Hedgehog signaling in the zebrafish embryo role of kif7 and DZIP1 2

Hedgehog Signaling in the zebrafish embryo: Role of Kif7 and DZIP1 Chapter INTRODUCTION 1.1 Origin of the Hedgehog (Hh) pathway How diverse cell types are generated and organized to form appropriate body patterns remains an important question in animal development Body segmentation and patterning of organs pose interesting questions for biologists to unravel One important concept that was gathered from these studies is that signaling molecules such as 2nd messenger molecules relay signals from the receptors and target cells to specify their fate (Pan and Rubin, 1995) or regulate metabolic activities (Hartl and Wolfe, 1990) Therefore, much of the early developmental research was on creating mutagenic screens to identify mutants and understand the pathways that govern these processes The use of Drosophila as a model in early genetics studies came from Morgan et al efforts (Morgan, 1911) The framework of Hh pathway comes from genetic and molecular analyses in Drosophila animal model owing to its ease of forward genetic approach such as the use of P and M strains for transposon tool (Kidwell et al., 1977) and discovery of balancer chromosome (Muller et al., 1937) ease the maintenance in keeping the flies in their heterozygous state The hedgehog (hh) gene was first identified in a genetic screen aimed at understanding body segmentation and development in Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980) Disrupted larval body plan resulting in a duplication of the projections from the larval cuticle was found in hh mutants These additional denticles gave the mutant embryos the appearance of a “spiky” phenotype which resembles the animal hedgehog, hence its name Hh proteins act as morphogens which have either short or long range effects (Caspary et al., 2002) They act in a concentration dependent manner and control multiple developmental processes such as neural patterning, limb development and muscle formation (McMahon et al., 2003; Schilling et al., 1999) Therefore, Hh signaling plays important roles in cell fate specification and animal development (Ingham and McMahon, 2001; Varjosalo and Taipale, 2008) Most of the known signaling pathways (such as Mitogen-Activated Protein Kinase and Chk1 checkpoint) are conserved across species (Sanchez et al., 1997; Widmann et al., 1999) To investigate if this is also true for the Hh pathway, genetic screens in vertebrates were performed The existence of vertebrate hh genes was first reported in 1993 A collaborative effort between three groups helped to identify hh genes in fish (Krauss et al., 1993) , chick (Riddle et al., 1993) and mouse (Echelard et al., 1993) Three mammalian hh genes were subsequently identified: Sonic, Indian and Desert hh (Shh, Ihh and Dhh) In the zebrafish model, multiple paralogs of hh genes exist due to an additional round of duplication For example, shh has a paralog shhb which is subsequently renamed as tiggy winkle hh (twhh) (Currie and Ingham, 1996; Ekker et al., 1995; Ingham and McMahon, 2001) Hh paralogs work dependently with each other, and can normally compensate for each other loss (Echelard et al., 1993; Marigo et al., 1995) The existence of the human homolog, SHH soon follows It is not surprising that the Hh pathway also has similar regulatory functions in human development (Jacob and Lum, 2007; McMahon et al., 2003) Mis-regulation of the pathway has been reported in a number of human genetic diseases For instance, down regulation of the Hh pathway has been implicated in birth defects such as craniofacial defects, holoprosencephaly and skeletal malformations (Bale, 2002; Hu and Helms, 1999) Up-regulation of Hh activity can result in basal cell carcinomas and other kinds of tumor formation (Bale and Yu, 2001) Examples of known human genetic illnesses where Hh have been implicated in are Bardet Biedl syndrome and Gorlin’s syndrome (Bale and Yu, 2001; Fan et al., 1997; Ruiz i Altaba et al., 2002) Patients suffering from these illnesses exhibit phenotypic traits as postaxial polydactyly, facial skeletal defects and multiple basal cell carcinomas of the skin that are highly resemblance to Hh signaling defects in animal models With the number of human diseases being linked to Hh regulation, it is important that this signaling pathway be thoroughly studied so that it can be manipulated to our advantage Although the Hh pathway has been well characterized in Drosophila, less is known of its components in the animal models, let