no. NM_130984 (25 April 2005)) (Figure 2.1), primers were
designed for both 5’ and 3’ RACE experiments to identify the additional 5’ and 3’ cDNA
sequences. The RACE experiments were performed using the SMART™ RACE cDNA
amplification kit (BD Biosciences Clontech).
Two non-overlapping reverse primers
complementary to 5’ region of the incomplete cDNA sequence (Twist1R: 5’- GTC TCT
CGT GCG CCA CAT AAC TG-3’ and Twist1Ra: 5’- GCT TCG GTT GTC GCC TGT
CGA GC-3’) were designed for nested 5’ RACE experiment.
Similarly, two non-
overlapping primers complimentary to the 3’ region of the incomplete cDNA sequence
(Twist1F: 5’GCC ACG ACC CGC AAT CTG-3’ and Twist1Fa: 5’-GTC CAT GTC
AAC ATC TCA CTA ACGC-3’) were designed for the nested 3’ RACE experiment.
Other primers that were provided by the kit and used were Universal Primer Mix (UPM),
Nested Universal Primer (NUP) and Control TFR primer. The PCR conditions for the
RACE experiments were 5 thermal cycles of 94oC for 10 sec and 72oC for 3 min, 5
thermal cycles of 94oC for 10 sec, 70oC for 20 sec and 72oC for 3 min followed by 5
thermal cycles of 94oC for 10 sec, 68oC for 20 sec and 72oC for 3 min. Amplified
products were analyzed by agarose gel electrophoresis (Figure 2.2). Sequencing was
carried out using BigDye® Terminator v3.1 cycle sequencing kit (on an ABI 3100
Genetic Analyzer (Applied Biosystems)).
18
NM_130984 (25-Apr-2005)
1 accgagcctc tgaccccatt ccgtcggact catttttgcc acgacccgca atctgagctt
Twist1F
61 ttccagaggt gatgtttgag gaagaggcga tgcacgagga ctccagctct ccagagtctc
121 cggtggacag tctgggaaac agcgaggagg agctcgacag gcgacaaccg aagcgcgtca
Twist1Ra
181
241
301
361
421
481
gcaggaaaaa
agaggagcaa
cgcagcgcgt
tcgcctccct
cgctcaaact
tggactccaa
acgcgccagc
gaagtgcagc
catggcgaac
gcgcaaaatc
cgcggcccgg
gatgtccagc
cgcaaaaacg
aacagcagca
gtgcgcgagc
atccccacct
tacattgact
tgcagttatg
ccgaggattc
gcagcccgca
gtcagaggac
taccctcgga
tcctctgtca
tggcgcacga
cgacagtccc
atctctggag
tcagtctctg
caaactcagc
ggtcctgcag
gagactcagc
acgcccggga
gacctgcaga
aacgaggcgt
aaaatacaga
agcgatgagc
tacgcgtttt
Twist1R
541 ctgtgtggag aatggagggc gcgtggtcca tgtcaacatc tcactaacgc acggatgcac
Twist1Fa
601 gcgttgatgc agcatggtat gcga
Figure 2.1:
The incomplete cDNA sequence of zebrafish twist1b gene. GenBank
accession number NM_130984 (25-Apr-2005). Primers used for RACE
experiments are indicated by arrows. Start (ATG) and stop (TAA) codons
are underlined.
A
300bp
Figure 2.2:
M1 1 2 3 4 5
B 1
2 3
4 5
M2
500bp
Agarose gel electrophoresis of 5’ and 3’ RACE experiment. (A) 5’RACE
and (B) 3’RACE. M1 and M2 are 50bp and DNA ladder mix from
19
Fermentas respectively. The bands were amplified with Twist1Ra
(A)/Twist1Fa (B) and NUP (lane 1), TFR and NUP (positive control) (lane
2), UPM primer only (lane 3), Twist1Ra (A)/Twist1Fa (B) only (lane 4).
PCR blank is added as a negative control (lane 5).
2.3.2
Assembly of zebrafish twist1b full-length cDNA
5’UTR sequences obtained from 5’RACE is as follows:
gagaaagccc
tttcgtcgga
aggaagaggc
acagcgagga
tccgtgacgc
ctcagggaag
gatgcacgag
ggagctcgac
aggaggagac
ccacgacccg
gactccagct
aggcgacaac
gcgctgagag ggaccgagcc tctgacccca
caatctgagc ttttccagag gtgatgtttg
ctccagagtc tccggtggac agtctgggaa
cgaagc
Twist1Ra
Figure 2.3:
5’ UTR sequences of the zebrafish twist1b gene obtained from 5’RACE
experiment. Arrow indicates the position of primer. Start (ATG) codon is
underlined.
3’UTR sequences obtained from 3’RACE is as follows:
gtccatgtca acatctcact aacgcacgga tgcacgcgtt tgatgcagca tgattctcgg
Twist1Fa
cctgaggagc
aaagggaaaa
catgggagca
tgcttccgtc
caacaaccag
gaaggaaatc
tatttattta
attttaaata
aaaa
Figure 2.4:
tgaactcact
ttctggagcg
ggaattacag
cacaaaacag
catggcgtca
ggctcatatc
ttgatgattg
tgatgtaaat
ggaaggagcg
tcatgacgtc
tcagatctgt
agactcgctg
tatttttttc
agtgttaaca
tcacaatgca
atgtgtatat
gctcaaaaca
gttgcaagca
gctgttgcga
caggaaaaga
tctgaaggaa
ttttctttga
gaatagatct
tttctgcaat
agggcgaaaa
cttacagttg
cggtgaatgt
cgctcctgcg
aacacacaca
tcggtccaag
ggtgtctaca
aaaacatgat
taaggattat
tgaactacga
ggaaaacatg
cttctgacag
ctcaacgaat
aaaatacttt
tgcattttct
ttgaaataca
3’ UTR sequences of the zebrafish twist1b gene obtained from 3’RACE
experiment. Arrow indicates the position of primer. Stop (TAA) codon is
underlined.
The complete sequence of the full-length cDNA of zebrafish twist1b was obtained
and has been deposited in GenBank under accession no. DQ351987 (Figure 2.5). The
20
open
reading
frame
(ORF)
was
translated
using
an
online
software
(http://www.tw.expasy.org/).
DQ351987:
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
gagaaagccc
tttcgtcgga
aggaagaggc
acagcgagga
gccgcaaaaa
gcaacagcag
acgtgcgcga
tcatccccac
ggtacattga
gctgcagtta
gcgcgtggtc
ttctcggcct
ggattataaa
actacgacat
aaacatgtgc
ctgacagcaa
aacgaatgaa
atacttttat
attttctatt
aaatacaaaa
Figure 2.5:
2.4
tccgtgacgc
ctcagggaag
gatgcacgag
ggagctcgac
cgccgaggat
cagcagcccg
gcgtcagagg
cttaccctcg
cttcctctgt
tgtggcgcac
catgtcaaca
gaggagctga
gggaaaattc
gggagcagga
ttccgtccac
caaccagcat
ggaaatcggc
ttatttattg
ttaaatatga
a
aggaggagac
ccacgacccg
gactccagct
aggcgacaac
tccgacagtc
caatctctgg
actcagtctc
gacaaactca
caggtcctgc
gagagactca
tctcactaac
actcactgga
tggagcgtca
attacagtca
aaaacagaga
ggcgtcatat
tcatatcagt
atgattgtca
tgtaaatatg
gcgctgagag
caatctgagc
ctccagagtc
cgaagcgcgt
ccacgcccgg
aggacctgca
tgaacgaggc
gcaaaataca
agagcgatga
gctacgcgtt
gcacggatgc
aggagcggct
tgacgtcgtt
gatctgtgct
ctcgctgcag
ttttttctct
gttaacattt
caatgcagaa
tgtatatttt
ggaccgagcc
ttttccagag
tccggtggac
cagcaggaaa
gaagaggagc
gacgcagcgc
gttcgcctcc
gacgctcaaa
gctggactcc
ttctgtgtgg
acgcgtttga
caaaacaagg
gcaagcactt
gttgcgacgg
gaaaagacgc
gaaggaaaac
tctttgatcg
tagatctggt
ctgcaataaa
tctgacccca
gtgatgtttg
agtctgggaa
aaacgcgcca
aagaagtgca
gtcatggcga
ctgcgcaaaa
ctcgcggccc
aagatgtcca
agaatggagg
tgcagcatga
gcgaaaataa
acagttgtga
tgaatgtgga
tcctgcgctt
acacacactc
gtccaagaaa
gtctacatgc
acatgatttg
The complete full-length cDNA sequence of the zebrafish twist1b gene.
Start (ATG) and stop (TAA) codons are underlined. Polyadenylation
signal is double-underlined.
Genomic sequence of zebrafish twist1b
An intron within the zebrafish twist1b gene was determined by polymerase chain
reaction (PCR) amplification of genomic DNA using the gene-specific primer pair 5′
GTTTGATTCTTGGTATAACG-3′ and 5′-GATCTATTCTGCATTGTGAC- 3′.
The
PCR reaction mixture contained 100 ng of genomic DNA template, 200 μM of each
deoxyribonucleotide triphosphate (dNTP), 0.2 μM of each primer, and 1 U of
HotStarTaq™ DNA polymerase (Qiagen) in 1X supplied PCR buffer containing 1.5 mM
21
MgCl2. The amplification reaction consisted of a 15-min polymerase activation at 95°C,
followed by 30 thermal cycles of 95°C denaturation for 45 sec, 50°C annealing for 1 min,
and 72°C extension for 5 min. A final 5-min extension at 72°C completed the reaction.
The amplified products were analyzed by gel electrophoresis. The amplified fragment
was excised from the gel and extracted using GFXTM PCR, DNA and Gel Band Purification
Kit (GE Healthcare). The extracted fragment was sequenced using BigDye® Terminator
v3.1 cycle sequencing kit on an ABI 3100 Genetic Analyzer (Applied Biosystems). The
genomic sequence of zebrafish twist1b has been deposited in GenBank under accession
no. DQ191169.
2.5
RT-PCR
Total RNA was extracted from embryos at various stages using TRIZOL® RNA
isolation reagent (Invitrogen): 1-cell (0.2 hour post fertilization (hpf)), 8-cell (1.25 hpf),
64-cell (2 hpf), 1000-cell (3 hpf), shield (6 hpf), bud (10 hpf), 14-somite (14 hpf), 1 day
post fertilization (dpf), 2 dpf and 3 dpf. Intron-spanning gene-specific primer pairs for
twist1a,
twist1b,
twist2
and
twist3
transcript
detection
were
5’-
AGGTTCTACAGAGTGACGAGC-3’ / 5’-GCACAGGATTCGAACTAGAGG-3’, 5’TGTGGCGCACGAGAGACT-3’
/
5’-GATCTATTCTGCATTGTGAC-3’,
5’-
CGCACGAGAGACTCAGTTAC-3’ / 5’-CCATACAGATAGCAGATAGCC-3’, and 5’CTGAATCCCGAACTCTGATCC-3’
/
5’-GTGTTACCCGTCACTGAAG-3’,
respectively. As a control for equivalent starting amounts of RNA template, β-actin
transcript
expression
was
detected
using
the
primer
pair
CGCACTGGTTGTTGACAACG-3’ / 5’-AGGATCTTCATGAGGTAGTC-3’.
5’Total
22
RNA was reverse transcribed using SuperScript™ III reverse transcriptase (Invitrogen),
followed by PCR. The PCR reaction mixture contained 1 μl of cDNA as template, 200
μM of each dNTP, 0.2 μM of primer, and 1 U of HotStarTaq™ DNA polymerase
(Qiagen) in 1X supplied PCR buffer containing 1.5 mM MgCl2. The amplification
reaction consisted of a 15-min polymerase activation at 95°C, followed by 35 thermal
cycles of 95°C denaturation for 45 sec, 60°C annealing for 1 min, and 72°C extension for
5 min. A final 5-min extension at 72°C completed the reaction.
2.6
Synthesis of RNA probes for in situ hybridization analysis
2.6.1
Identification of unique 3’UTR sequences of the zebrafish twist gene family
Alignment of zebrafish twist gene family full-length cDNA was carried out using
Vector NTI® Suite 7.0 to identify unique sequences used as probes for in situ
hybridization.
2.6.2
Isolation of unique 3’UTR sequences of zebrafish twist gene family
Zebrafish twist1a, twist1b, twist2 and twist3 3’UTR were amplified from reverse-
transcribed
RNA
of
24
hpf
GAAAACACGAGGACCAATG-3’
embryos
/
CTGAACTCACTGGAAGGAGC-3’
/
using
the
primer
pairs
5’-
5’-GAATTGTACTAAAGCTTTGTA-3’,
5’-
5’-GATCTATTCTGCATTGTGAC-3’,
5’-
GAACGGACTGTTTACTTCCAC-3’ / 5’-CCATACAGATAGCAGATAGCC-3’ and
5’-CTGAATCCCGAACTCTGATCC-3’
/
5’-CGACATCTCATCCTATTAGCG-
3’respectively (Figure 2.6 to Figure 2.9)
23
EF620930 (cDNA sequence of twist1a):
1
61
121
181
241
301
361
421
481
541
601
661
721
ctcctctcaa
cacaaagttg
tgtatttcac
aagcgagatg
cagtctcagc
acggtcgagc
aaagtctagc
gatggcgaac
acgcaaaatc
cgccgccagg
gatggcaagt
gatggagggc
tttgttttta
acactttacc
cttggaatac
cctcatgctg
cccgaagagc
aacagcgacg
aagaaaaacg
aacagcagca
gtgcgcgagc
atccccactt
tacatcgatt
tgtagttatg
gcttggtcca
atggtcaacc
agactataag
ctagtgatct
gaataacgtc
ccgcgcgaga
gagagcccga
gggaggattc
acagccctca
gacagaggac
taccttccga
ttctctgtca
ttgctcacga
tgtctgcatc
cgtgagctgg
agctccctaa
tctccagaac
cttattcgca
ctcctccagc
caggccacca
cgatagctcg
gtctttcgag
gcagtcgctc
taaactgagc
ggttctacag
gcgtttgagc
tcactagtgt
gaaaacacga
cttttttcct
acgaaacgta
cgcgctttca
tcccccgtgt
aaaaggtgcg
acccttggga
gagctgcaga
aacgaagcgt
aaaatacaga
agtgacgagc
tacgcgttct
gcagggaaac
ggaccaatgc
ttcaacctaa
cgcgtggaat
gcagagactt
ctcccgcgga
caaggaaaag
aaagggggaa
cgcagcgcgt
ttgcggcttt
cgctgaaact
tggactccaa
cggtttggag
tttttcttgt
taattccatc
Ztwist1a-F
781
841
901
961
1021
ataatcttgg
ccacggacag
aatatgtgca
taactttctg
gaagcctcta
ggaaaacggc
cgaattgtca
tatgcatgtt
gaagaaagtg
gttcgaatcc
aaatgttcca
tatggatttc
tttttttttt
aatttgcatt
tgtgcaaata
acagaggtca
ctcccgagtc
tttttttttt
acaaggactg
caaagcttta
tggctgttac
ttatgacgac
ttggaagact
tcgataagaa
gtacaattct
cgagagaagg
gaatgttgga
cagatgtgca
aatgggaatt
atttatttat
Ztwist1a-R
1081 tgatgacaca ctttttgaaa tgaaagtaaa tgtatcaaat gtgttgaaat gcattattat
1141 tttttattac ttttgtaaat aaatgtgtat ttctgtaata aaaaaatgaa gaattttaag
1201 aaata
Figure 2.6:
cDNA sequence of zebrafish twist1a. Positions of primers are indicated
by arrows and the 3’UTR region used for the synthesis of RNA probe for
in situ hybridization is highlighted in grey. Start (ATG) and stop (TAG)
codons are underlined. Polyadenylation signal is double-underlined.
24
DQ351987 (cDNA sequence of twist1b):
1
61
121
181
241
301
361
421
481
541
601
661
gagaaagccc
tttcgtcgga
aggaagaggc
acagcgagga
gccgcaaaaa
gcaacagcag
acgtgcgcga
tcatccccac
ggtacattga
gctgcagtta
gcgcgtggtc
ttctcggcct
tccgtgacgc
ctcagggaag
gatgcacgag
ggagctcgac
cgccgaggat
cagcagcccg
gcgtcagagg
cttaccctcg
cttcctctgt
tgtggcgcac
catgtcaaca
gaggagctga
aggaggagac
ccacgacccg
gactccagct
aggcgacaac
tccgacagtc
caatctctgg
actcagtctc
gacaaactca
caggtcctgc
gagagactca
tctcactaac
actcactgga
gcgctgagag
caatctgagc
ctccagagtc
cgaagcgcgt
ccacgcccgg
aggacctgca
tgaacgaggc
gcaaaataca
agagcgatga
gctacgcgtt
gcacggatgc
aggagcggct
ggaccgagcc
ttttccagag
tccggtggac
cagcaggaaa
gaagaggagc
gacgcagcgc
gttcgcctcc
gacgctcaaa
gctggactcc
ttctgtgtgg
acgcgtttga
caaaacaagg
tctgacccca
gtgatgtttg
agtctgggaa
aaacgcgcca
aagaagtgca
gtcatggcga
ctgcgcaaaa
ctcgcggccc
aagatgtcca
agaatggagg
tgcagcatga
gcgaaaataa
tgacgtcgtt
gatctgtgct
ctcgctgcag
ttttttctct
gttaacattt
caatgcagaa
gcaagcactt
gttgcgacgg
gaaaagacgc
gaaggaaaac
tctttgatcg
tagatctggt
acagttgtga
tgaatgtgga
tcctgcgctt
acacacactc
gtccaagaaa
gtctacatgc
Ztwist1-F
721
781
841
901
961
1021
ggattataaa
actacgacat
aaacatgtgc
ctgacagcaa
aacgaatgaa
atacttttat
gggaaaattc
gggagcagga
ttccgtccac
caaccagcat
ggaaatcggc
ttatttattg
tggagcgtca
attacagtca
aaaacagaga
ggcgtcatat
tcatatcagt
atgattgtca
Ztwist1-F
1081 attttctatt ttaaatatga tgtaaatatg tgtatatttt ctgcaataaa acatgatttg
1141 aaatacaaaa a
Figure 2.7:
cDNA sequence of zebrafish twist1b. Positions of primers are indicated
by arrows and the 3’UTR region used for the synthesis of RNA probe for
in situ hybridization is highlighted in grey. Start (ATG) and stop (TAA)
codons are underlined. Polyadenylation signal is double-underlined.