alone homo sapiens Some vertebrate orthologs of the Drosophila Hh components have similar conserved functions but existence of paralogs and novel components suggest that vertebrate Hh signaling is much more complicated 1.2 Drosophila Hedgehog Signaling Pathway In the Drosophila Hh pathway, there is one Hh ligand which binds to its receptor Patched (Ptc) (Fig 1.1) Binding of Hh to Ptc results in alleviation of its repression on a seven transmembrane protein Smoothened (Smo) whose domain resembles that of G-protein coupled receptors (GPCR) (Alcedo et al., 1996) Smo acts as a positive regulator of Hh pathway and activation of this protein regulates the transcription factor Cubitus interruptus (Ci) In response to Hh activation, the full length transcription factor, Ci-155 is activated and translocates into the nucleus to drive Hh-dependent target genes such as engrailed (eng) (Aza-Blanc et al., 1997) However, in the absence of Hh, Ci-155 becomes phosphorylated and proteolysed into a shorter fragment, Ci-75 Ci-75 acts as a repressor, repressing the transcription of Hh target genes (Dominguez et al., 1996; Ingham and McMahon, 2001) The processing and nuclear translocation of Ci is regulated by the Hedgehog signaling complex (HSC) in the cytoplasm The HSC is made up of a serine threonine kinsase Fused (Fu), a kinesin protein Costal2 (Cos2), the PEST domain containing protein Suppressor of Fused (Su(fu)) (Robbins et al., 1997; Sisson et al., 1997) and Ci HSC remains bound to microtubules in the absence of Hh but dissociates when Hh signaling is activated In the absence of Hh, Su(Fu) is thought to prevent the nuclear translocation of Ci, thereby dampening the transcription of Hh target genes (Monnier et al., 1998) Thus, the HSC is a central determinant of Hh signaling activity Fig1.1 The Hedgehog pathway in Drosophila and vertebrates The Hedgehog (Hh) pathway in Drosophila (A,B) and in vertebrates (C,D) in the absence (A,C) or presence (B,D) of the Hh ligand (A) In the absence of Hh, Ptc prevents the cell-surface localization of Smo, and Ci forms a complex with Cos2, Fu and Sufu, which targets Ci for proteolytic processing into the repressor form (CiR) (B) In the presence of high levels of Hh ligand, Ptc inhibition is relieved; Smo accumulates at the plasma membrane and forms a complex with Cos2 and Fu through its C-terminal tail; Ci is activated (C) In the absence of Hh, Ptc1 prevents the accumulation of Smo in cilia, possibly through the action of a small molecule Gli3 is processed into a repressor form (Gli3R) in a cilia-dependent manner The activation of all Gli proteins is inhibited by Sufu, Iguana (for zebrafish) and probably Cos2 (D) In the presence of high levels of Hh ligand, Ptc1 inhibition is relieved; Smo is targeted to cilia and activates Gli proteins in a cilia-dependent manner Gli3 processing is also inhibited p, phosphorylation; PKA, protein kinase A Taken from: (Huangfu and Anderson, 2006) 1.3 Vertebrate Hedgehog Signaling Pathway 1.3.1 Genetics in Vertebrate Models Mus musculus (mouse) and Danio rerio (zebrafish) are two models which have been widely used to study the genetics of vertebrate homologs of Drosophila Hh signaling The use of gene targeting in mouse (Lanske et al., 1996) and forward genetic approaches in zebrafish (Haffter et al., 1996) have helped to identify a number of conserved as well as novel components In addition, reverse genetics using antisense morpholino oligonucleotide (MO) knockdown (Draper et al., 2001) in zebrafish also provided further insights into the Hh pathway Morpholinos bind to either the translational start site or splicing junction of the pre-mRNA of interest and prevent its translation or proper splicing The mis-spliced mRNAs created by the splice MO are presumably degraded through the non-sense mediated decay pathway before translation can occur or proteins translated as a result of the start MO are normally truncated which renders them nonfunctional This technique creates an efficient knockdown of the target gene In situations where mutants are available, morpholino-injected embryos can phenocopy the mutants and be used for loss-of-function studies One obvious difference in the components of Hh signaling between vertebrates and Drosophila is the number of paralogs found in vertebrates In vertebrates, three Hh proteins (Bitgood et al., 1996; Chiang et al., 1996; St-Jacques et al., 1999), two Ptc paralogs (Ptc1 and Ptc2) (Goodrich et al., 1997) and three Ci paralogs (Gli1, Gli2 and Gli3) (Bai and Joyner, 2001; Hui and