25
NM_001005956 (cDNA sequence of twist2):
1
61
121
181
241
301
361
421
481
541
601
661
aaaaaaaatt
agtaggggac
acggatatta
ctttaaaaaa
gaccagcgag
aaggaagtcg
gagcccgagc
acgcgagcgg
ccccacgctc
cattgatttc
cagctacgtc
gtggtcgatg
aggaaaaaac
gcgcgcagtg
ctagtctaaa
gaaatggaag
gaggagctgg
agcgaggaca
agcactcagt
caacggactc
ccctcggata
ctctgtcagg
gcgcacgaga
tctgcgtccc
ttcctgcggg
tcaggacttc
ccacggaacc
agagttctag
acagacagca
gcagcagccc
ccttcgagga
aatcgctgaa
aactcagcaa
tgctgcagag
gactcagtta
actagcagcg
aggaaaaccg
aggaggacag
aaaagtggag
ctctcccgtc
gaaaaggttc
gagctccgtc
gctccagaac
cgaagccttc
gatccagacg
cgacgagatg
cgcgttttca
agacgcgtcc
tgccaagctc
acaaaatccc
atcgtatttt
tccccagtgg
gggaggaaaa
aataaacgta
cagcgcgtcc
gcgtctttgc
ctcaaactcg
gacaacaaga
gtgtggagga
tgataatgcc
tacttctgga
caacaacaaa
ctcctcgttt
acagcctggt
ggaggcaagg
acaaaaagcc
tggcgaacgt
gcaaaatcat
catccaggta
tgtccagttg
tggagggcgc
gaacggactg
721 tttacttcca ctaattttga ggatgccaaa ggattattcg atgaacctct aaacctcagt
Ztwist2-F
781
841
901
961
gacgtggcca
acaataaaag
caaatacatt
aaggctatct
aaggacattc
ggacttttta
taatcttttt
gctatctgta
agtggataca
atatacttag
cacaagttat
tggaaactat
tctacggact
aggaaaagta
tcttaaatgt
atttcatgtc
tgagcatcct
gatgacgtct
cgccatgatg
tgtctctaaa
catgaaggac
tctgtgtcgt
atagtacagg
cagaagagtt
Ztwist2-R
1021 tatatatata taagaacaaa aaaaaaaaaa aaaa
Figure 2.8:
cDNA sequence of zebrafish twist2. Positions of primers are indicated by
arrows and the 3’UTR region used for the synthesis of RNA probe for in
situ hybridization is highlighted in grey. Start (ATG) and stop (TAG)
codons are underlined.
26
NM_130985 (cDNA sequence of twist3)
1
61
121
181
241
301
361
421
481
541
601
661
721
781
ggggacaaac
attgcatttg
ccacagagct
acacggagga
gatccttccc
tccaccggcg
cgacaaacca
tccgtcatcc
agttcccgga
acgccagcgc
cttgccttcg
cttcctctac
tctggcccac
catgtccgcc
agccacatga
atttctaaca
cgcttcagtg
ctgatcgtca
attgaagaag
ggcgcacgca
tccctggaca
tcatcgtctt
cagcccttcg
acacagtccc
gacaagctga
caggtcctgc
gagaggctga
actcactaaa
cacacacaca
acaaacccaa
tttaactttg
tgcgagagga
agcaggagcg
agcggctgac
acccatcgaa
cttcgtcctc
aagacctcca
tgaacgacgc
gcaagatcca
agagcgacga
gctacgcctt
catccacccg
catacacaca
agcaatggct
acaccgctca
acagacttgt
gcgccccaat
gggtcccaaa
tctggctccc
tctggtgccc
cacgcaacga
cttcgcctcc
gatcctcaaa
gatggacgcc
ctccgtctgg
ttcctcatct
cacacacaca
gtataactct
caggtacagc
ggagattttc
aagtgtgcgg
aaagagcccg
aaacgtccca
gtcgtgagtt
gtgatcgcca
ctcagaaaga
ctggcctcca
aagctggcca
aggatggagg
caaaaactga
cactgcagag
cacagagatc
aggagcactg
ctgaaagtgg
ttgtggtttc
tctcacaaga
aaagaagctc
cggtttctcc
acgttcggga
tcatccccac
gatacatcga
gctgcaacta
gcgcctggtc
atcccgaact
841 ctgatcctga ttttcatctc tatatccact ccacacggcc tccgactcac cctgccatga
Ztwist3-F
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
ccccaccctg
gtttcccaca
cctaatggag
agagcatttc
tcagatcagt
tgttctgagg
gcttcattat
attcctgctt
taatgcttca
gtggctttaa
aagacattta
atagcagggt
ctgtttgagt
cctaaagatg
accccagagg
ctttaccagc
cacaagactg
gttcctccat
actcactaac
gcagatgtga
cagtgacggg
agattcactc
tgggggaatg
gaaagatgct
tcatgtgagt
acatctttac
acctctgacc
acttcatgct
aaacgcagga
aatccagcac
gttgactgtt
cctgacctga
gagtgattgt
taacactgtg
tattcctcta
aggagctgta
ctgcttcagg
ggacttggtg
agcagatcca
tctctcactt
tcatctacaa
gagaaagagt
tgagctgagt
ctctggactc
tggatcaagg
gcttgtgttt
taaaccgcca
ttaaaagaga
catcaataat
attcagtcaa
attataaata
ggcagcggtc
ttccaaatca
gccgacagtt
cttgaggagc
ggaagtgact
atgtttgggc
cttcctccgc
ctgctgtata
gttcatgtaa
ctttaactgc
gatgtgtcgg
acgtctgcaa
tttctacaat
gctaatagga
agccttcgca
ttcccctttc
gcagatgagc
gctgctgatc
agatattgac
tctttatatt
ttagccattc
aataatcaaa
agaagtgtta
agtacagagg
acgtataaag
aatgaagaag
tgagatgtcg
Ztwist3-R
1681
1741
1801
1861
gctaaacttg
actcgatgag
tcaataaaca
aaaaaaaaaa
Figure 2.9:
gtgttggtga
tagctaacag
aacaataaga
aaaaaaaaaa
tggtgtgaaa
actaagagtt
tgaaaaaaaa
aattaaaaaa
ataaactgaa atgaagcgta cagcaggaga
tacgctgatt atcaagagct tgaaaattca
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa
cDNA sequence of zebrafish twist3. Positions of primers are indicated by
arrows and the 3’UTR region used for the synthesis of RNA probe for in
situ hybridization is highlighted in grey. Start (ATG) and stop (TAA)
codons are underlined. Polyadenylation signal is double-underlined.
27
The amplified products were analyzed by gel electrophoresis. Each amplified
fragment was excised from the gel and extracted using GFXTM PCR, DNA and Gel Band
Purification Kit (GE Healthcare). Amplified fragments of twist1b and twist3 were cloned
into a pBlueScript®II KS(+) vector (Stratagene) and amplified fragments of twist1a and
twist2 were cloned into pCR®2.1-TOPO vector (Invitrogen). The inserts region of the
plasmid DNA were sequenced using BigDye® Terminator v3.1 cycle sequencing kit on
an ABI 3100 Genetic Analyzer (Applied Biosystems) to confirm the clones.
2.6.3
Linearization of plasmid DNA
For generation of antisense RNA probes, 3’UTR-twist1a and twist2 plasmid DNA
were digested with EcoRV restriction enzyme; 3’UTR-twist1b and twist3 plasmid DNA
were digested with HindIII restriction enzyme. For generation of sense RNA probes,
3’UTR-twist1a and twist2 plasmid DNA were digested with XbaI restriction enzyme;
3’UTR-twist1b and twist3 plasmid DNA were digested with BamHI restriction enzyme.
The linearized products were analyzed by gel electrophoresis. RNase free reagents, tips
and tubes are used subsequently.
The linearized plasmids were purified once with
phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 13,000 rpm for 5 mins
and residual phenol was removed by chloroform:isoamyl alcohol(24:1) extraction. The
aqueous phase was transferred to a fresh tube and precipitated with 0.1 volume of 3M
sodium acetate (pH5.2), 2.5 volumes of cold absolute ethanol and 20 μg of glycogen
(Invitrogen) and incubated at -20oC for 20 min. The solution was centrifuged at 13,000
rpm for 20 min. The supernatant was removed and DNA pellet was washed with 250 μl
28
of cold 70% ethanol and centrifuged at 13,000 rpm for 10 min. The supernatant was
removed, pellet was air-dried and dissolved in 0.1% DEPC water.
2.6.4 RNA labeling with Digoxenin/Fluorescein RNA labeling kits (SP6/T7/T3)(Roche)
One μg of linearized plasmid was transferred to a 200 μl PCR tube. The labeling
reaction contained 2 μl of 10X NTP labeling mixture (Digoxenin or Fluorescein), 2 μl of
10X transcription buffer, 1μl of RNase inhibitor and 2 μl of RNA polymerase. DEPC
treated water was used to top up the reaction to final volume of 20 μl. The mixture was
mixed and incubated at 37oC for 2 hours. Plasmid DNA was degraded with 2 μl of
DNase I (Roche) at 37oC for 1 hour.
Antisense twist1b and twist3 RNA probes were synthesized using T7 RNA
polymerase, while negative control sense twist1b and twist3 RNA probes were
synthesized using T3 RNA polymerase. Antisense twist1a and twist2 RNA probes were
synthesized using SP6 RNA polymerase and negative control sense twist1a and twist2
RNA probes were synthesized using T7 polymerase.
2.6.5
Purification of RNA probe
The RNA probes were precipitated with 0.5 volume of 10M ammonium acetate, 3
volumes of cold absolute ethanol and 20 μg of glycogen (Invitrogen) at -80oC for 30 min
or longer. The mixture was centrifuged at 13,000 rpm at 4oC for 30 min. The pellet was
washed with 300 μl of cold 70% ethanol, air-dried and dissolved in 40-75 μl of 0.1%
DEPC water. The RNA probes were further purified through the Chroma SpinTM-100
29
columns (Clontech). The RNA probes were kept at -80oC and the quality of the RNA
probes were checked by gel electrophoresis.
2.7
Whole mount in situ hybridization
4 g paraformaldehyde (Sigma) was added into 100 ml of 1X PBST (1/10 dilution
from 10X PBS (1st Base) and 0.1% Tween 20) to achieve final concentration of 4%. The
fixative was incubated at 70oC until it is completely dissolved. Embryos younger than
24-somite stage were fixed with 4% paraformaldehyde and incubated overnight at room
temperature prior to dechorionation.
Embryos older than 24-somite stage were
dechorionated prior to fixation. After fixation, the embryos were washed twice with 1X
PBST to remove the fixative and dehydrated by three washes in absolute methanol for 5
min each. The dehydrated embryos were stored in absolute methanol at -20oC until next
use.
The embryos were rehydrated in 75% methanol/25% 1X PBST, followed by 50%
methanol/50% 1X PBST, 25% methanol/75% 1X PBST and four washes with 100% 1X
PBST, 5 min each.
Proteinase K was used to digest protein on the embryos aiding in the penetration
of RNA probes into tissues. Embryos at and older than 18-somite stage were treated with
5 or 20 μg/ml proteinase K prepared in 1X PBST at room temperature. The incubation
time with proteinase K varied with the stage of the embryos. Embryos at 18-somite stage
were treated with 5μg/ml proteinase K for 5 min; embryos at 24-hpf stage were treated
30
with 5 μg/ml proteinase K for 12 min; embryos at 48-hpf stage were treated with 5 μg/ml
proteinase K for 25 min; embryos at 72-hpf were treated with 20 μg/ml proteinase K for
20 min. Proteinase K activity was inactivated by 20 min wash in 4% paraformaldehyde.
The embryos were then washed in 1X PBST for five times, 5 min each.
Prehybridization was performed by immersing the embryos in hybridization
buffer (50-60% formamide (Merck), 5X SSC (150mM NaCl and 15mM sodium citrate),
500 μg/ml yeast RNA (sigma), 50μg/ml heparin (sigma) and 0.1% Tween 20, adjusted to
pH 7.0 with 1M citric acid) for 4 hours at 55-68oC. The prehybridized embryos were
transferred into fresh hybridization buffer containing 500 ng/ml labeled RNA probe. The
embryos were incubated overnight at the same temperature used for prehybridization
with gently shaking. The formamide concentration in the hybridization buffer and the
hybridization temperature were used at the optimized condition.
After hybridization, the embryos were washed at the hybridization temperature
with 50% or 60% formamide/2X SSC, 37.5 or 45% formamide/2X SSC, 25% or 30%
formamide/2X SSC, 12.5 or 15% formamide/2X SSC for 10 min each, followed by two
washes in 0.2X SSC/0.1% Tween 20 for 30 min each. Embryos were then washed at
room temperature with 0.15X SSC/1X PBST, 0.1X SSC/1X PBST and 0.05X SSC/ 1X
PBST for 10 min each.
The hybridized embryos were blocked in blocking buffer prepared with 2%
Boehringer Blocking Reagent (Roche) and 150 mM NaCl in DEPC water for 2 hours.
31
After blocking, the embryos were incubated overnight at 4oC in 1:4000 dilution of antiDigoxenin (Roche) or 1:2500 dilution of anti-Fluorescein Fab (Roche) in blocking buffer.
The embryos were washed four times with maleic acid buffer (150 mM maleic acid
(Sigma) and 100 mM NaCl) for 20 min each.
The embryos were washed three times in alkaline phosphatase buffer (100 mM
Tris-HCl at pH9.5 (detection of anti-Dig AP Fab fragments) or pH8.2 (detection of antiFluorescein-AP Fab fragments), 50 mM MgCl2, 100 mM NaCl and 0.1% Tween 20) for 5
min each. The embryos were the incubated in the dark in the substrate solution for color
signal development. There were three types of substrates used in this study: 4-Nitroblue
tetrazolium chloride/ 5-Bromo-4-chloro-3-indolylphosphate (NBT/BCIP) and BM-Purple
substrate (Roche) were used for the detection of anti-Dig-AP Fab fragments.
The
staining buffer for NBT/BCIP was prepared with 4.5 μl of NBT (100 mg/ml in
dimethylformamide (Roche) and 3.5 μl of BCIP (50mg/ml in dimethylformamide (Roche)
in 1 ml of alkaline phosphatase buffer. The staining buffer was replaced with freshlymade substrate solution upon turning purple. As for BM-purple substrate, it was added
directly to stain the embryos. The other substrate, Fast Red (Roche) was used to detect
anti-Fluorescein-AP Fab fragments. One tablet of Fast-red was dissolved in 4 ml of
alkaline phosphatase buffer (100 mM Tris-HCl at pH8.2, 50 mM MgCl2, 100 mM NaCl
and 0.1% Tween 20). The solution was centrifuged to remove the particles. In another
tube, 40 μl of NAMP stock (Naphthol AS-MX phosphate (Sigma) prepared in dimethyl
sulfoxide (50 ng/ml) was added into 4ml of alkaline phosphatase (pH8.2). The Fast Red
staining solution was prepared by mixing the 4ml of Fast red solution and 4ml of alkaline
32
phosphatase with 40 μl of NAMP (50 mg/ml). The embryos were stained for not more
than 48 hours in the dark at room temperature until the desired signal was developed.
For double color in situ hybridization, both Dig-labeled and Fluorescein-labeled
RNA probes were added into the hybridization buffer after the embryos were
prehybridized. After blocking, the embryos were incubated with one antibody, either
anti-Dig Fab fragments or anti-Fluorescein Fab fragments. After the first color detection
was completed, the staining was stopped by washing the embryos three times in 1X
PBST, 5 min each. The embryos were incubated in 0.1M Glycine (pH2.2 (Sigma)) for 1
hour to inactivate the first antibody. The embryos were washed three times in maleic
acid buffer for 5 min each. After which, the embryos were blocked with blocking agent,
followed by incubation with the second antibody. The embryos were washed and stained
as mentioned above.
When the color development was completed, the staining was terminated by
washing the embryos thrice in 1X PBST for 5 min each and the embryos were re-fixed in
4% paraformaldehyde for half an hour and stored in 30% glycerol/1X PBST at 4oC.
The table below describes the restriction enzymes and RNA polymerases used to
generate RNA probes and in situ hybridization conditions used in this study.
33
Table 1: The synthesis of RNA probes and in situ hybridization conditions
Gene name
Restriction enzyme for RNA polymerase
In situ hybridization
linearization of plasmid
conditions
EcoRV (antisense)
SP6 (antisense)
50% formamide, 60oC
twist1a
XbaI (sense)
T7 (sense)
HindIII (antisense)
T7 (antisense)
50% formamide, 68oC
twist1b
BamHI (sense)
T3 (sense)
EcoRV (antisense)
SP6 (antisense)
50% formamide, 60oC
twist2
XbaI (sense)
T7 (sense)
EcoRV (antisense)
T7 (antisense)
60% formamide, 68oC
twist3
XbaI (sense)
T3 (sense)
SacII (Cfr42I)
T7
50% formamide, 68oC
pax2.1
BamHI
T7
50% formamide, 68oC
dlx2a
NotI
T7
50% formamide, 68oC
wt1
XbaI
T7
50% formamide, 60oC
fli1a
2.8
Cryosection
Zebrafish embryos were fixed in 4% paraformaldehyde/1X PBS for half an hour
and washed three times in 1X PBS. Agar-sucrose solution (1.5% Bacteriological agar
(Sigma) in 5% sucrose) was prepared and cooled to 40-50oC. An embryo was transferred
with a drop of 1X PBS to a cap of 1.5 ml microfuge tube (as a mold for making agar
block). Excess fluid was removed and agar-sucrose solution was added to cover the
embryo. A syringe needle was used to manipulate the embryos to ensure even coating
and correct position. The agar block was cooled to solidify. The agar blocked was
removed, trimmed into a “pyramid” shape and immersed into 30% sucrose solution at
4oC overnight. A raised platform was made with a layer of tissue freezing medium to the
cryostat chuck and left at -20oC for 1hr. A small drop of tissue freezing medium was
added to the raised platform. The agar block was positioned onto the raised platform of
the cryostat chuck and lowered into liquid nitrogen surface.
The block/chuck was
34
equilibrated in the cryostat machine prior to sectioning for 30 min or longer. The agar
block was cryosection at 10-15 μm thickness using a cryostat machine (Microtome
cryostat HM525 (Microm International)). The sections were overlaid onto poly-lysine
coated slides (Sigma) and heated at 42oC for 30 mins on a hot plate. The slides were left
to air-dry overnight. The section slides were washed three times with 1X PBS, 5 min
each. The sections were mounted in 50% glycerol/1X PBS solution by a large coverslip
with nail polish.
2.9
Image processing
All images were digitally captured using the MicroPublisher™ 5.0 color digital
camera (QImaging™) and processed using Image-Pro® Plus version 5 software (Media
Cybernetics).
2.10
Phylogenetic analysis
2.10.1 Alignment and phylogenetic tree
Multiple sequence alignments of Twist protein/ Twist cDNA sequences were
generated using Vector NTI® Suite 7.0.
Inter-species sequence comparisons and
similarity calculations were performed using the AlignX software in Vector NTI® Suite
7.0.
Phylogenetic tree was generated by the Neighbor-Joining method (Saitou and Nei,
1987) using PHYLIP software, with default gap opening penalty of 10 and gap extension
35
penalty of 0.2, and with bootstrap analysis of 1000 replicates. Gaps that were present in
the alignment were removed before phylogenetic tree generation. Phylogenetic analysis
was performed with four zebrafish Twist peptides and 29 other Twist peptides from 11
species, based on GenBank cDNA sequences NM_000474 (human TWIST1),
NM_057179 (human TWIST2), NM_053530 (rat Twist1), NM_021691 (rat Twist2),
NM_011658 (mouse Twist1), NM_007855 (mouse Twist2), NM_204739 (chick Twist1),
NM_204679 (chick Twist2), BK006265 (chick Twist3), NM_204084 (frog twist1),
NM_001103209 (frog twist3), BK006281 (spotted green pufferfish twist1a), BK006282
(spotted green pufferfish twist1b), BK006283 (spotted green pufferfish twist2),
NM_001104599 (fugu twist1a), NM_001104598 (fugu twist1b), NM_001104600 (fugu
twist2), BK006268 (medaka twist1a), BK006269 (medaka twist1b), BK006270 (medaka
twist2), BK006271 (medaka twist3a), BK006272 (medaka twist3b), BK006276
(stickleback twist1a), BK006277 (stickleback twist1b), BK006278 (stickleback twist2),
BK006279 (stickleback twist3a), BK006280 (stickleback twist3b), EF620930 (zebrafish
twist1a),
NM_130984
(zebrafish
twist1b),
NM_001005956
(zebrafish
twist2),
NM_130985 (zebrafish twist3), AF097914 (Japanese lancelet twist), and ABW34714 (sea
urchin twist).
2.10.2 Calculation of genetic distances
Coding cDNA sequences of zebrafish, medaka, stickleback and fugu twist1a and
twist1b used were taken from GenBank and the accession numbers were as above.
Kimura 2-parameter nucleotide distances and Tajima-Nei corrected number of
36
substitutions per site at third codon positions were computed using MEGA 4.0 software
(Tamura et al., 2007).
2.10.3 Comparative synteny analysis
The map locations of the orthologous genes in human and zebrafish were obtained from
NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/projects/mapview/), Human genome
view (Build 36.3, March 2008) and Zebrafish genome view (Zv 7, July 2008)
respectively. The map locations of Medaka twist1a (BK006268), twist1b (BK006269),
twist2 (BK006270), twist3a (BK006271) and twist3b (BK006272) were determined using
the
search
engine
on
the
Ensembl
Medaka
Blast
View
(http://www.ensembl.org/Oryzias_latipes/blastview ). Other orthologous gene locations
in Medaka were obtained from the Ensembl Oryzias latipes genome website
(http://www.ensembl.org/Oryzias_latipes/index.html) (Ensembl release 50, July 2008).
37
Chapter 3: Results
_____________________________________________________________
3.1
Characterization of zebrafish full-length cDNA of twist1b
Using the incomplete cDNA sequence of twist1b, 5’ and 3’ RACE experiments
was performed. The complete full-length cDNA of the zebrafish twist1b was obtained.
The 1,151 bp full-length cDNA of zebrafish twist1b comprises of a 113 bp 5’UTR, a 516
bp ORF encoding a predicted 171 amino acid protein, and a 522 bp 3’UTR. The deduced
amino acid sequence has a bHLH domain (Gln78 to Ser134) and a WR motif (Glu150 to
Gly163). The sequence has been deposited to GenBank under the accession number
DQ351987 using the old name of twist1.
38
DQ351987:
1 gagaaagccc tccgtgacgc aggaggagac gcgctgagag ggaccgagcc tctgacccca
61 tttcgtcgga ctcagggaag ccacgacccg caatctgagc ttttccagag gtgatgtttg
M F
121 aggaagaggc gatgcacgag gactccagct ctccagagtc tccggtggac agtctgggaa
E E E A
M H E
D S S
S P E S
P V D
S L G
181 acagcgagga ggagctcgac aggcgacaac cgaagcgcgt cagcaggaaa aaacgcgcca
N S E E
E L D
R R Q
P K R V
S R K
K R A
241 gccgcaaaaa cgccgaggat tccgacagtc ccacgcccgg gaagaggagc aagaagtgca
S R K N
A E D
S D S
P T P G
K R S
K K C
301 gcaacagcag cagcagcccg caatctctgg aggacctgca gacgcagcgc gtcatggcga
S N S S
S S P
Q S L
E D L Q
T Q R
V M A
361 acgtgcgcga gcgtcagagg actcagtctc tgaacgaggc gttcgcctcc ctgcgcaaaa
N V R E
R Q R
T Q S
L N E A
F A S
L R K
421 tcatccccac cttaccctcg gacaaactca gcaaaataca gacgctcaaa ctcgcggccc
I I P T
L P S
D K L
S K I Q
T L K
L A A
481 ggtacattga cttcctctgt caggtcctgc agagcgatga gctggactcc aagatgtcca
R Y I D
F L C
Q V L
Q S D E
L D S
K M S
541 gctgcagtta tgtggcgcac gagagactca gctacgcgtt ttctgtgtgg agaatggagg
S C S Y
V A H
E R L
S Y A F
S V W
R M E
601 gcgcgtggtc catgtcaaca tctcactaac gcacggatgc acgcgtttga tgcagcatga
G A W S
M S T
S H *
661 ttctcggcct gaggagctga actcactgga aggagcggct caaaacaagg gcgaaaataa
721 ggattataaa gggaaaattc tggagcgtca tgacgtcgtt gcaagcactt acagttgtga
781 actacgacat gggagcagga attacagtca gatctgtgct gttgcgacgg tgaatgtgga
841 aaacatgtgc ttccgtccac aaaacagaga ctcgctgcag gaaaagacgc tcctgcgctt
901 ctgacagcaa caaccagcat ggcgtcatat ttttttctct gaaggaaaac acacacactc
961 aacgaatgaa ggaaatcggc tcatatcagt gttaacattt tctttgatcg gtccaagaaa
1021 atacttttat ttatttattg atgattgtca caatgcagaa tagatctggt gtctacatgc
1081 attttctatt ttaaatatga tgtaaatatg tgtatatttt ctgcaataaa acatgatttg
1141 aaatacaaaa a
Figure 3.1:
3.2
Full-length cDNA sequence of zebrafish twist1b (in lowercase) and its
deduced amino acid sequence (in UPPERCASE single letter code). The
ATG start and TAA stop codon are underlined, and the polyadenylation
signal is double-underlined. The bHLH domain is highlighted in light
grey and the WR motif is highlighted in dark grey.
Genomic organization of zebrafish twist1b
The zebrafish twist1b gene, as in its orthologs in other organisms has a dual-exon
genomic structure. A 884 bp intron was amplified and the sequence was generated by
39
sequencing. The genomic DNA sequence of twist1b has been deposited to GenBank
under the accession number DQ191169 (Figure 3.2).
DQ191169:
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
2281
2341
2401
2461
gtagttgtat
tttgtcatga
atcactattc
ttctttgaca
caacagcgtg
cgtttgattc
ttacatcact
ctataagatc
cgcgctgaga
gcaatctgag
tctccagagt
ccgaagcgcg
cccacgcccg
gaggacctgc
ctgaacgagg
agcaaaatac
cagagcgatg
agctacgcgt
cgcacggatg
aaaacttgca
gttagattga
aaataattat
tgaatttaaa
atatagattg
cccatcaaac
atgttttcac
ataattttaa
taatgtagaa
cagattttta
aataatctgg
ctttttatga
cttgaataat
aattaatgca
cttcgttttt
caagggcgaa
cacttacagt
gacggtgaat
gacgctcctg
aaaacacaca
gatcggtcca
ctggtgtcta
ataaaacatg
tcacagtaca
tgaatcagtc
tgacatgtca
caaaaaatgg
cctatgttgt
ttggtataac
cagcttcttg
ctctgaactt
gggaccgagc
cttttccaga
ctccggtgga
tcagcaggaa
ggaagaggag
agacgcagcg
cgttcgcctc
agacgctcaa
agctggactc
tttctgtgtg
cacgcgtttg
cttgaatatc
ttatgaaaca
tcgttgtatt
gaaacaaatt
tttgcgactt
tcgttcttga
gtgttttggt
atgtttggcc
ctaaattaaa
aaaacatatt
tgttatttat
actgttgtct
gtttttttat
taataattat
ttagattctc
aataaggatt
tgtgaactac
gtggaaaaca
cgcttctgac
cactcaacga
agaaaatact
catgcatttt
atttgaaata
tctgcatctg
tacaattaat
tgaatggatg
ctttcaattc
gtttatttta
ggaacccttt
tccaatcgca
cccttcgagt
ctctgacccc
ggtgatgttt
cagtctggga
aaaacgcgcc
caagaagtgc
cgtcatggcg
cctgcgcaaa
actcgcggcc
caagatgtcc
gagaatggag
atgcagcatg
aagggttttg
gtttcacatt
tgttttttaa
aaataatgtt
accttaaaca
aaatctcatg
ggctccataa
ataataaaga
acatatttaa
caaaatttaa
aaacaatctt
agtaataatt
ttactgtata
tagataaaca
ggcctgagga
ataaagggaa
gacatgggag
tgtgcttccg
agcaacaacc
atgaaggaaa
tttatttatt
ctattttaaa
caaaaa
agtctaaaat
ttgtcagcgc
atggatgctc
tattttaatg
ttttaaatga
ggatcgcggt
ggccagccca
tgagaaagcc
atttcgtcgg
gaggaagagg
aacagcgagg
agccgcaaaa
agcaacagca
aacgtgcgcg
atcatcccca
cggtacattg
agctgcagtt
ggcgcgtggt
gtatgcgatg
gataaccagt
cacgttgtag
ggtaagtgat
cagcttaatt
cgtaaataca
aaaatggttg
tcctcaactt
atgagtagtt
gcatacttta
aataccgaat
ttccacttca
taaaagcagt
aaataagtac
ttgcatcaat
gctgaactca
aattctggag
caggaattac
tccacaaaac
agcatggcgt
tcggctcata
tattgatgat
tatgatgtaa
atcagcagat
aagtctcagt
atttagatat
catttcaatg
agcgctttga
cccctcccgc
aaaagttagc
ctccgtgacg
actcagggaa
cgatgcacga
aggagctcga
acgccgagga
gcagcagccc
agcgtcagag
ccttaccctc
acttcctctg
atgtggcgca
ccatgtcaac
caaaactttt
caaaagtgtg
acaattatac
tgcaaacaat
tgtttgttaa
atgtgattta
atatcacaac
ttgaagtttc
gtgtccaaac
tgctttaaat
ttgcacctaa
gacaaaccag
caatggtagc
tgttattgtt
gtctaaacta
ctggaaggag
cgtcatgacg
agtcagatct
agagactcgc
catatttttt
tcagtgttaa
tgtcacaatg
atatgtgtat
catcagttca
atttcgttgg
atttggctat
atattgacat
ttggctcaga
ctctcccttc
tccttccaga
caggaggaga
gccacgaccc
ggactccagc
caggcgacaa Exon 1
ttccgacagt
gcaatctctg
gactcagtct
ggacaaactc
tcaggtcctg
cgagagactc
atctcactaa
taaaggctca
gacacaatac
atactctgga
ttatatgggc
aattcagccc
tctatattta Intron 1
attgattttc
tcaagtttta
ttttgtctgc
gtagctaatt
aatgcttcat
tatttacaga
tattttatca
tagtaaaata
aaatattttc
cggctcaaaa
tcgttgcaag
gtgctgttgc Exon 2
tgcaggaaaa
tctctgaagg
cattttcttt
cagaatagat
attttctgca
40
Figure 3.2:
3.3
Genomic DNA sequence of zebrafish twist1b. Exons are enclosed by
brackets. The ATG start and TAA stop codons are underlined, and the
polyadenylation signal is double-underlined.
Alignment of TWIST family peptides
The alignment of the zebrafish twist family amino acid sequences against those
from human, mouse, rat, chick, Xenopus, medaka, stickleback, fugu, green spotted
pufferfish, sea urchin and lancelet revealed a high degree of inter-species sequence
conservation (Figure 3.3).
3.4
Comparison and alignment of zebrafish twist gene family
Comparison of the coding cDNA sequences showed that zebrafish twist genes are
highly conserved and a higher similarity can be observed in the bHLH region and the WR
domain (Table 2).
Table 2:
Nucleotide identity of the coding region, bHLH domain, and WR domain
of the zebrafish twist genes.
Coding cDNA
Dr1a
Dr1b
Dr2
bHLH region only
WR domain only
Dr1b
Dr2
Dr3
Dr1b
Dr2
Dr3
Dr1b
Dr2
Dr3
81
76
53
85
83
74
81
81
86
76
55
52
86
80
77
83
83
83
With the knowledge that the zebrafish twist genes have high coding nucleotide
similarity, an alignment was performed on the full-length cDNA of the zebrafish twist
41
gene family, to identify unique regions that can be used as specific probes for in situ
hybridization to minimize cross-hybridization (Figure 3.4).
42
Mm1
Rn1
Hs1
Gg1
Xt1
Tn1a
Tr1a
Ga1a
Ol1a
Dr1a
Dr1b
Ol1b
Ga1b
Tr1b
Tn1b
Bb
Lv
Ga3a
Ol3a
Ol3b
Ga3b
Xt3
Gg3
Dr3
Dr2
Ol2
Tr2
Ga2
Tn2
Gg2
Hs2
Rn2
Mm2
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPA-----SGKRGARKRRSSRRSAGGSAGPGG-ATGGGIGGGDEPGSPAQGKRGKKS-AGGGGGGGAGGGG
------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPA-----SGKRGARKRRSSRRSAGGSAGPGG-ATGGGIGGGDEPGSPAQGKRGKKS-AGGGGGAGGGGGG
------------------------MMQDVSSSPVSPADDSLSNSEEEPDRQQPP-----SGKRGGRKRRSSRRSAGGGAGPGG-AAGGGVGGGDEPGSPAQGKRGKKS-AGCGGGGGAGGGG
-----------------------MMQQDESNSPVSPADDSLSNSEEEPDRQQLP-----SAKRGGRKRRSSRR-----------SAGGAVGAADEPCSPAQGKRGKK----CGAGAGGGGGG
------------------------MMQEESSSPVSP-VDSLSNSEEELDRQ--------HSKRGCRKKRSTRKS------------------PEDPDSPISVKRNKK---------------------------------MSEENLGEESGSSPVSP-VDSLSNSEGELDR---------QPKRSGRKRRPSRKN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEESGSSPVSP-VDSLSNSEGELER---------QPKRSGRKRRPSRKN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEESSSSPVSP-ADSLSNSEGELDR---------QPKRCGRKKRPSRRN------------------GEDSDSPTPGKRGKK---------------------------------MSEENLGEDSASSPVSP-VDSVSNSEGELDR---------PPKRCGRKRRPSRKN------------------GEDSDSPTPGKRGKK----------------------------------MPEEPARDSSSSPVSP-ADSLSNSDGEPDR---------PPKRCARKRRSSKKN------------------GEDSDSSTLGKRGKK---------------------------------MFEEEAMHEDSSSPESP-VDSLGNSEEELDRR--------QPKRVSRKKRASRKN------------------AEDSDSPTPGKRSKK-------------------------------------------MREENSPPMDSAGNSEEETERQ--------LPRRGARKRRIARRSA------------DEDDEADMESSTCPGKKKCRK-SCEEGGGSAGSGS
-----------------------------MREEDSSPMDSAGNSEEENDRQ--------RPRRGARKRRATRRRAG----------DDEEEEEEDIEIPSPEKKQCRK-SCDAGGGSAGSG-----------------------------MRDEDCSPMDSAGNSEEETERQ--------LPRRGARKRRPTRRSS------------GGEEEEGDTESPSPGTKKCRK-SSEGGGGSAGSGG
-----------------------------MREEDSSPMDSAGNSEEETERL--------LPRRGARKRRPTRRSS------------GEEEEEGDTESPSSGKKKCRK-SSEGGGGSAGSAG
-----MAADVVTYTTLETFSHEDLVKPDPDASPLADHSSSGPEAEEDPSR----------SKRYERKRRYSKSSA------------SSDDGSTGGGSVGGGKSGKN--------------MVHAPKMNHDNVMTNGGGGMKYLNGRCDDLDVKREPQLDLREHFELEDDPM--------KQHRYGRKRRYRQSEGE--------DECQLGDSSDDGSSTGSGSLSHK----------------------------------------MREEVSCTNSPEGGLGASEEELERGSKK-----TSQAGNRKRSPYPKKD------------SLVQAEESSTGSPTSLLPTGP-KKVKKSPMTVVSL
--------------------------MREEVSCTSAPEGGMGASEEELERGSKK-----SLQQGNRKRSPYPKKE-------------S--LSQAEESCTGSPSSLLP-SGPKRPKKSPTAV
------------------MRKEQASRNEHPEERVAVSEGKTGKPFSNTCPVVVA-----TPGLTGRKRQMGSHTEDDLTPVTP-PSSVDNQVKDGDGFQVYSPSGPKRLKRSPQHPSSSSGP
------------------MREEEQSNEDHPEGGVVSSEENTVRPPSNTCPVVVA-----TPGVTGRKRHTGSHPEDHVTTVTPTTSAHNGKTKPVEDIQIHALTGPKRHKRSSQDRSPSSGT
--------------------------MKEEGVCPDSPEGSMVTSEEEADRQ---------QRKAIRKRTILVGKP----------------SEGRVPLSPPCKRNKR----------------------------------------MKEENMCPESPEGSLVTSEEEGERL---------HKKCLRKRGQAGKAL-----------------EDGGAASPQGKRCKR----------------------------------------MREEQTCGDFPESGILPIEEEQERRPNKCAVVVSPPAGARKRLTGPKKE------------PVSQDDKPSLDNPSNLAPKRPKRSSPSSSSSSSSS
--------------------------MEESSSSPVSPVDSLVTSEEELDRQ---------QKRFGRKRRQGRKSS-----------------EDSSSPSSVNKRNKKP---------------------------------------MEEGSGSPVSPVDSLVTSEEELDRQ---------QKRFARKRRHSKKFS----------------EDSSGSSPGPVKRAKKP---------------------------------------MEEGSSSPVSPVDSLVTSEEELDRQ---------QKHFSKKRRHSKKSS----------------EDSSGSSPGRVKRGKKA---------------------------------------MEEGSSCPGSPVDSLGTSEEELDRQ---------QKRFPAKRRQSKKSS----------------GDSSGSSPGPVKRAKKP---------------------------------------MEEGSSSPVSPVDSLVTSEEELDRQ---------QKRFAKKRRHSKKSS----------------EDSSGSSPGPLKRGKK----------------------------------------MEESSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPNPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK----------------------------------------MEEGSSSPVSPVDSLGTSEEELERQ---------PKRFGRKRRYSKKSS-------------------EDGSPTPGKRGKK--------------EE SS SSPVDSL SEEELDRQ
PKR GRKRR SRKSS
DD SP GKRGKK
43
Mm1
Rn1
Hs1
Gg1
Xt1
Tn1a
Tr1a
Ga1a
Ol1a
Dr1a
Dr1b
Ol1b
Ga1b
Tr1b
Tn1b
Bb
Lv
Ga3a
Ol3a
Ol3b
Ga3b
Xt3
Gg3
Dr3
Dr2
Ol2
Tr2
Ga2
Tn2
Gg2
Hs2
Rn2
Mm2
Consensus
(92)
(92)
(92)
(80)
(57)
(61)
(61)
(61)
(61)
(60)
(62)
(73)
(74)
(73)
(73)
(81)
(92)
(79)
(76)
(99)
(100)
(57)
(56)
(85)
(57)
(58)
(58)
(58)
(57)
(54)
(54)
(54)
(54)
(123)
Figure 3.3:
GGGGGSSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----GG---SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----G----SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGA------------G----SSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------SSSTGSSPQSFEELQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------SN--SSSPQSFEDIQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SN--SSSPQSFEDIQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SS--SSSPQSFEDLQSQRVMANVRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SS--SSSPQSFEELQSQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKLSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH----------SSNSSNSPQSFEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMAS-CSYVAHERLSYAFSVWRMEGAWSMSASH----------CSNSSSSPQSLEDLQTQRVMANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSTSH-----E----ASSSSSPARSFDDLQTQRVMANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----D----SETSSSPAPSFDDLQTQRVMANVRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLESDELDARGTS-CSYVAHERLSYAFSVWRMGGSWSLSTTTH---------SEGSSSPELSFDDLQTQRVLANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLESDELDGRGTS-CSYVAHERLSYAFSVWRMGGAWSLSTTSH---------SEGSSSPELSFDDLQTQRVLANIRERQRTQSLNEAFTSLRKIIPTLPSDKLSKIQTLKLAARYIDFLCQVLQSDELDGRGTS-CSYVAHERLSYAFSVWRMGGAWSLSTTSH----------RKKTSKAESFEDLQNQRVLANVRERQRTQSLNEAFSSLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLRSDDTDTKMASSCSYVAHERLSYAFSVSRQELYFGLGAPSSGPNHF
-------IRRKGPQSFEELQNQRVLANVRERQRTQSLNDAFANLRKIIPTLPSDKLSKIQTLKLASRYIDFLFQVLKSDEEDQKMVGSCTYMAHERLSYAFSVWRMEGAWNSMASHR----AP---TSLGPRLDQPFEDLHSQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----VSLAPTSLGPRSEPPFEELHSQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWAMSTSH-----SLSPVPANSPGGLSALEDPHAQRVIANIRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQSDQMDSKLAG-CNYLAHERLSYAFSVWRMEGAWSSMSAGH----SL---SPGPDPSPGDLEDPHGQRVIANIRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQNDEMDTKLAG-CNYLAHERLSYAFSVWRMEGAWSTMSAGH-------------SPHIETFEDVHTQRIIANVRERQRTQSLNDAFAELRKIIPTLPSDKLSKIQTLKLASRYIDFLYQVLQSDELDHKIAS-CNYLAHERLSYAFSVWRMEGAWSMSTTH--------------SPVPQSFEDVHTQRVIANVRERQRTQSLNDAFAELRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDHKITS-CNYLAHERLSYAFSVWRMEGAWSMSASH-----LVPVVSSVSPVPGQPFEDLHTQRVIANVRERQRTQSLNDAFASLRKIIPTLPSDKLSKIQILKLASRYIDFLYQVLQSDEMDAKLAS-CNYLAHERLSYAFSVWRMEGAWSMSATH-------------SPSSTQSFEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSTQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDSKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSSQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SPSSNQSYEELQNQRCLANVRERQRTQSLNEAFSSLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH------------ASPSSSQSYEELQNQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLASRYIDFLCQVLQSDEMDNKMSS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------SSPSSQSYEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDSKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-------------GSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS-CSYVAHERLSYAFSVWRMEGAWSMSASH-----S
S S QSFEELQTQRVLANVRERQRTQSLNEAFASLRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS CSYVAHERLSYAFSVWRMEGAWSMSASH
Alignment of predicted Twist proteins. Zebrafish Twist1a (Dr1a), Twist1b (Dr1b), Twist2 (Dr2) and Twist3
(Dr3) predicted amino acid sequences were aligned with human (Hs), rat (Rn), mouse (Mm), chick (Gg), frog
(Xt), fugu (Tr), medaka (Ol), stickleback (Ga), pufferfish (Tn), lancelet (Bb) and sea urchin (Lv) Twist proteins.
The consensus bHLH and WR domains are highlighted in blue and red, respectively.
44
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1)
(1)
(1)
(1)
(1)
1
100
-------------------------------------------------------------------CTCCTCTCAAACACTTTACCAGACTATAAGAGC
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AAAAAAAATTAGGAAAAAACTTCC
GGGGACAAACAGCCACATGACACACACACACATACACACACACACACACACACTGCAGAGATTGCATTTGATTTCTAACAACAAACCC-AAAGCAATGGC
T T TC AACAA AAACC GAAAA AAT GC
Dr1a
Dr1b
Dr2
Dr3
Consensus
(34)
(1)
(25)
(100)
(101)
101
200
TCCCTAACTTTTTTCCTTTCAA-CCTAACACAAAGTTGCTT-GGAATACCTAGTGATCTTCTCCAGAACACGAAACGTACG--CGTGGAATTGTATTTCA
---------------GAGA-AAGCCCTCCGTGACGCAGGAG-GAGACGCGCTGAGAG--------------GGACCGAG----CCTCTGACCCCATTTCG
TGCGGGAGGAAAACCGTGCCAAGCTCTACTTCTGGAAGTAG-GGGACGCGCGCAGTGTCAGGACTTCAGGAGGACAGACAAAATCCCCAACAACAAAACG
TGTATAACTCTCACAGAG--ATCCCACAGAGCTCGCTTCAGTGTTTAACTTTGACACCGCTCACAGGTACAGCAGGAGCACTGACACGGAGGACTGATCG
TGC TAACT T ACCGTG CAAGCCCTACATCTCGCTGCAG GGGACGCGTTGAGAGC
ACAG A AGGACCGACA
CCTCGGAC ACATTTCG
Dr1a
Dr1b
Dr2
Dr3
Consensus
(130)
(66)
(124)
(198)
(201)
201
300
CCCTCATGCTGGAATAACGTCCTTATTCGCACGCGC-----TTTCAGCAGAGACTTAAGCGA-GATGCCCGAAGAGCCCGCGCGAGACTCCTCCAGCTCC
---TCGGACTCAGGGAAGCCACG-ACCCGCAATCTG-----AGCT----TTTCCAGAGGTGATGTTTGAGGAAGAGGCGATGCACGAGGACTCCAGCTCT
GA-TATTACTAGTCTAAACCACGGAACCAAAAGTGG-----AGATCGTATTTTCTCCTCGTTTCTTTAAAAAAGAA---ATGGAAGAGAGTTCTAGCTCT
---TCATGCGAGAGGAACAGACTTGTGGAGATTTTCCTGAAAGTGGGATCCTTCCCATTGAAGAAGAGCAGGAGCGGCGCCCCAATAAGTGTGCGGTTGT
TCATGCTAGAGTAACCCACTTATCCGCAATTTG
AGTT G ATTTTCTCATGGGATGTTTGCAGAAGAGGCGATGCAAGAGG CTCCAGCTCT
Dr1a
Dr1b
Dr2
Dr3
Consensus
(224)
(153)
(215)
(295)
(301)
301
400
CCCGTGTCT-CCCGCGGACA---------GTCTCAGCAACAGCGACGGAGAGCCCGACAGG---CCACCAAAAAGGTGCG-CAAGGAAAAGACGGTCGAG
CCAGAGTCT-CCGGTGGACA---------GTCTGGGAAACAGCGAGGAGGAGCTCGACAGGCGACAACCGAAGCGCGTCA-GCAGGAAAAAACGCGCCAG
CCCGTCTCC-CCAGTGGACA---------GCCTGGTGACCAGCGAGGAGGAGCTGGACAG---ACAGCAGAAAAGGTTCG-GGAGGAAAAGGAG-GCAAG
GGTTTCTCCACCGGCGGGCGCACGCAAGCGGCTGACGGGTCCCAAAAAAGAGCCCGTCTCACAAGACGACAAACCATCCCTGGACAACCCATCGAATCTG
CCCGTGTCT CCGGTGGACA
GTCTGGGGAACAGCGAGGAGGAGCTCGACAGGC ACAACCGAAACGGTTCG GGAGGAAAAGACG GCCAG
Dr1a
Dr1b
Dr2
Dr3
Consensus
(310)
(242)
(300)
(395)
(401)
401
500
CAAGAAAAACGG-------GGAGGATTCCGATAGCTCGACCCTTGGGAAAA--------GGGGGAAAAAGTCT-AGCAACAGCAGCAACAGCCCT-CAGT
CCGCAAAAACGC-------CGAGGATTCCGACAGTCCCACGCCCGGGAAGA--------GGAGCAAGAAGTGC-AGCAACAGCAGCAGCAGCCCG-CAAT
GAAGGAAGTCGAG------CGAGGACAGCAGCAGCCCGAGCTCCGTCAATA--------AACGTAACAAAA---AGCCG-AGCCCGAGCAGCACT-CAGT
GCTCCCAAACGTCCCAAAAGAAGCTCTCCGTCATCCTCATCGTCTTCTTCGTCCTCTCTGGTGCCCGTCGTGAGTTCGGTTTCTCCAGTTCCCGGACAGC
GCAGAAAAACG
GGAGGATTCCGACAGCCCGACCCTCGTGAA A
GG GCAAGAAGTG AGCAGCAGCAGCAGCAGCCCT CAGT
Dr1a
Dr1b
Dr2
Dr3
Consensus
(393)
(325)
(381)
(495)
(501)
501
600
CTTTCGAGGAGCTGCAGACGCAGCGCGTGATGGCGAACGTGCGCGAGCGACAGAGGACGCAGTCGCTCAACGAAGCGTTTGCGGCTTTACGCAAAATCAT
CTCTGGAGGACCTGCAGACGCAGCGCGTCATGGCGAACGTGCGCGAGCGTCAGAGGACTCAGTCTCTGAACGAGGCGTTCGCCTCCCTGCGCAAAATCAT
CCTTCGAGGAGCTCCAGAACCAGCGCGTCCTGGCGAACGTACGCGAGCGGCAACGGACTCAATCGCTGAACGAAGCCTTCGCGTCTTTGCGCAAAATCAT
CCTTCGAAGACCTCCACACGCAACGAGTGATCGCCAACGTTCGGGAACGCCAGCGCACACAGTCCCTGAACGACGCCTTCGCCTCCCTCAGAAAGATCAT
CTTTCGAGGAGCTGCAGACGCAGCGCGTGATGGCGAACGTGCGCGAGCG CAGCGGACTCAGTCGCTGAACGAAGCGTTCGCGTCTTTGCGCAAAATCAT
Dr1a
Dr1b
Dr2
Dr3
Consensus
(493)
(425)
(481)
(595)
(601)
601
700
CCCCACTTTACCTTCCGATAAACTGAGCAAAATACAGACGCTGAAACTCGCCGCCAGGTACATCGATTTTCTCTGTCAGGTTCTACAGAGTGACGAGCTG
CCCCACCTTACCCTCGGACAAACTCAGCAAAATACAGACGCTCAAACTCGCGGCCCGGTACATTGACTTCCTCTGTCAGGTCCTGCAGAGCGATGAGCTG
CCCCACGCTCCCCTCGGATAAACTCAGCAAGATCCAGACGCTCAAACTCGCATCCAGGTACATTGATTTCCTCTGTCAGGTGCTGCAGAGCGACGAGATG
CCCCACCTTGCCTTCGGACAAGCTGAGCAAGATCCAGATCCTCAAACTGGCCTCCAGATACATCGACTTCCTCTACCAGGTCCTGCAGAGCGACGAGATG
CCCCACCTTACCTTCGGATAAACTGAGCAAGATCCAGACGCTCAAACTCGCCTCCAGGTACATTGATTTCCTCTGTCAGGTCCTGCAGAGCGACGAGCTG
45
Dr1a
Dr1b
Dr2
Dr3
Consensus
(593)
(525)
(581)
(695)
(701)
701
800
GACTCCAAGATGGCAAGTTGTAGTTATGTTGCTCACGAGCGTTTGAGCTACGCGTTCTCGGTTTGGAGGATGGAGGGCGCTTGGTCCATGTCTGCATCTC
GACTCCAAGATGTCCAGCTGCAGTTATGTGGCGCACGAGAGACTCAGCTACGCGTTTTCTGTGTGGAGAATGGAGGGCGCGTGGTCCATGTCAACATCTC
GACAACAAGATGTCCAGTTGCAGCTACGTCGCGCACGAGAGACTCAGTTACGCGTTTTCAGTGTGGAGGATGGAGGGCGCGTGGTCGATGTCTGCGTCCC
GACGCCAAGCTGGCCAGCTGCAACTATCTGGCCCACGAGAGGCTGAGCTACGCCTTCTCCGTCTGGAGGATGGAGGGCGCCTGGTCCATGTCCGCCACTC
GACTCCAAGATGTCCAGTTGCAGTTATGTGGCGCACGAGAGACTGAGCTACGCGTTTTC GTGTGGAGGATGGAGGGCGCGTGGTCCATGTCTGCATCTC
Dr1a
Dr1b
Dr2
Dr3
Consensus
(693)
(625)
(681)
(795)
(801)
801
900
ACTAGTGTGCAGGGAAACTT-TTTCTTGTTTTGTTTTTAATGGTCAACCCGTGAGCTGGGAAAACACG-AGGACCAATGCTAATTCCA-TCATAATCTTACTAACG--CACGGATGCAC-GCGTTTGATGCAGCATGATTCTCGGCCTGAGGAGCTGAACTCACTGGAAGGAGCGGCTCAAAACAAGGGCGAAAATAAACTAGCA--GCGAGACGCGT-CCTGATAATGCCGAA-------CGGACTGTTTACTTCCACTAATTTTGAGGAT---------------GCCAAA----ACTAAAC---ATCCACCCGTTCCTCATCTCAAAAACTGAATCCCGAACTCTGATCCTGATTTTCATCTCTATATCCACTCCACACGGCCTCCGACTCACC
ACTAGCG CAGGGACGCGT CCTCTTGTTGCAGAATGAATC CGGACTGTTGAGCTGAACTAACTCT AGGATC ACTC AAAC
TCCAAATCA
Dr1a
Dr1b
Dr2
Dr3
Consensus
(789)
(721)
(751)
(892)
(901)
901
1000
GGGGAAAACGGCAAATGT---TCCAACAGAG----GTCATGGCTGT--TACCGAGAGAAGG---CCACGGACAGCGAATTGTCATATGGATTT-----CC
GGATTATAAAGGGAAAATT-CTGGAGCGTCAT---GACGTCGTTGC---AAGCACTTACAG---TTGTGAACTACGACATGGGAGCAGGAATTACAGTCA
GGATTATTCGATGAACCT--CTAAACCTCAGT---GACGTGGCCA----AAGGACATTCAG---TGGATACAT-CTAC---------GGACTT------CTGCCATGACCCCACCCTGCCTAAAGATGACCTCTGACCTCTCTCACTTTTCCAAATCAAGCCTTCGCAGTTTCCCACAACCCCAGAGGACTT------GGGTTATACGGCGAACCT CTAAAGCTGAGT
GACGTGGCTG
TAAGGACATACAG
TCGCGGACT CGACATG CA AGGACTT
C
Dr1a
Dr1b
Dr2
Dr3
Consensus
(872)
(811)
(822)
(985)
(1001)
1001
1100
TCCCGAGTCTTATG--ACGACGAATGTTGGAAATATGTGCATATGCATGTTTTTTTTTTTTTTTTTTTTTTTTGGAAGACTCAGATGTGCATA----ACT
GATCTGTGCTGTTGCGACGGTGAATGTGGAAAACATGTGCTTCCG--TCCACAAAACAGAGACTCGCTGCAGGAAAAGACGCTCCTGCGCTTCTGACAGC
GAGCA-TCCTCATG--AAGGACACAAT----AAAAGGGACTT-----TTTAATATACTTAGA---GGAAAAGTAGATGACG-TCTTCTGTGTC-GTCAAA
CATGCTTCATCTAC--AAGCCGACAGTTTTCCCCTTTCCCTAATG---GAGCTTTACCAGCAAACGCAGGAGAGAAAGAGT--CTTGAGGAGC--GCAGA
GATC TCCTCTTG ACGGCGACTGTTG AAACATGTGCTTATG TGTACTTTACTTAGA TCGCTG AGTGGAAGACTCTCTTGTGCATC G CAGA
Dr1a
Dr1b
Dr2
Dr3
Consensus
(966)
(909)
(905)
(1076)
(1101)
1101
1200
TTCTGGAAGAAAGTG--AATTTGCATTACAAGGACTGTC--GATAAGAAAATGGGAA--TTGAAGCCT-CTAGTTCGAATCCTGTGCAAATACAAAGCTT
AACAACCAGCATGGCGTCATATTTTTTTCTCTGAAGGAA--AACACACACACTCAACGAATGAAGGAA-ATCGGCTCATATCAGTGTTAACATTTTCTTT
TACATTTAATCTTTT-TCACAAGTTATTCT-TAAATGTC--GCCATGATGATAGTAC--AGGAAGGCT-ATCTGCT---ATCTGTATGGAAACTATATTT
TGAGCAGAGCATTTCCACAAGACTGAATCCAGCACTGAGCTGAGTGGAAGTGACTGCTGCTGATCTCAGATCAGTGTTCCTCCATGTTGACTGTTCTCTG
TACA
AGCATTTC TCATATGTTTTTCTATGACTGTC GACA GAAGATAGTAC ATGAAGGCT ATCGGTT A ATCTGTGTTGACACTTT TTT
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1059)
(1006)
(995)
(1176)
(1201)
1201
1300
TA---GTACAATTCTATTTATTTATTGATGACACACTTTTTGAAATGAAAGTAAATGTATCAAATGTGTTGAAATGCATTATTATTTTTTATTACTTTTG
GATCGGTCCAAGAAAATACTTTTATTTATTTATTGATGATTGTCACAATGCAGAATAGATCTGGTGTCT--ACATGCATTTTCTATTTTAAATA-TGATG
CATGTCTGTCTCTAAACAGAAGAGTTTATATATATATAAGAACAAAAAAAAAAAAAAAAA---------------------------------------GACTCATGTTTGGGCAGATATTGACTGTTCTGAGGACTCACTAACCCTGACCTGATGGATCAAGGCTTC----CTCCGCTCTTTATATTGCTTCATTATG
GAT GTGTATGTAAATATATTTATTTAT TAT GATTATTGAAACAAAACAAAATGGATCAAGTGT T A ATGCATT TTTATTTT ATTA TTATG
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1156)
(1103)
(1055)
(1272)
(1301)
1301
1400
TAAATAAATGTGTATTTCTGTAATAAAAAAATGAAGAATTTTAAGAAATA-------------------------------------------------TAAATATGTGTATATTTTCTGCAATAAAACATGATTTGAAATACAAAAA-----------------------------------------------------------------------------------------------------------------------------------------------------CAGATGTGAGAGTGATTGTGCTTGTGTTTC-TGCTGTATATTAGCCATTCATTCCTGCTTCAGTGACGGGTAACACTGTGTAAACCGCCAGTTCATGTAA
TAAATATGTGTGTATTT TG A TAAAACATGATGTATATTA AAAT
46
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1371)
(1401)
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1471)
(1501)
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1571)
(1601)
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1671)
(1701)
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1771)
(1801)
Dr1a
Dr1b
Dr2
Dr3
Consensus
(1206)
(1152)
(1055)
(1871)
(1901)
Figure 3.4:
1401
1500
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AATAATCAAATAATGCTTCAAGATTCACTCTATTCCTCTATTAAAAGAGACTTTAACTGCAGAAGTGTTAGTGGCTTTAATGGGGGAATGAGGAGCTGTA
1501
1600
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CATCAATAATGATGTGTCGGAGTACAGAGGAAGACATTTAGAAAGATGCTCTGCTTCAGGATTCAGTCAAACGTCTGCAAACGTATAAAGATAGCAGGGT
1601
1700
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TCATGTGAGTGGACTTGGTGATTATAAATATTTCTACAATAATGAAGAAGCTGTTTGAGTACATCTTTACAGCAGATCCAGGCAGCGGTCGCTAATAGGA
1701
1800
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TGAGATGTCGGCTAAACTTGGTGTTGGTGATGGTGTGAAAATAAACTGAAATGAAGCGTACAGCAGGAGAACTCGATGAGTAGCTAACAGACTAAGAGTT
1801
1900
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TACGCTGATTATCAAGAGCTTGAAAATTCATCAATAAACAAACAATAAGATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
1901
1930
---------------------------------------------------------------------------------------AAAAAAAAAAAATTAAAAAAAAAAAAAAAA
Alignment of zebrafish full-length twist cDNAs. Start (ATG) and stop (TAA/TAG) codons are highlighted in
black. Identical regions are highlighted in grey. The consensus bHLH and WR domains are highlighted in blue
and red, respectively. Sequences used for synthesis of antisense RNA in situ hybridization probes are
underlined. Sequences were obtained from GenBank: twist1a (EF620930), twist1b (DQ351987), twist2
(NM_001005956) and twist3 (NM_130985).
47
3.5
Identification and confirmation of the true orthologs of zebrafish twist genes
3.5.1
Comparison of zebrafish twist gene family with other species
A detailed comparison of the coding cDNA and amino acid sequence of the
zebrafish twist gene family against other species was performed (Table 3).
Table 3: Comparison of zebrafish twist gene sequences with TWIST sequences from
other species
Coding nucleotides/amino acid similarity (%)
Zebrafish
Zebrafish
Zebrafish
Zebrafish
twist1a
twist1b
twist2
twist3
Human TWIST1
60/66
62/63
59/63
61/51
Mouse Twist1
58/65
61/61
58/60
61/50
Rat Twist1
59/66
62/63
59/62
61/52
Chick Twist1
66/70
68/69
65/68
59/54
77/84
80/82
77/86
57/55
Xenopus twist1
Fugu twist1a
84/88
82/87
78/82
53/51
84/90
83/87
78/83
54/50
Medaka twist1a
Stickleback twist1a 78/85
78/86
73/78
56/53
Tetradon twist1a
81/86
79/85
73/52
56/53
Fugu twist1b
64/61
63/63
64/64
58/55
Medaka twist1b
63/61
63/65
63/64
58/61
65/62
62/58
59/52
Stickleback twist1b 60/58
Tetradon twist1b
61/59
64/62
62/61
60/52
Human TWIST2
70/73
74/74
80/86
54/53
Mouse Twist2
70/73
73/74
78/86
53/53
Rat Twist2
70/73
73/74
78/86
54/53
75/86
49/54
Chick Twist2
68/73
69/75
Fugu twist2
68/72
71/75
84/90
51/54
Medaka twist2
69/74
72/75
84/90
52/54
49/58
Chick Twist3
66/73
73/72
77/76
64/69
71/69
74/75
48/59
Xenopus Twist3
Medaka twist3a
50/57
61/57
65/61
71/70
68/65
Stickleback twist3a 55/53
63/56
58/58
Medaka twist3b
48/41
50/42
48/44
64/51
Stickleback twist3b 52/49
52/47
52/50
66/60
The comparison table clearly revealed that zebrafish twist1a and twist1b have the
highest percentage of coding cDNA and amino acid sequence similarity compared to
48
other species TWIST1 and zebrafish twist2 has the highest percentage of coding cDNA
and amino acid sequence similarity compared to other species TWIST2. Interestingly,
zebrafish twist1 genes (twist1a and twist1b) have a higher percentage similarity to Twist2
of other species compared to Twist1 of other species. In addition, zebrafish twist1a,
twist1b and twist2 showed higher sequence similarity to chick and Xenopus Twist3
compared to zebrafish twist3.
However, zebrafish twist3 is highly homologous to
medaka and three spined stickleback twist3a.
3.5.2
Phylogenetic analysis
To understand the relationships of the zebrafish twist genes to each other and to
those of a non-chordate, a cephalochordate, and other vertebrates, a phylogenetic tree was
constructed using multiple alignments of homologous proteins belonging to human
(Homo sapiens / Hs), rat (Rattus norvegicus / Rn), mouse (Mus musculus / Mm), chick
(Gallus gallus / Gg), frog (Xenopus tropicalis / Xt), zebrafish (Danio rerio / Dr), medaka
(Oryzias latipes / Ol), fugu (Takifugu rubripes / Tr), spotted green pufferfish (Tetraodon
nigroviridis / Tn); stickleback (Gasterosteus aculeatus / Ga), Japanese lancelet
(Branchiostoma belcheri / Bb), and sea urchin (Lytechinus variegatus / Lv). The Twist
proteins clustered into three clades, with the zebrafish Twist1 (Twist1a and Twist1b),
Twist2 and Twist3 peptides falling into different clades (Figure 3.5). Interestingly,
zebrafish Twist1b did not cluster with the Twist1b of the other fish species medaka, fugu,
pufferfish, and stickleback (Figure 3.5A and 3.5B).
49
Tn1a
999
601 Tr1a
Ol1a
901
Ga1a
434
Dr1a
270
Dr1b
A
II
Tn1
I
Rn1
809
Mm1
818
Hs1
998
Gg
687
Xt1
767
735
960
932
918
489
976
Rn
Ga3
Ol3
98
L
B
Gg
91
35
99
Gg
84
97
40
Xt
96
83
Mm
93
Dr
96
I
Tn
100
48
73
II
53
100
Hs
92
43
76
68
90
99
60
27
Dr1
Dr1
Ga1 Ol1
Tn1
Tr1
99
46
81
Xt
Ga3b
1000
Ol3b
Tr1b
998
Tn1b
982
Ga1b
1000
Ol1b
Dr
100
Ol3a
1000
Ga3a
530
Dr3
Xt3
843
Gg3
Ga3
Ol1
99
Tr2
928
Tn2
Mm2
354
Hs2
997
Rn2
967
Gg2
Ol3
Ga1
Tr1
Ga2
467
Ol2
Dr2
839
405
B
Tr
III
Ol
Ga
80
G
I
Rn
Hs
Mm
Bb
Lv
Figure 3.5:
Rooted cladogram (A) and unrooted radial tree (B) of Twist proteins generated by the neighbor-joining method,
with bootstrap values shown. The cladogram is rooted using sea urchin (Lv) as an outgroup. Zebrafish Twist1
(Dr1a and Dr1b), Twist2 (Dr2) and Twist3 (Dr3) proteins are clustered into the three major clades. Twist
protein members from human (Hs), rat (Rn), mouse (Mm), chick (Gg), frog (Xt), fugu (Tr), medaka (Ol),
stickleback (Ga), pufferfish (Tn), lancelet (Bb) and sea urchin (Lv) were used to produce the phylogenetic trees.
50
3.5.3
Calculation of genetic distances
The genetic distance between twist1a and twist1b among these fish was compared
using the Kimura 2-parameter model of nucleotide substitution, transitions and
transversions. The calculated Kimura value between zebrafish twist1a and twist1b was
0.26, whereas it was almost double in the fugu, stickleback, pufferfish and medaka (0.46,
0.47, 0.55 and 0.55 respectively).
These values suggest a higher nucleotide
substitution/mutation rate between the twist1a and twist1b sequences in the other fish
species compared to the zebrafish.
The Tajima-Nei-corrected number of substitutions per site at third codon positions
between twist1a and twist1b sequences of zebrafish, fugu, pufferfish, stickleback and
medaka were also determined. Since most third-codon position substitutions do not
result in an amino acid change, mutations at these positions are assumed to be under little
or no selective pressure and the calculated mutation rates are thus more likely to reflect
neutral evolution. The Tajima-Nei value of 0.718 (± 0.118) for zebrafish twist1a and
twist1b was lower compared to fugu (1.113, ±0.248), stickleback (0.794, ±0.145),
pufferfish (1.243, ±0.273) and medaka (0.992, ±0.183). These results again suggest a
lower substitution/mutation rate between the twist1a and twist1b genes in the zebrafish
compared with the other four fish species.
51
3.5.4
Comparative synteny analysis
Zebrafish twist1a (EF620930) is located on linkage group (LG) 16 at ~13.484 Mb,
while twist1b (NM_130984) is located on LG19 at ~ 4.245 Mb, based on the Zv7
assembly,
July
2007
release
(http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=7955). The comparative
synteny analysis showed that many genes on human chromosome 7, including TWIST1
(chromosome 7p21 (~19.1 MB), were found to be duplicated on zebrafish LG16 and
LG19 (Table 4).
Medaka twist1a (BK006268) is located on LG16 at ~12.859 Mb, while twist1b
(BK006269) is located on LG11 at ~8.931 Mb, based on the HdrR assembly, July 2008
release (http://www.ensembl.org/Oryzias_latipes/index.html). The comparative synteny
analysis of the chromosomal regions around zebrafish twist1a (LG16) with medaka
twist1a (LG16) and twist1b (LG11) showed a significant higher number of genes in
medaka LG16 compared to LG11 (Table 5).
Conversely, the comparative synteny
analysis of the chromosomal regions around zebrafish twist1b (LG19) against medaka
twist1a and twist1b revealed comparatively a higher number of genes in medaka LG11
compared to LG16 (Table 6).
52
Table 4. Comparative synteny analysis of chromosomal regions around zebrafish twist1a
and twist1b and human TWIST1.
D.rerio LG16
hdac9b (17.0Mb)
twist1a (13.5Mb)
sp8l (13.9Mb)
dfna5 (15.4Mb)
cbx3b (15.8Mb)
hoxa11b (16.0Mb)
hoxa10b (16.0Mb)
hoxa9b (16.0Mb)
hoxa2b (16.0Mb)
tax1bp1 (16.0Mb)
creb5 (16.3Mb)
cpvl (16.4Mb)
tbx20 (34.3Mb)
sept7 (49.7Mb)
psma2 (32.9Mb)
nacad (22.2Mb)
ddc (5.0Mb)
abcb4 (21.1Mb)
adam22 (33.0Mb)
sri (3.9Mb)
cldn12 (33.1Mb)
Hsa Chr 7
GLCCI1 (7.9Mb)
NXPH1 (8.5Mb)
BZW2 (16.5Mb)
TSPAN13 (16.5Mb)
AGR2 (16.8Mb)
HDAC9 (18.7Mb)
TWIST1 (19.1Mb)
TMEM196 (19.6Mb)
SP8 (20.8Mb)
TRA2A (23.5Mb)
NPY (24.3Mb)
MPP6 (24.5Mb)
DFNA5 (24.6Mb)
CBX3 (26.1Mb)
SNX10 (26.3Mb)
HOXA11 (27.3Mb)
HOXA10 (27.3Mb)
HOXA9 (27.3Mb)
HOXA5 (27.3Mb)
HOXA3 (27.3Mb)
HOXA2 (27.3Mb)
HOXA1 (27.2Mb)
TAX1BP1 (27.6Mb)
CREB5 (28.5Mb)
CPVL (29.1Mb)
TBX20 (35.2Mb)
SEPT7 (35.8Mb)
ELMO1 (37.1Mb)
PSMA2 (42.9Mb)
NACAD (45.0Mb)
DDC (50.4Mb)
FKBP9L (55.5Mb)
GRB10 (50.6Mb)
ABCB4 (86.7Mb)
ADAM22 (87.4Mb)
SRI (87.5Mb)
CLDN12 (89.8Mb)
PEX1 (91.8Mb)
COL1A2 (93.7Mb)
GNGT1 (93.7Mb)
CASD1 (93.8Mb)
SGCE (94.1Mb)
ASB4 (94.9Mb)
SHFM1 (96.2Mb)
ASNS (97.2Mb)
D.rerio LG19
glcci1 (16.1Mb)
nxph1 (34.9Mb)
bzw2 (27.9Mb)
tspan13 (28.6Mb)
agr2 (28.6Mb)
hdac9a (4.2Mb)
twist1b (4.2Mb)
tmem196 (45.6Mb)
sp8 (45.5Mb)
tra2a (11.0Mb)
npy (10.9Mb)
mpp6 (10.9Mb)
cbx3a (46.0Mb)
snx10 (46.0Mb)
hoxa11a (10.5Mb)
hoxa10ap (10.5Mb)
hoxa9a (10.5Mb)
hoxa5a (10.5Mb)
hoxa3a (10.5Mb)
hoxa1a (10.5Mb)
elmo1 (31.5Mb)
psma2 (32.5Mb)
fkbp9 (39.5Mb)
grb10 (14.0Mb)
pex1 (31.5Mb)
col1a2 (35.4Mb)
gngt1 (35.1Mb)
casd1 (37.7Mb)
sgce (37.7Mb)
asb4 (37.9Mb)
shfm1 (38.0Mb)
asns (16.4Mb)
53
Table 5. Comparative synteny analysis of chromosomal regions around zebrafish twist1a
and medaka twist1a and twist1b.
O. latipes LG16
dctn3 (24.1Mb)
ctnnb1 (11.1Mb)
bat5 (17.7Mb)
nt5c3 (23.3Mb)
ddc (9.8Mb)
entpd3 (9.8Mb)
m6pr (17.0Mb)
atp1a3 (17.8Mb)
gsk3a (16.2Mb)
ceacam1 (2.4Mb)
usp5 (17.5Mb)
tex10 (14.6Mb)
epn1 (16.8Mb)
u2af2 (16.7Mb)
ntd5 (16.5Mb)
rpp38 (14.8Mb)
gapdh (17.2Mb)
twist1a (12.8Mb)
sp8 (12.8Mb)
rxrb (12.6Mb)
dfna5 (13.3Mb)
hoxa13b (13.1Mb)
hoxa11b (13.1Mb)
hoxa10b (13.1Mb)
hoxa9b (13.1Mb)
hoxa2b (13.1Mb)
cpvl (13.0Mb)
ctsk (24.5Mb)
hapln2 (23.9Mb)
mef2d (23.7Mb)
tmem67 (14.6Mb)
mcl1 (24.6Mb)
styk1 (17.0Mb)
ptpn6 (17.3Mb)
psma2 (11.6Mb)
cldn12 (14.9Mb)
thbs3 (29.3Mb)
tbx20 (29.1Mb)
hfe2 (29.4Mb)
sh3bp5 (23.6Mb)
snip1 (20.9Mb)
otud7b (26.8Mb)
dnase2 (26.3Mb)
gapdhs (11.4Mb)
pbx2 (26.3Mb)
etfb (22.4Mb)
ubr5 (2.4Mb)
D. rerio LG16
dctn3 (1.0Mb)
ctnnb1 (2.1Mb)
ndufb9 (2.3Mb)
bat5 (3.6Mb)
nt5c3 (4.9Mb)
ddc (5.0Mb)
entpd3 (5.1Mb)
m6pr (6.3Mb)
atp1a3b (7.5Mb)
gsk3a (7.8Mb)
ceacam1 (8.3Mb)
usp5 (8.4Mb)
tex10 (9.3Mb)
epn1 (9.4Mb)
u2af2a (9.5Mb)
ntd5 (10.3Mb)
rpp38 (10.9Mb)
gapdh (12.9Mb)
twist1a (13.5Mb)
sp8l (13.9Mb)
rxrbb (14.9Mb)
dfna5 (15.50Mb)
hoxa13b (16.0Mb)
hoxa11b (16.0Mb)
hoxa10b (16.0Mb)
hoxa9b (16.0Mb)
hoxa2b (16.0Mb)
cpvl (16.4Mb)
ctsk (20.3Mb)
hapln2 (20.5Mb)
mef2d (20.6Mb)
tmem67 (22.2Mb)
gdf6a (25.0Mb)
ptpru (26.5Mb)
mcl1b (26.6Mb)
styk1 (27.5Mb)
ptpn6 (28.5Mb)
psma2 (32.9Mb)
cldn12 (33.0Mb)
thbs3a (33.9Mb)
tbx20 (34.3Mb)
hfe2 (37.5Mb)
sh3bp5 (39.5Mb)
snip1 (40.4Mb)
otud7b (42.8Mb)
dnase2 (43.3Mb)
gapdhs (43.6Mb)
ubin (44.4Mb)
pbx2 (45.4Mb)
etfb (46.2Mb)
ubr5 (47.2Mb)
O. latipes LG11
ctnnb1 (8.4Mb)
ndufb8 (0.9Mb)
atp1a3 (16.0Mb)
gsk3a (16.2Mb)
twist1b (8.9Mb)
sp8 (8.8Mb)
rxrb (15.1Mb)
dfna5 (10.7Mb)
gdf6 (16.8Mb)
ptpru (17.8Mb)
ubin (22.0Mb)
54
Table 6. Comparative synteny analysis of chromosomal regions around zebrafish twist1b
and medaka twist1a and twist1b.
O. latipes LG16
mcl1 (24.6Mb)
pbx2 (26.3Mb)
gnb3 (17.4Mb)
atp1a3 (17.8Mb)
twist1a (12.8Mb)
flot1 (17.6Mb)
ptp4a3 (6.8Mb)
rxrb (12.6Mb)
cnot3 (16.3Mb)
crabp2 (15.8Mb)
vamp1 (17.3Mb)
ing4 (17.2Mb)
u2af2 (16.7Mb)
MF‐hoxa11b (13.1Mb)
hoxa10b (13.1Mb)
hoxa9b (13.1Mb)
npy (13.2Mb)
rab5a (27.7Mb)
grb10 (9.7Mb)
thbs3 (29.3Mb)
glcci1 (28.3Mb)
spag1 (1.9Mb)
sox4 (7.8Mb)
ndrg1 (6.7Mb)
azin1 (2.5Mb)
psma2 (11.6Mb)
D. rerio LG19
mcl1a (0.1Mb)
msto1 (0.3Mb)
ehmt2 (0.3Mb)
hnrpr (0.6Mb)
pbx2 (0.7Mb)
edn1 (1.0Mb)
gnb3 (2.5Mb)
atp1a3a (3.5Mb)
twist1b (4.2Mb)
flot1 (4.5Mb)
psmb9a (4.8Mb)
ptp4a3 (4.8Mb)
rxrba (4.9Mb)
cnot3 (5.0Mb)
skiv2l (5.0Mb)
gtf2h4 (5.0Mb)
pygo2 (5.3Mb)
mylip (5.5Mb)
crabp2b (6.7Mb)
oprd1 (6.8Mb)
vamp1 (7.1Mb)
ing4 (7.2Mb)
u2af2b (8.0Mb)
vps52 (9.5Mb)
slc39a7 (9.7Mb)
mrpl3 (10.0Mb)
hoxa11a (10.5Mb)
hoxa10ap (10.5Mb)
hoxa9a (10.5Mb)
cbx3a (11.0Mb)
npy (11.0Mb)
rab5a (11.5Mb)
mbp (12.6Mb)
gdf6b (13.8Mb)
grb10 (14.0Mb)
thbs3b (15.2Mb)
txnip (15.2Mb)
eya3 (15.6Mb)
glcci1 (16.1Mb)
spag1 (17.3Mb)
sox4a (19.5Mb)
yars (28.0Mb)
mrps18b (28.5Mb)
ndrg1 (28.6Mb)
col1a2 (30.0Mb)
dlx6a (30.1Mb)
rrm2b (30.5Mb)
rpl30 (30.5Mb)
azin1 (30.6Mb)
anp32e (30.7Mb)
elmo1 (31.5Mb)
trps1 (32.5Mb)
psma2 (32.5Mb)
nxph1 (35.0Mb)
gngt1 (35.2Mb)
col1a2 (35.4Mb)
sgce (37.7Mb)
shfm1 (38.0Mb)
O. latipes LG11
msto1 (19.2Mb)
ehmt2 (4.4Mb)
hnrpr (5.6Mb)
edn1 (18.9Mb)
atp1a3 (16.0Mb)
twist1b (8.9Mb)
psmb9 (15.3Mb)
rxrb (15.1Mb)
cnot3 (14.1Mb)
skiv2l (4.4Mb)
gtf2h4 (21.1Mb)
pygo2 (0.3Mb)
mylip (23.3Mb)
crabp2 (5.8Mb)
oprd1 (18.3Mb)
vamp1 (14.9Mb)
vps52 (2.1Mb)
slc39a (13.9Mb)
mrpl3 (8.5Mb)
hoxa11a (10.5Mb)
hoxa10a (10.5Mb)
hoxa9a (10.5Mb)
cbx3a (10.4Mb)
npy (9.8Mb)
rab5a (22.1Mb)
mbp (18.7Mb)
gdf6 (16.8Mb)
txnip (21.2Mb)
eya3 (19.2Mb)
spag1 (6.0Mb)
yars (26.6Mb)
mrps18b (21.0Mb)
ndrg1 (8.4Mb)
col1a2 (9.5Mb)
dlx6 (9.8Mb)
rrm2b (23.6Mb)
rpl30 (23.5Mb)
azin1 (22.6Mb)
anp32e (3.3Mb)
elmo1 (7.7Mb)
trps1 (7.4Mb)
nxph1 (20.7Mb)
gngt1 (9.4Mb)
col1a2 (9.5Mb)
sgce (9.5Mb)
shfm1 (9.8Mb)
55
sp8 (12.8Mb)
fkbp9 (39.5Mb)
angpt1 (42.2Mb)
sp8 (45.5Mb)
cbx3 (46.0Mb)
snx10 (46.0Mb)
fkbp9 (21.8Mb)
angpt1 (20.3Mb)
sp8 (8.8Mb)
cbx3 (10.4Mb)
snx10 (13.2Mb)
Zebrafish twist2 (NM_001005956) is located on LG9 at ~39.145 Mb on the
zebrafish Zv7 genome assembly. The zebrafish twist2 chromosomal segment is syntenic
to the human and medaka twist2 locus on chromosome 2 and LG21 respectively (Table 7).
56
Table 7. Comparative synteny analysis of chromosomal regions around twist2 from
zebrafish, human and medaka.
O. latipes LG21
dlx1 (24.1Mb)
slc9a2 (11.7Mb)
rab20 (28.4Mb)
tmem41a (22.1Mb)
fzd7 (18.9Mb)
als2 (18.9Mb)
gdap2 (0.9Mb)
tbx15 (1.8Mb)
txndc9 (11.6Mb)
arl5a (6.0Mb)
ccnt2 (29.7Mb)
ndufa10 (30.4Mb)
cxcr7 (26.8Mb)
ebi2 (9.4Mb)
ercc1 (21.7Mb)
olig2 (6.7Mb)
gabpa (5.2Mb)
dirc2 (6.5Mb)
ptplb (6.0Mb)
ddx18 (0.4Mb)
mcm3ap (18.4Mb)
pms1 (18.5Mb)
asnsd1 (18.7Mb)
sestd1 (8.0Mb)
twist2 (28.7Mb)
ofd1 (26.2Mb)
ttc21b (23.4Mb)
prpf40a (22.3Mb)
pou1f1 (19.9Mb)
D. rerio LG9
dlx1a (3.0Mb)
slc9a2 (5.8Mb)
fev (7.3Mb)
rab20 (7.5Mb)
tmem41a (9.4Mb)
fzd7a (10.3Mb)
als2 (10.5Mb)
nrp2b (11.2Mb)
gdap2 (14.3Mb)
tbx15 (14.4Mb)
txndc9 (15.2Mb)
arl5a (16.3Mb)
ccnt2 (16.7Mb)
ndufa10 (16.8Mb)
cxcr7b (17.4Mb)
acvr2a (18.9Mb)
ebi2 (25.0Mb)
sf3b1 (26.0Mb)
rftn2 (26.2Mb)
ercc1 (26.6Mb)
olig2 (26.8Mb)
gabpa (29.1Mb)
dirc2 (31.5Mb)
ptplb (32.2Mb)
ddx18 (32.6Mb)
mcm3ap (32.7Mb)
pms1 (34.9Mb)
asnsd1 (35.1Mb)
sestd1 (37.2Mb)
ube2e3 (37.7Mb)
twist2 (39.2Mb)
ofd1 (43.6Mb)
ttc21b (45.0Mb)
prpf40a (45.6Mb)
pou1f1 (47.6Mb)
Hsa chr2
DLX1 (172.7Mb)
SLC9A2 (102.7Mb)
FEV (219.5Mb)
FZD7 (202.6Mb)
ALS2 (202.3Mb)
NRP2 (206.3Mb)
TXNDC9 (1.0Mb)
ARL5A (152.4Mb)
CCNT2 (135.4Mb)
NDUFA10 (240.6Mb)
CXCR7 (237.2Mb)
ACVR2A (148.4Mb)
SF3B1 (198.0Mb)
RFTN2 (198.2Mb)
DDX18 (0.1Mb)
PMS1 (0.2Mb)
ASNSD1 (190.2Mb)
SESTD1 (179.7Mb)
UBE2E3 (181.6Mb)
TWIST2 (239.5Mb)
TTC21B (166.5Mb)
PRPF40A (153.2Mb)
Zebrafish twist3 is located on LG23 at ~0.105 Mb on the zebrafish Zv7 genome
assembly. In medaka, there are two twist3 genes, twist3a on LG5 and twist3b on LG7.
Comparative synteny analysis revealed that the zebrafish LG23 region around twist3
shows a greater number of shared genes with medaka LG7 compared with medaka LG5
(Table 8).
57
Table 8. Comparative synteny analysis of chromosomal regions around zebrafish twist3
and medaka twist3a and twist3b.
O. latipes LG5
twist3a (30.2Mb)
mitf (5.2Mb)
csrp1 (13.1Mb)
eif4b (30.3Mb)
foxp1 (5.0Mb)
eif4e3 (21.0Mb)
ppp4r2 (21.2Mb)
ndrg3 (26.8Mb)
trpc4ap (26.8Mb)
slmap (33.5Mb)
tkt (3.7Mb)
adnp (27.0Mb)
dhrs3 (32.5Mb)
phc2 (13.8Mb)
slc2a1 (13.9Mb)
D. rerio LG23
twist3 (0.1Mb)
ctnnbl1 (0.2Mb)
mitfb (0.8Mb)
plcg1 (1.5Mb)
zhx3 (1.5Mb)
mip2 (1.6Mb)
spata2 (2.6Mb)
tnnc1 (3.3Mb)
slc9a8 (3.6Mb)
csrp1 (4.2Mb)
c20orf14 (5.0Mb)
slco4a1 (5.1Mb)
gata5 (5.4Mb)
tm9sf4 (5.5Mb)
tcea2 (6.4Mb)
adrm1b (7.2Mb)
eif4b (8.0Mb)
foxp1a (8.2Mb)
eif4e3 (8.3Mb)
ppp4r2a (8.4Mb)
ndrg3b (11.2Mb)
ddx23 (11.8Mb)
cacnb3a (11.9Mb)
adcy6 (12.0Mb)
ard1a (12.2Mb)
smc1a (13.3Mb)
fbxo44 (14.6Mb)
rnd1 (15.6Mb)
prim1 (15.7Mb)
hsd17b10 (16.2Mb)
ssr4 (16.4Mb)
trpc4apb (16.6Mb)
nsfl1c (16.6Mb)
pard6b (16.9Mb)
slmap (17.5Mb)
emd (17.7Mb)
atp2b3b (17.7Mb)
tkt (17.9Mb)
tnnc (18.4Mb)
dpm1 (18.5Mb)
adnp (18.5Mb)
her12 (19.9Mb)
dhrs3 (20.1Mb)
phc2 (20.5Mb)
slc2a1 (20.6Mb)
eno1 (21.1Mb)
samd11l (21.4Mb)
her9 (21.8Mb)
pa2g4b (23.7Mb)
hnf4a (24.3Mb)
ada (24.4Mb)
gdi1 (24.5Mb)
lzic (25.1Mb)
clstn1 (25.1Mb)
ube4b (25.2Mb)
kif1b (25.3Mb)
pgd (25.3Mb)
casz1 (25.7Mb)
O. latipes LG7
twist3b (1.7Mb)
ctnnbl1 (1.3Mb)
mitf (20.7Mb)
plcg1 (15.0Mb)
zhx3 (15.1Mb)
mip (19.4Mb)
spata2 (15.4Mb)
tnnc1 (18.1Mb)
scl9a8 (15.7Mb)
c20orf14 (15.8Mb)
slco4a1 (5.4Mb)
gata5 (5.1Mb)
tm9sf4 (5.0Mb)
tcea2 (27.2Mb)
adrm1 (22.2Mb)
foxp1 (20.7Mb)
eif4e3 (20.7Mb)
ppp4r2 (20.7Mb)
ddx23 (19.5Mb)
cacnb3 (19.5Mb)
adcy6 (19.6Mb)
ard1a (11.8Mb)
smc1a (12.1Mb)
fbxo44 (21.0Mb)
rnd1 (19.5Mb)
prim1 (19.4Mb)
hsd17b10 (26.6Mb)
ssr4 (11.8Mb)
nsfl1c (5.6Mb)
pard6b (4.4Mb)
emd (10.7Mb)
atp2b3 (4.6Mb)
tkt (7.3Mb)
tnnc1 (18.1Mb)
dpm1 (4.4Mb)
adnp (4.4Mb)
her12 (7.7Mb)
eno1 (10.6Mb)
samd11 (10.3Mb)
her9 (10.2Mb)
pa2g4 (12.5Mb)
hnf4a (25.9Mb)
ada (25.8Mb)
gdi1 (25.5Mb)
lzic (10.0Mb)
clstn1 (10.0Mb)
ube4b (10.0Mb)
kif1b (9.9Mb)
pgd (9.9Mb)
casz1 (9.7Mb)
58
arf3 (2.3Mb)
pfkm (2.9Mb)
itga5 (23.9Mb)
bin2 (23.9Mb)
racgap1 (31.4Mb)
rarg (28.0Mb)
src (12.8Mb)
mafb (26.9Mb)
dhh (27.0Mb)
wnt10b (27.1Mb)
arf3a (27.1Mb)
phf8 (27.1Mb)
pfkm (27.2Mb)
asb8 (27.2Mb)
atf7a (27.2Mb)
dip2b (27.3Mb)
ptges3 (27.7Mb)
uts2a (28.8Mb)
cd63 (30.9Mb)
taf8 (31.2Mb)
itga5 (32.3Mb)
dazap2 (32.5Mb)
bin2 (32.5Mb)
racgap1 (32.5Mb)
sars (32.8Mb)
rarga (33.6Mb)
hoxc13a (33.7Mb)
hoxc12a (33.7Mb)
hoxc11a (33.7Mb)
cbx5 (33.9Mb)
copz1 (34.0Mb)
csad (34.1Mb)
src (38.1Mb)
mafb (38.2Mb)
dhh (12.5Mb)
wnt10b (12.5Mb)
arf3 (12.5Mb)
phf8 (12.0Mb)
pfkm (11.4Mb)
asb8 (11.4Mb)
atf7 (11.4Mb)
dip2b (11.5Mb)
ptges3 (19.4Mb)
uts2 (21.4Mb)
cd63 (22.6Mb)
taf8 (25.5Mb)
itga5 (22.9Mb)
dazap2 (22.8Mb)
bin2 (22.7Mb)
racgap1 (22.8Mb)
sars (3.5Mb)
hoxc13a (12.8Mb)
hoxc12a (12.8Mb)
hoxc11a (12.9Mb)
cbx5 (13.0Mb)
copz1 (13.0Mb)
csad (13.0Mb)
src (25.4Mb)
mafb (15.3Mb)
3.6
Embryonic expression patterns of the zebrafish twist gene family
3.6.1
RT-PCR analysis
To detect the presence of the four zebrafish twist transcripts at different
embryonic stages, an RT-PCR analysis of total RNA extracts using gene-specific intronspanning amplification primers was performed (Figure 3.6).
59
twist1a
37
193
510
twist1b
472
30
113
516
twist2
492
34
175
467
250
twist3
191
600
Figure 3.6:
16
879
Gene structure of twist1a, twist1b, twist2 and twist3 showing positions of
intron-spanning primers (arrows) for RT-PCR and extent of unique 3’UTR
probe targets (blue underline) for in situ hybridization. Empty boxes
represent untranslated regions. Black boxes represent coding regions.
GenBank accession numbers of zebrafish twist1a, twist1b, twist2 and
twist3 are NM_001017820, NM_130984, NM_001005956 and
NM_130985, respectively.
Zebrafish twist1b and twist3 transcripts could be detected from as early as the 1cell stage, indicating the presence of maternally expressed and deposited transcripts in the
early embryo prior to zygotic transcript expression (Figure 3.7). In contrast, zebrafish
twist1a and twist2 transcripts were detected only at or after the 1-K cell stage, suggesting
that twist1a and twist2 transcripts are expressed exclusively from the zygote (Figure 3.7).
60
M
1cell
8cell
64cell
1Kcell
shield
bud
14som
1dpf
2dpf
3dpf
No
RNA
twist1a
476bp
twist1b
516bp
twist2
372bp
twist3
520bp
β-actin
561bp
Figure 3.7:
3.6.2
RT-PCR of zebrafish twist genes. Using RT-PCR, twist1b and twist3 are
detectable at the 1-cell stage, indicating the presence of maternally
deposited transcripts. Zebrafish twist1a and twist2 are detectable only
from the 1K-cell stage onwards, when zygotic transcription begins.
In situ hybridization analysis
The observations from RT-PCR analysis were further confirmed by whole-mount
in situ hybridization analysis (Figure 3.8A-D).
Given the high nucleotide identity
between the coding regions of the four zebrafish twist genes, especially in the highly
conserved bHLH and WR domains (Figure 3.4 & Table 2), RNA hybridization probes
were synthesized to target unique 3’UTR of each transcript (Figure 3.6).
During gastrulation (5.25-10 hpf), twist1b and twist3 transcripts are ubiquitously
expressed (Figure 3.9B, D, F, H), whereas there was no detectable expression of twist1a
61
(Figure 3.9A, E). Interestingly, twist2 was specifically expressed in the organizer and the
axial mesoderm at the shield and bud stage respectively (Figure 3.9C, G).
During early segmentation, twist1a transcripts were found to be concentrated in
the premigratory neural crest cells (Figure 3.10A & E). At around 2-somite stage,
zebrafish twist1b expression was detected in the head mesenchyme and intermediate
mesoderm (Figure 3.10B). Expression in the intermediate mesoderm was confirmed by
two-color hybridization with pax2.1 molecular marker for intermediate mesoderm
(Majumdar et al., 2000) (Figure 3.10B). Expression of twit1b in the developing somites
was first detected in all somites of the five-somite stage embryo in a clearly segmental
pattern (Figure 3.10F). Expression of twist2 was observed in the axial mesoderm (Figure
3.10C & G). Zebrafish twist3 was expressed in the somites (Figure 3.10D & H).
62
twist1b
twist1a
A
Figure 3.8:
B
twist2
C
twist3
D
Expression of zebrafish twist genes during the cleavage period, 16-cell stage (A-D). Lateral (A-D) views are
shown. Zebrafish twist1b and twist3 are maternally transcribed (B & D respectively) and no twist1a and twist2
expression was detected at the 16-cell stage (A & C respectively).
63
twist1b
twist1a
A
B
twist2
twist3
C
D
or
E
F
G
H
am
Figure 3.9:
Expression of zebrafish twist genes during the gastrula period. The shield (A-D) and bud (E-H) stages are
shown. Lateral (A-H) views are shown. During the gastrula stage, twist1b and twist3 are homogeneously
expressed (B, D, F, H) while twist2 is specifically expressed in the organizer (C) and later in the axial
medsoderm (G). No expression was detected for twist1a during this period (A, E). am, axial mesoderm; or,
organizer.
64
twist1b
twist1a
A
B
twist2
twist3
D
C
hm
pnc
so
im
am
5S twist1b/pax2.1
E
2S
H
G
F
5S
5S
hm
pnc
so
am
im
5S
sc
im
5S
5S
5S
Figure 3.10: Expression of zebrafish twist genes during the early segmentation period (2-5 somite stage). Lateral (A-D) and
dorsal (E-H) views are shown. At the 5-somite stage, twist1a transcript is detected in the premigratory neural
crest cells (A & E). At the 2-somite stage, twist1b and pax2.1 double color hybridization showed that twist1b is
expressed in the intermediate mesoderm (B). At the 5-somite stage, expression of twist1b is also observed in the
head mesenchyme and sclerotome (F). Expression of twist2 is observed in the axial mesoderm (C & G) and
twist3 expression is detected in the somite (D & H). am, axial mesoderm; hm, head mesenchyme; im,
intermediate mesoderm; pnc, premigratory neural crest cells; sc, sclerotome; so, somites.
65
Later in the segmentation period (11-22 hpf), both distinct and overlapping
expressions was observed in the zebrafish twist gene family. At 14-somite stage, twist1a
transcripts were detected in the neural crest cells (Figure 3.11A & E) and also in the
sclerotome by late somitogenesis (Figure 3.12A & B). At the 18-somite stage, expression
of twist1a was also observed in the neural crest cells that delaminates from the neural
tube at the dorsal region of the somites (Figure 3.12A)
Zebrafish twist1b was expressed in the head mesenchyme, intermediate mesoderm,
sclerotome and caudal gut (Figure 3.11B & F, 3.13A-C).
Double-color in situ
hybridization with dlx2a revealed that twist1b is expressed in the cranial neural crest
derived head mesenchyme (Figure 3.13A). The expression in the intermediate mesoderm
declined through early somitogenesis to the 18-somite stage and double-color in situ
hybridization with wt1, a molecular marker for glomerulus showed that twist1b is not
expressed in the glomerulus (Figure 3.15K)
The expression in the somite was restricted to the anterior portion of each formed
somite (Figure 3.14A). Analysis of sagittal cryosections further revealed that twist1b
expression was concentrated in the sclerotome, with stronger expression throughout the
anterior region of each formed somite (Figure 3.14B & C). This pattern of expression
was maintained until the 11-somite stage (14 hpf) (Figure 3.14D). With continuing
somitogenesis, twist1b expression appeared to shift from the anterior to the more
posterior somites. From the 14-somite stage onward, twist1b expression decreased in the
66
more anterior somites and was concentrated around the more posterior somites (Figure
3.13E-I).
Zebrafish twist2 was expressed in the presumptive vasculature and axial
mesoderm (Figure 3.11C & G). With progressing somitogenesis, its expression in the
axial mesoderm was greatly reduced, and was restricted to the caudal notochord by the
18-somite stage (18 hpf) (Figure 3.12D). Two-color in situ hybridization, using fli1a as a
molecular marker for the presumptive vasculature, confirmed localization of twist2 in this
tissue (Figure 3.12C-E).
Expression of twist3 was observed in the head mesenchyme adjacent to the
forebrain region (Figure 3.11D & 3.13D) as well as in the somites (Figure 3.11H).
67
twist1b
twist1a
twist2
B
A
twist3
D
C
hm
hm
cn
F
E
im
nc
H
G
im
sc
so
pv
Figure 3.11: Expression of zebrafish twist genes during mid-somitogenesis (14-somite stage). Lateral (A-D) and dorsal (EH) views are shown. During somitogenesis, twist1a is expressed in the premigratory neural crest cells (A, E),
twist1b is expressed in the head mesenchyme, intermediate mesoderm and sclerotome (B, F), twist2 is
expressed in the presumptive vasculature and caudal notochord (C, G), , and twist3 is expressed in the head
mesenchyme and somites (D, H). cn, caudal notochord; hm, head mesenchyme; im, intermediate mesoderm; nc,
neural crest cells; pv, presumptive vasculature; sc, sclerotome; so, somites.
68
A
B
sc
sc
nc
18S twist1a
twist1a
C
D
pv
pv
cn
cn
twist2/fli1a
E
18S
14S
twist2/fli1a
18S
twist2/fli1a
18S
Figure 3.12: Zebrafish twist expression along the trunk. Lateral (A, C & D), dorsal (B)
and sagittal section (E) views are shown. At 18-somite stage, expression
of twist1a is detected in the sclerotome and neural crest delaminated from
the neural tube (arrowhead) (A & B). Zebrafish twist2 is expressed in the
presumptive vasculature (C, D & E)), as shown by colocalization with
molecular marker fli1a. cn, caudal notochord; nc, neural crest; pv,
presumptive vasculature; sc, sclerotome.
69
twist1b/dlx2a
A
B
14S
16S
twist1b/dlx2a D
C
twist3/dlx2a
hm
hm
hm
hm
fb
fb
18S
18S
Figure 3.13: Zebrafish twist1b and twist3 expression during somitogenesis. Ventral flat
mount (A), lateral (B) and transverse section (C-D) views are shown.
Zebrafish twist1b and dlx2a double color hybridization shows twits1b
expression in the cranial neural crest-derived head mesenchyme (A). At
the 16-somite stage, twist1b expression is detected in the head
mesenchyme (arrowheads) and caudal gut (arrow) (B). Zebrafish twist1b
(C) and twist3 (D) are not expressed in the forebrain but in the head
mesenchyme adjacent to forebrain.
70
A
6S B
C
9S
10S D
11S
14S F
14S
no
E
no
G
18S H
18S I
21S
no
Figure 3.14:
Zebrafish twist1b expression in the somites. Embryos are shown with
anterior to the left (A, B, D, F, H & I). Dorsal flat mount (A, D, F),
transverse section (C, E & G), sagittal section (B), and lateral flat mount
(H & I) views are shown. During earlier stages of somitogenesis (A-D),
twist1b transcripts are strongly expressed in the sclerotome (arrows). No
expression is observed in the posterior half of the somite. At the 14-somite
stage (E & F), weak expression is restricted to the anterior portion of the
successively more posterior somites. During late somitogenesis (G – I),
weak twist1b expression is confined to the last seven somites from the tail
bud, again, only in the anterior portion of each somite.
71
At the prim-5 stage (24-hpf), twist1a was expressed in the sclerotome,
delaminated neural crest cells from the neural tube, and weakly in the fin bud (Figure
3.15A, E & I). Zebrafish twist1b transcripts were localized in the head mesenchyme
between the eye and diencephalon, cranial neural crest stream 1 to 3, posterior somites,
and the tail bud (Figure 3.15B, F & J). Two-color in situ hybridization with wnt1, a
marker for glomerulus showed that twist1b is not expressed in the glomerulus (Figure
3.15K). Zebrafish twist2 transcripts were observed in the hypochord, dorsal aorta and
caudal notochord (Figure 3.15C, G & L). Strong expression of twist3 was detected in the
head mesenchyme, fin bud, and tail bud, with weak expression in the somites (Figure
3.15D, H & M).
By the long-pec stage (48hpf), transcripts of all four zebrafish twist genes were
detected in the pharyngeal arches (Figure 3.16A-H). In addition, twist1a and twist3
transcripts were also observed in the pectoral fins, specifically in the actinotrichs for
twist1a (Figure 3.16I) and in the proximal (strong) and distal (weak) endochondral disc
for twist3 (Figure 3.16K).
Expression of twist1a was also detected in the head
mesenchyme, fin fold and intermediate cell mass (Figure 3.16E & J). Weak expression
of twist2 was detected in the dorsal aorta (Figure 3.16C).
72
twist1a
A
twist1b
D
C
B
fb
twist3
twist2
fb
fb
fb
E
nc
F
sc
s2
H
G
s3
fb
s1
cn
tb
hm
tb
K
J
I
M
L
nc
no
no
s1 s2
s3
no
gl
sc
twist1a
hc
da
twist1b
twist1b/wt1
twist2
twist3
Figure 3.15: Expression of zebrafish twist genes during the prim-5 stage. Dorsal (A-D), lateral (E-H) and transverse section (IL) views are shown. At 24 hpf, twist1a is expressed in the sclerotome, neural crest cells delaminated from the
neural tube and fin bud (A, E and I). Zebrafish twist1b is expressed in the neural-crest derived head mesenchyme
(s1, s2 & s3), tail bud and three preceding somites (B, F). No expression is observed along the trunk (J) and in the
glomerulus (K), as shown by double color in situ with wt1. twist2 expression is observed in the hypochord, dorsal
aorta and caudal notochord (C, G, and L). twist3 is expressed in the head mesenchyme, fin bud, and tail bud, with
low expression in the somites (D, H, and M). Dotted lines denote the location of the transverse sections. cn,
caudal notochord; da, dorsal aorta; fb, fin bud; ff, fin fold; gl, glomerulus; hc, hypochord; hm, head mesenchyme;
nc, neural crest; no, notochord; s1 – s3, neural crest streams 1 through 3; sc, sclerotome; tb, tail bud.
73
twist1a
A
fb
twist3
twist2
twist1b
B
C
da
pa
fb
D
pa
fb
fb
ff
E
G
F
icm
pa
I
pa
H
pa
pa
48hp
36hp
K
J
fb
ed
no
twist1a
at
hm
twist1a
twist3
Figure 3.16: Expression of zebrafish twist genes during the long-pec stage. Dorsal (A-D), lateral (E-H), transverse section (J),
and flat mount (I, K) views are shown. At 48 hpf, all four twist genes are expressed in the pharyngeal arch (A-H).
Only twist1a and twist3 transcripts are detected in the fin buds (A, E, D, H, I, K). twist1a is also expressed in the
head mesenchyme (arrow), fin fold and intermediate cell mass (E, J), and twist2 is weakly expressed in the dorsal
aorta (C, G). Dotted lines denote the location of the transverse sections. at, actinotrichs; da, dorsal aorta; ed,
endochondral disc; fb, fin bud; ff, fin fold; hm, head mesenchyme; icm, intermediate cell mass; pa, pharyngeal
arches; no, notochord; tb, tail bud.
74
At the protruding mouth stage (72hpf), all four zebrafish twist transcripts
continued to be observed in the pharyngeal arches (Figure 3.17A-H). Additionally,
twist1a was expressed in the head mesenchyme, heart valve, pectoral fin, fin fold and
intermediate cell mass (Figure 3.17A & E). Expression in the heart was not observed for
the other twist genes (Figure 3.17B-D). Zebrafish twist1b was also expressed in the
olfactory placode (Figure 3.17F). Expression of twist3 was also detected in the pectoral
fin (Figure 3.17D & H).
75
twist1a
twist1b
ff
A
B
twist3
twist2
D
C
icm
pa
pa
pa
hv
E
F
pa
H
G
pf
pf
pa
of
hm
pf
Figure 3.17:
pa
pa
pa
pf
Expression of zebrafish twist genes during the hatching period. Lateral (A-D) and dorsal (E-H) views are shown. At
72hpf, all four twist genes are expressed in the pharyngeal arch (A-H). Zebrafish twist1a and twist3 are expressed in the
pectoral fins (E, H). twist1b is also found to be expressed in the olfactory placode (F), and twist1a is expressed in the
head mesenchyme, heart valve (arrow head), fin fold and intermediate cell mass (A, E). Insets show enlarged heart views.
ff, fin fold; hm, head mesenchyme; hv, heart valve; icm; intermediate cell mass; of, olfactory placode; pa, pharyngeal
arch; pf, pectoral fin.
76
Chapter 4: Discussion
_____________________________________________________________
4.1
The zebrafish twist gene family
The zebrafish twist gene family consists of four members: twist1a, twist1b, twist2
and twist3 (Gitelman, 2007). All four members are highly conserved transcription factors
that shared the bHLH domain and the WR motif. Within the conserved bHLH domain
and WR motif, high nucleotide similarity of up to 86% is observed. Regions outside the
conserved domains are also relatively similar (52-81% similarity) (Table 2).
Prior to the Gitelman (2007) publication, there were three members of the
zebrafish twist gene family reported in GenBank (Accession no: NM_130984 for twist1,
NM_130985 for twist2 and NM_001005956 for dermo1). However, there was some
confusion in the identity of the zebrafish orthologs as the namings by Gitelman (2007)
were different from GenBank.
The first step in identifying and confirming the true zebrafish orthologs was to
compare them against the sequences in other species. Zebrafish twist1a and twist1b show
a higher sequence similarity to Twist1 in other species compared to zebrafish twist2 and
twist3 with other species Twist1. This suggests that zebrafish twist1a and twist1b are coorthologs of mammalian Twist1. Interestingly, zebrafish twist1a and twist1b have higher
similarity to mammalian Twist2 compared to mammalian Twist1.
Nevertheless, the
ortholog of human TWIST2 is zebrafish twist2 because zebrafish twist2 has higher
similarity (compared to zebrafish twist1a and twist1b with Twist2 of other species) and
the alignment also showed that zebrafish twist2 peptide is highly similar to mammalian
77
Twist2 peptide (Figure 3.4 and Table 3). The high sequence similarity of zebrafish
twist1a and twist1b to mammalian TWIST2 suggests that the functions of zebrafish
twist1a and twist1b could be more similar to the functions of mammalian TWIST2
compared to the functions of mammalian TWIST1. Based on the amino acid analysis,
TWIST2 is highly conserved not just in mammals but also in chick suggesting conserved
functions.
Interestingly, zebrafish twist3 has a lower sequence similarity to chick and
Xenopus Twist3 than zebrafish twist1 and twist2. However, the sequence similarity of
zebrafish twist3 compared to the other Acanthopterygiian twist3 (medaka and stickleback)
is significantly higher compared to zebrafish twist1 or twist2.
4.2
Phylogenetic relationships of the twist genes in fish
Using peptide sequences from 33 homologous Twist proteins belonging to 12
different species, a clear phylogenetic clustering of the vertebrate Twist proteins into 3
major clades was observed, each of which contained both teleost and tetrapod orthologs.
Zebrafish Twist1a (Dr1a) and Twist1b (Dr1b) fall within clade I, together with Twist1a
from medaka, stickleback, fugu, and pufferfish (Figure 3.6). However, Twist1b from
medaka, stickleback, fugu, and pufferfish did not cluster within clade I, but instead
grouped together with the Twist3 proteins within clade III in the rooted and unrooted
trees. These observations reflect the very high degree of peptide sequence similarity
between Twist1a and Twist1b in zebrafish, a member of the superorder Ostariophysi,
whereas the Twist1b peptides in the other four fish species, which all belong to the
78
superorder Acanthopterygii, exhibit greater peptide similarity with Twist3 than with
Twist1a (Figure 3.4).
While the Twist1b peptides of the acanthopterygii are tightly clustered together,
their presence within clade III raises questions regarding their true phylogenetic
relationships. A recent phylogenetic analysis of a similar set of Twist peptides, using
multiple phylogenetic reconstruction methods, placed the acanthopterygian Twist1b
peptides outside of the 3 major clades (Gitelman, 2007). The author postulated that this
outgroup was likely an artifact of long-branch attraction, and suggested that their
placement within clade I was the most parsimonious choice. I now provide evidence
from the analysis of conserved synteny between the zebrafish and medaka twist1a and
twist1b chromosomal regions that despite the observed phylogenetic grouping of the
acanthopterygian Twist1b, these genes are the true orthologs of ostariophysian twist1b, as
Germanguz et al. (2007) and Gitelman (2007) had predicted. The comparative synteny
studies also demonstrate that the ostariophysian and acanthopterygian twist1a and twist1b
genes are co-paralogs and co-orthologs of mammalian Twist1/TWIST1. Thus, although
phylogenetic analysis algorithms provide reasonable estimations of phylogenetic and
evolutionary relationships of gene families among species, ambiguities may arise which
could be resolved through other methods such as comparative analysis of conserved
synteny.
4.3
Genetic distance analysis of twist1a and twist1b among the fishes
The Kimura and Tajima-Nei analyses of the nucleotide substitution rates between
twist1a and twist1b cDNA sequences are consistent with the observed closer evolutionary
79
relationship among the four acanthopterygian fish as compared to the ostariophysian
zebrafish.
They also account for the observed phylogenetic clustering of the
acanthopterygian Twist1b peptides into clade III. Given the substantial Twist1b peptide
sequence difference between zebrafish and the other four fish species, twist1b expression
and functional observations made in the zebrafish may not necessarily be extrapolatable
to other species. Germanguz et al. (2007) have hypothesized that Twist1b in medaka,
stickleback, fugu, and pufferfish may be involved in building the interarcual cartilage, a
cephalic neural crest derivative that defines all four fish. It would be interesting to see if
the medaka twist1b expression profile has indeed diverged significantly from zebrafish.
4.4
Comparative synteny analyses
A detailed comparative synteny analysis was performed to confirm the zebrafish
orthologs of human TWIST1 (Table 4).
The comparative table clearly showed that
zebrafish twist1a and twist1b are co-orthologs of the human TWIST1 as the genes around
human TWIST1, on chromosome 7, were found to be duplicated or located on either
LG16 or LG19 where zebrafish twist1a and twist1b are located respectively (Table 4).
Both genes are linked to the hoxa gene cluster, whose duplication in ray-finned fishes
was inferred to occur by whole genome duplication (Amores et al., 1998).
Thus
zebrafish twist1a and twist1b are co-paralogs and co-orthologs of human TWIST1 that
most likely arose through a fish-specific genome duplication event.
Due to some confusion in the naming of the zebrafish LG16 and LG19 located
twist1 co-paralogs in the genomic databases, a more in-depth comparative synteny
analysis of the zebrafish and medaka twist1a and twist1b chromosomal regions was
80
performed to resolve their orthologies. Comparison of the chromosomal region around
zebrafish twist1a (on LG16) against the chromosomal regions around medaka twist1a (on
LG16) and twist1b (on LG11), revealed that a large number of genes were present on
both zebrafish LG16 and medaka LG16. Although there were some genes that were in
common between zebrafish LG16 and medaka LG11, these were comparatively fewer
(Table 5). When the chromosomal region around zebrafish twist1b (on LG19) was
compared against the chromosomal regions around medaka twist1a and twist1b, a large
number of genes were observed to be present on both zebrafish LG19 and medaka LG11
region, with fewer shared genes between zebrafish LG19 and medaka LG16 (Table 5 &
6). These observations provide further confirmation that the twist1a and twist1b genes
arose from a fish-specific genome duplication event and are true co-paralogs.
Furthermore, the results indicate that the zebrafish LG16 and LG19 twist1 genes are
orthologous to the medaka LG16 and LG11 twist1 genes, respectively.
The comparative synteny analysis of the zebrafish twist2 chromosomal segment
clearly revealed that zebrafish twist2 is the ortholog of human and medaka Twist2 as it
shows chromosomal synteny with the human and medaka twist2 loci on chromosome 2
and LG21, respectively (Table 7).
Zebrafish twist3 has no known mammalian ortholog, and lacks chromosomal
synteny with any of the other twist loci in mammals, consistent with its placement in a
clade separate from twist1 or twist2. The comparative synteny analysis of zebrafish
twist3 with medaka twist3a and twist3b show that the sole zebrafish twist3 gene is
orthologous to both medaka twist3a and twist3b, although there are more shared genes
81
between the zebrafish twist3 chromosomal region and the medaka twist3b chromosomal
region (Table 8).
The two medaka twist3 genes appear to have arisen through a
chromosomal or genome duplication event, as suggested by the presence of multiple
shared genes between LG5 and LG7.
Unlike twist1, however, the number of twist3 genes is variable, even within the
acanthopterygii. There are two copies in medaka and stickleback, one in zebrafish, and
none in fugu and pufferfish.
Combining the data from our phylogenetic and comparative synteny analyses, a
model for the evolutionary history of the twist genes has been reconstructed (Figure 4.1).
This model assumes the occurrence of three genome duplication events in the
evolutionary history of the teleosts. The first two events occurred prior to the divergence
of the teleost ancestors the ray-finned fishes (Actinopterygii) from the tetrapod ancestors
the lobe-finned fishes (Sarcopterygii). In the first duplication event, the ancestral Twist
gene probably gave rise to the Twist3 gene and a Twist1/Twist2 ancestral gene. This
latter ancestral gene then split to give rise to Twist1 and Twist2 during the second
duplication event. Subsequently during the teleost-specific third duplication event, which
occurred approximately 350 million years ago (Ravi and Venkatesh, 2008), the twist1 and
twist3 genes were duplicated into twist1a and twist1b and into twist3a and twist3b,
respectively. The absence of a complete set of 8 twist genes in any of the fish lineages
that have been examined suggests that some of the duplicated genes were lost over time
through functional redundancy.
82
D
Tr1a
Tn1a
Ol1a
Ga1a
Dr1a
Dr1b
Ga1b
Ol1b
Tn1b
Tr1b
Twist1
Mm1
Rn1
Hs1
Gg1
Xt1
Tr2
Tn2
Ol2
Ga2
D
Dr2
Mm2
Rn2
D
Twist2
Hs2
Gg2
Ol3a
Ga3a
D
Dr3
Ol3b
Ga3b
Twist3
Gg3
Xt3
Bb
Lv
Figure 4.1:
A model for the evolutionary history of twist genes. Circle with a 'D' in the middle represent gene or genome duplication.
83
4.5
Comparison of zebrafish twist family expression patterns with other species.
4.5.1
Zebrafish twist1a and twist1b genes
Zebrafish twist1a and twist1b are co-orthologs of mammalian Twist1.
Interestingly, the expression patterns of this duplicated gene revealed shared and unique
expression sites. Zebrafish twist1b is present as a maternal transcript in early zebrafish
embryos prior to the mid-blastula transition; this observation is consistent with mouse
and Xenopus Twist1 (Stoetzel et al., 1995; Stoetzel et al., 1998). The transcripts of
zebrafish twist1a were first detected at 1K-cell stage by RT-PCR (Figure 3.8). This
observation is similar in Drosophila, where twist is expressed as a zygotic transcript
(Thisse et al., 1987; Thisse et al., 1988).
During early somitogenesis from the 2-somite to the 10-somite stage, zebrafish
twist1b is expressed in the intermediate mesoderm, also known as the presumptive
pronephric duct.
The 2-5 somite stage coincides with the first of 4 stages in the
development of the zebrafish pronephros, when the intermediate mesodermal cells
become committed and a nephrogenic field is established.
The other 3 stages are
between the 6-somite and prim-5 stages, when the pronephric duct and nephron
primordia are formed, from the prim-5 to high-pec stages, when cell patterning events
take place leading to the formation of the pronephric glomeruli and tubules, and from the
high-pec to hatching (long-pec) stages, when the pronephros becomes functional with the
formation of the glomerular capillary tuft and onset of blood filtration (Solnica-Krezel,
2002). Lee et al. (2000) reported that expression of human TWIST1 and/or DERMO1
(TWIST2) protein has been observed in the renal collecting and secretory tubules.
Interestingly, there have been reports of patients with SCS who had renal tubular
84
dysfunction or agenesis (Russo et al., 1991; Oktenli et al., 2002), although the
relationship between the renal condition and the TWIST1 gene in these patients remains
to be proven. Two color in situ hybridization of twist1b and wt1, a marker for glomerulus,
showed the absence of twist1b expression in the glomerulus. The presence of twist1b
transcripts in the intermediate mesoderm only during stage 1 and early stage 2 of
zebrafish pronephros development suggests that twist1b may be important in the
commitment of the undifferentiated mesodermal cells to a nephrogenic fate.
The localized expression of twist1a and twist1b to the sclerotome of the
developing somites is consistent with similar observations in mouse, Xenopus, chick and
medaka (Table 10) (Hopwood et al., 1989; Wolf et al., 1991; Tavares et al., 2001;
Yasutake et al., 2004). In zebrafish, the sclerotome is located at the ventromedial region
of each somite and gives rise to connective tissue and vertebral cartilages. The presence
of twist1a and twist1b transcripts in the sclerotome suggests a role in somitogenesis and
vertebral cartilage formation. Knockdown studies in the medaka fish reveal that twist1a
is not involved in the migration of sclerotomal cells but in regulating the differentiation
of the sclerotomal cells into the vertebral column (Yasutake et al., 2004). Furthermore,
cervical spine abnormalities have been described in patients with SCS, including fusion
of the vertebral bodies and posterior elements (Anderson et al., 1997; Trusen et al., 2003).
Together, these observations strongly suggest that tight regulation of TWIST1 gene
expression is necessary for normal development of vertebral bodies.
The zebrafish cranial neural crest cells appear to be specified at an early stage and
migrate in three distinct streams (Schilling and Kimmel, 1994). Stream 1 will eventually
give rise to the first or mandibular branchial/pharyngeal arch, stream 2 will form the
85
second or hyoid arch, and stream 3 will give rise to the 5 ceratobranchial arches
(Lumsden et al., 1991; Piotrowski and Nusslein-Volhard, 2000). Consistent with Twist1
expression in mouse and Xenopus, zebrafish twist1a and twist1b was expressed at the
neural crest cells and later in the pharyngeal arches (Hopwood et al., 1989; Fuchtbauer,
1995).
Twist1-/- knockout mouse embryos showed merging of the first and second
branchial arches (Soo et al., 2002), suggesting that Twist1 activity is required for proper
migration of the different streams of neural crest cells and differentiation of the branchial
arches.
4.5.2
Zebrafish twist2
Consistent with the observation of Dermo-1 (Twist2) expression in the mouse,
zebrafish twist2, whose transcript was detected at the 1K-cell stage, is present as a
zygotic transcript (Li et al., 1995).
During the pharyngula period, twist2 is also expressed in the pharyngeal arches.
The expression of zebrafish twist2 in the pharyngeal arches is consistent with the
expression of mouse twist2 (Li et al., 1995). Studies performed in mouse showed that
Twist2 was expressed in osteoblastic cells and it may act as a negative regulator of the
osteoblast differentiation, i.e. to inhibit osteoblast maturation and maintain the cells in a
preosteoblast phenotype (Tamura and Noda, 1999; Lee et al., 2000). I was unable to
detect the expression in osteoblastic cells because I was only looking at early embryonic
expression up to 72 hpf and the osteoblast cells are not formed yet.
86
4.5.3
Zebrafish twist3
The Twist3 gene is absent in mammals but found in Xenopus, chick, medaka,
stickleback and zebrafish (Gitelman, 2007). No expression data of the Twist3 gene is
available in other species.
4.6
Shared and unique expression sites of the zebrafish twist genes
4.6.1
Importance of using unique 3’UTR sequences as riboprobes
Two reports in the 1990s used zebrafish twist1 (twist1b) as a molecular marker for
the sclerotome and the axial mesoderm (Halpern et al., 1995; Morin-Kensicki and Eisen,
1997). However, in this study, the expression in the axial mesoderm is from zebrafish
twist2 and not zebrafish twist1b. It is possible that the apparent twist1b expression in
axial mesoderm could have arisen from use of a riboprobe that included coding
sequences, which could have cross-hybridized to twist2 transcript.
As mentioned above and shown in the results section, the zebrafish twist genes
have high sequence similarity especially in the coding region. Thus, in this study, the
design of unique riboprobes is of great importance and 3’UTR sequences were chosen to
be used as riboprobes to exclude and minimize any possible cross-hybridization among
the family members.
4.6.2
Comparison of zebrafish twist genes expression sites with other publications
The expression patterns of the zebrafish twist gene family have been previously
described by Rauch (2003) and Thisse and Thisse (2004) in the ZFIN database, and
87
recently by Germanguz et al (2007). Comparing my in situ results and those previously
reported, I found differences in the expression patterns and the differences could be
attributed by the targeting RNA probes used for in situ hybridization.
Previously
reported expression profiles were performed using either expressed sequence tags (ESTs)
or full-length cDNA, either exclusively or in combination with shorter gene-specific
probe outside of conserved domains.
In profiling of the twist gene expression,
hybridization probes targeting unique 3’ UTR sequences of each of the four twist genes
were utilized to completely avoid the highly homologous bHLH and WR domains (Table
2). Members of the twist gene family are highly homologous and this present study may
provide a good example to illustrate that the use of probes targeting at different sequences
of highly homologous gene family members can give rise to different expression profiles.
Zebrafish twist1a and twist1b are co-orthologs of mammalian Twist1. Using both
RT-PCR and whole-mount in situ hybridization analyses, maternal transcripts of
zebrafish twist1b are detected in early zebrafish embryos prior to the mid-blastula
transition. In contrast, zebrafish twist1b expression was detected only at the gastrula
period by two previous groups (Rauch, 2003; Germanguz et al., 2007). Using RT-PCR,
zebrafish twist1a transcripts is detected at 1K-cell stage and subsequenct in situ
hybridization shows its expression in the neural crest cells at the bud stage (10hpf). This
zygotic expression pattern of zebrafish twist1a is consistent with previously published
data (Germanguz et al., 2007; Gitelman, 2007). Similarly, zebrafish twist2 transcripts are
detected at 1K-cell stage by RT-PCR and previous reports in zebrafish also show that
twist2 is zygotically transcribed (Thisse and Thisse, 2004; Germanguz et al., 2007;
Gitelman, 2007). For zebrafish twist3, its expression is detected from as early as in the 1-
88
cell zygote stage (0.2 hpf) by both RT-PCR and in situ hybridization, strongly suggest
that it is a maternal transcript. In contrast, this was not previously reported and the twist3
expression is only detected from the 13-somite stage (16 hpf) onwards (Germanguz et
al., 2007; Gitelman, 2007).
During early somitogenesis, Twist1 is expressed in the lateral plate mesoderm in
mouse, chick and Xenopus (Hopwood et al., 1989; Wolf et al., 1991; Tavares et al., 2001).
Similarly, previous report also showed that zebrafish twist1a (early- to midsomitogenesis) and twist3 (mid-somitogenesis) are expressed in the lateral plate
mesoderm Germanguz et al. (2007). However, I did not detect any expression in the
lateral plate mesoderm except for zebrafish twist1a and twist3 in the fin bud, which is
derived from the posterior lateral plate mesoderm (Table 8). A previous report also
found twist1a, twist1b, and twist3 expression in the pectoral fin bud (Germanguz et al.,
2007; Gitelman, 2007). Contrastingly, I observed pectoral fin bud expression only for
twist1a and twist3 and this observation corroborates with Rauch (2003) data. I also
detected twist1b expression in the intermediate mesoderm until late somitogenesis and
this was confirmed by two-color WISH using pax2.1 molecular marker.
Zebrafish twist1a and twist1b is expressed in the sclerotome and twist3 in the
somite. I also detected twist1b expression in the caudal gut at around the 16-somite stage
(17hpf). However, a previous study showed zebrafish twist1b expression in the myotome
and ventral gut (Germanguz et al., 2007). Besides the expression in the somites or
sclerotome expression, I also detected twist1a expression in the intermediate cell mass
during the pharyngula period (24-48 hpf), which was not reported previously.
89
Additionally, zebrafish twist1a, twist1b, and twist2 were previously reported to be
expressed in the blood vessels of the developing brain at around 36 hpf (Germanguz et al.,
2007), but I did not observe this. In contrast, I found that only zebrafish twist2 is
expressed in the presumptive vasculature along the body from the 14- to 18-somite stage
(16-18 hpf), and I further confirmed this localization by two-color in situ hybridization
with fli1a, a molecular marker for presumptive vasculature. This expression has not been
previously reported.
Furthermore, I observed that twist2 expression in the axial
mesoderm during mid-somitogenesis was concentrated not in the tail bud as previously
reported (Germanguz et al., 2007; Gitelman, 2007), but in the caudal notochord.
The Twist genes are also found to be expressed in the pharyngeal arches in mouse,
chick and Xenopus (Hopwood et al., 1989; Wolf et al., 1991; Li et al., 1995; Scaal et al.,
2001; Tavares et al., 2001).
Similarly, we also observed expression of all the four
members of the zebrafish twist gene family in the pharyngeal arches which corroborates
with previously reported data for zebrafish twist gene family (Thisse and Thisse, 2004;
Germanguz et al., 2007). Table 9 summarizes the essential similarities and differences in
twist expression patterns between this study and the previous data reported in Germanguz
et al (2007) and Gitelman (2007). It is not entirely clear why there are substantive
differences in twist expression sites observed between my study and those previously
reported but I note that the differences may be due to the different segments of cDNA
used as probes by the different groups.
90
Table 9: Expression domains of the four zebrafish twist genes
Period
Stage (time)
Tissue
Present study; Yeo et al. (2007)
Dr1a
Dr1b
Dr2
Dr3
(LG16) (LG19)
(LG9) (LG23)
Germanguz et al. / Gitelman (2007)
Dr1a
Dr1b
Dr2
Dr3
(LG16) (LG19) (LG9)
(LG23)
Cleavage
Zygote (0 hpf)
‐
1K‐cell (3 hpf)
‐
Shield (6 hpf)
Whole embryo
Areas lateral to embryonic axis
Organizer
Whole embryo
Axial mesoderm
Anterior neural plate & mesodermal tissues
Whole embryo
Head mesenchyme
Axial mesoderm / Chordamesoderm
Intermediate mesoderm
Lateral plate mesoderm
Cephalic neural crest / Premigratory crest cells
Cranial/cephalic neural crest derived head
mesenchyme
Neural rod
Axial mesoderm / Chordamesoderm
Intermediate mesoderm
Lateral plate mesoderm
Premigratory crest cells
Somite
Sclerotome
Myotome
Tail bud
Cranial/cephalic neural crest derived head
mesenchyme
Olfactory placode
Dorsal forebrain
Migratory neural crest cells
Neural crest cells (delaminated from neural tube)
Intermediate mesoderm
Lateral plate mesoderm
Nephric duct/pronephric duct
Somite
Sclerotome
Myotome
Presumptive vasculature
Caudal gut (16‐somite)
Ventral gut (15‐somite)
Caudal notochord
Tail bud
Forebrain/Midbrain/Hindbrain area
Pharyngeal arches
Nephric duct/ pronephric duct
Hypochord
Somite
Sclerotome
Myotome
Pectoral fin bud
Tail bud
a
a
Head mesenchyme (cranial neural crest derived)
Brain (forebrain, midbrain, hindbrain)
Neural crest cells (delaminated from neural tube)
Dorsal aorta
Blastula
Gastrula
Bud (10 hpf)
Segmentation
2‐somite (10.7 hpf)
5‐10 somite (11.5‐14 hpf)
13‐18 somite (15.5‐18 hpf)
24 somite
Pharyngula
Prim‐5 (24 hpf)
Not analyzed
91
30 hpf
36 hpf
Long‐pec (48 hpf)
Hatching
Protruding mouth (72 hpf)
a
Not reported
Head mesenchyme
Olfactory placode
Pharyngeal arch
Pectoral fin
Fin fold
Heart valve
Intermediate cell mass
Not reported
Not analyzed
Not analyzed
a
Hypochord
Nephric duct/pronephric duct
Somite
Sclerotome
Pectoral fin bud
Caudal notochord
Tail bud
Nephric duct/pronephric duct
Tail bud
Vasculature of developing forebrain / midbrain
Olfactory placode
Dorsal aorta
Cranial neural crest derivatives (pharyngeal arch)
Pectoral fin bud
Dorsal fin bud
Head mesenchyme
Pharyngeal arch
Pectoral fin
Fin fold
Intermediate cell mass
dorsal somite
4.7
Evolutionary fates of the zebrafish twist gene family
The distinct and overlapping expression sites that I observed for the different
members of the twist gene family indicate that twist gene members may perform both
redundant and non-redundant functions during embryonic development. The functions of
each gene member in a gene family may evolve over time through non-, neo-, sub- and/or
synfunctionalization (Postlethwait et al., 2004; Gitelman, 2007; Roth et al., 2007). For
example, the mouse twist1 expression domains in the nasal placode and cells of the limb
bud are partitioned between zebrafish twist1b (olfactory placode) and zebrafish twist1a
and twist3 (pectoral fin) (Table 10). However, until the definitive expression patterns of
these genes are confirmed, it would not be easy to make meaningful conclusions about
the evolutionary changes that have occurred in the various zebrafish twist genes. For
example, Gitelman (2007) attributed the unique expression of zebrafish twist1b in the
92
myotome as a neofunctionalization event. However, I have not been able to reproduce
the twist1a expression in the myotome in our study. Also, if indeed none of the zebrafish
twist genes are expressed in the lateral plate mesoderm, this may represent a
nonfunctionalization event in the fish lineage, or conversely a neofunctionalization of
Twist1 in the tetrapod lineage, which exhibits lateral plate mesoderm expression in mouse,
chick and Xenopus.
However, I note that zebrafish twist1a is expressed in the
intermediate cell mass, a derivative of the lateral plate mesoderm (Table 10). Additional
detailed and careful expression profiles in other species are necessary in order to
construct a comprehensive understanding of the evolution of the vertebrate, and teleost,
twist genes. Nevertheless, what is clear is that despite the still significant degree of
peptide similarity among the four zebrafish twist genes, their regulatory control has
diverged to the point that there is now minimal overlap of their developmental expression
profiles, and thus minimal functional redundancy among them.
93
Table 10: Twist expression sites in selected species.
Mm1a
Mm2b
Lateral plate mesoderm
Head mesenchyme (neural crest derived)
Intermediate mesoderm
Somite
Caudal gut
Gg2d
Xl1e
Dr1a
Dr1bf
Dr2
Dr3
Neural crest cells
Olfactory placode
Pharyngeal arch/branchial arch
Axial mesoderm/caudal notochord
Tail bud
Pectoral fin bud / limb buds
Hypochord
Dorsal aorta
Fin fold /body wall
Presumptive vasculature
Heart valve
Intermediate cell mass
a
Gg1c
b
c
d
e
f
(Wolf et al., 1991); (Li et al., 1995); (Tavares et al., 2001); (Scaal et al., 2001); (Hopwood et al., 1989); (Yeo et al., 2007)
x
94
Chapter 5: Conclusion
________________________________________________________________________
In this study, I have clarified and confirmed the evolutionary orthology of the four
zebrafish twist genes with their mammalian counterparts and other fish species by
nucleotide similarity comparison, phylogenetic analysis as well as comparative synteny
analysis.
Using unique 3’UTR sequence probes for the four twist genes, I have documented
their shared and unique developmental expression profiles, which suggests minimized
functional redundancy among them.
95
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[...]... analysis of the chromosomal regions flanking the zebrafish, medaka, and human twist genes showed that the zebrafish twist1 a and twist1 b are coparalogs and co-orthologs of human TWIST1 Furthermore, zebrafish twist1 a and twist1 b are orthologous to medaka twist1 a and twist1 b, respectively, despite the different phylogenetic clusterings of zebrafish and medaka twist1 b The orthology of zebrafish twist2 to... structure, and map location of the zebrafish twist genes 2 Sort out the confusion in evolutionary orthology of the zebrafish twist genes with their mammalian counterparts through comparative gene structure and linkage synteny analyses 3 Characterize and compare the developmental and tissue-specific expression profiles of the zebrafish twist genes during embryogenesis and in adult zebrafish 4 Compare expression. .. and developmental expression patterns of the zebrafish twist gene family (reference in press) xii Abstract _ Four members of the twist gene family (twist1 a, 1b, 2 and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split Phylogenetic analysis groups most of the. .. structures, including the forebrain and area of the nasal placodes, the diencephalon and the optical vesicles, the rhombencephalon and around the otic vesicles Furthermore, a high level of expression was observed in the branchial arches In the trunk, Twist1 expression is detected in the sclerotome and somatopleura In addition, Twist1 expression is also found in the posterior limb buds and tail, the mesenchyme... 2007) Phylogenetic and gene expression studies are invaluable tools that aid in our understanding of the regulation and function of conserved genes and gene families such as the TWIST gene family Together, they provide important clues to the evolutionary events and functional changes that have occurred in these genes in different species 14 1.5 Aims In this study, I aimed to: 1 Determine the complete... (Samonte and Eichler, 2002) In the TWIST gene family, gene duplication is observed TWIST1 and TWIST3 genes are found to be duplicated in some species Duplication of this Twist1 gene (twist1 a and twist1 b) has been observed specifically in Actinopterygii (ray-fined fishes) (Gitelman, 2007) In stickleback and medaka, there are also two copies of the twist3 gene (Gitelman, 2007) 1.4.1 Evolutionary fates of duplicate... Zebrafish twist1 a and twist1 b were expressed in the sclerotome and twist3 in the somite during the segmentation period Zebrafish twist1 b and twist3 were found to be present as maternal transcript Many expression sites were unique Transcripts of twist1 a were detected specifically in the premigratory neural crest cells during early somitogenesis and in the heart valve at the hatching period Zebrafish twist1 b... and medaka TWIST2 /twist2 , was also confirmed Finally, zebrafish twist3 showed greater chromosomal synteny to medaka twist3 b than to the medaka twist3 a Based on these results, a model for the evolutionary history of the twist genes has been reconstructed I also performed a comprehensive developmental expression analysis of all four twist genes All four genes were expressed in the pharyngeal arches Zebrafish. .. synfunctionalization, one copy of the duplicate gene acquires a unique expression domain of the other and hence, all unique functions of this gene can be found on one copy of the duplicates Therefore, the other copy of the duplicate gene becomes redundant and leads to gene loss (Figure 1.2) (Gitelman, 2007) If both copy of the duplicate gene are retained due to their remaining unique functions, function shuffling 13... expressed in the intermediate mesoderm during segmentation period and in the olfactory placode at the hatching period Zebrafish twist2 expression was observed in the organizer at the shield stage, presumptive vasculature during the segmentation period, and in the hypochord and dorsal aorta during the prim-5 stage Zebrafish twist1 a and twist3 were expressed in the fin bud, with twist3 expression concentrated ... pattern with other species 83 4.5.1 Zebrafish twist1 a and twist1 b genes 83 4.5.2 Zebrafish twist2 85 4.5.3 Zebrafish twist3 86 4.6 Shared and unique expression sites of the zebrafish twist genes 86... proteins generated by the neighbor-joining method Figure 3.6: Gene structure of twist1 a, twist1 b, twist2 and twist3 Figure 3.7: RT-PCR of zebrafish twist genes Figure 3.8: Expression of zebrafish twist. .. medaka, and human twist genes showed that the zebrafish twist1 a and twist1 b are coparalogs and co-orthologs of human TWIST1 Furthermore, zebrafish twist1 a and twist1 b are orthologous to medaka twist1 a