Joyner, 1993) have been identified 1.3.2 The Signaling Cascade The scaffold of the Hh pathway appears to be conserved from Drosophila to vertebrates (Fig 1.1) Vertebrate Ptc1 functions as the receptor for Hh ligand and as a negative regulator of the Hh pathway (Marigo et al., 1996) When Hh binds to Ptc1, Smo will in turn transduce Hh signal into the cytoplasm to activate the Gli transcription factors and drive Hh-dependent target genes (Stone et al., 1996) Unlike the dual role of Drosophila Ci, the function of the three Gli proteins have distinct transcriptional activating and repressing roles Gli1 is thought to be the main activator for the pathway (Aza-Blanc et al., 2000; Bai and Joyner, 2001) while Gli3 is the transcriptional repressor (Wang et al., 2000a) Gli2 possesses both activating and repressing roles The levels of activators and repressors ultimately determine the differentiation of Hh-dependent cell types 1.3.3 Role of Sonic Hedgehog Amongst the Hh ligands, Shh is the most well-studied protein because of its widespread effects on animal development Shh plays an important role because it is widely expressed and has great effects in both embryonic and adult animal development The expression of Shh in the notochord, floor plate and zone of polarizing activity (ZPA) in the posterior of limb mesenchyme makes it an ideal candidate for studying patterning of the neural tube and limb (Chang et al., 1994; Riddle et al., 1993) As such, genetic diseases such as holoproensephaly, neural tube and limb patterning defects are linked to mutation in the shh gene that leads to aberrant Hh signaling (Pepicelli et al., 1998) Over expression of Hh can induce ectopic Hh signaling in cells which normally not respond to Hh (Concordet et al., 1996; Wolff et al., 2004) Hh signaling occurs via a negative feedback loop which helps to restrict the spread of Hh signal, limiting its range of effect (Jeong and McMahon, 2005) Activation of Hh signaling will turn on Hhdependent target genes, including the gene for its receptor ptc1 Ptc1 in turn internalizes Hh into the lysosomes for degradation which results in down regulation of the pathway In the vertebrate Hh pathway, a transmembrane protein Hh-interacting protein (HIP) was identified (Chuang and McMahon, 1999) Similar to Ptc1, it functions via a negative regulatory feedback loop to attenuate the Hh pathway by binding to the Hh proteins, thus restricting the spread of Hh ligand 1.3.4 Regulation of Smoothened Activity Naturally occurring compounds like the alkaloid, Cyclopamine, found in the plant Veratrum californicum was identified to inhibit the activity of Smo by binding to the protein (Chen et al., 2002a; Taipale et al., 2000) Its properties were first discovered in the 1950s during an outbreak of cyclopia in sheep in United States The phenotype of their offspring was similar to that of mouse embryos when Hh signaling is inhibited Based on this similarity, it was discovered that the compound cyclopamine can antagonize Hh signaling Consequently, it is now used routinely to inhibit Hh signaling in research The structural similarity between cyclopamine and sterols led to further findings that endogenous sterols like cholesterol and vitamin D3 derivatives can modulate Smo activity (Bijlsma et al., 2006) All the experiments were done using mammalian Smo and it was also shown that Drosophila Smo is not responsive towards these sterols These observations seems to suggest that there may be a difference in the regulation of Smo activity between vertebrate and Drosophila Smo by small molecules (Taipale et al., 2000) 1.4 Activation of Hedgehog protein Hh proteins need to undergo post-translational modification before being activated These steps are essential for the release of the ligand from producing cells to the receiving cells in order to activate the pathway (Buglino and Resh, 2008; Burke et al., 1999; Chamoun et al., 2001) This modification is a conserved process across species The Hh molecule can catalyze its own cleavage, forming a N-terminal Hh signaling domain of approximately 19 kDa that has an ester linked cholesterol at its C-terminal The functional N-terminal domain with its cholesterol modification is able to bind to the plasma membrane of receiving cells Subsequently, a palmitic acid moiety is then added to the N terminal of Hh to form a fully active Hh signaling molecule On the other hand, the cleaved Cterminal product contains an axon-targeting signal that targets it to the growth cones in retina cells, thought to be important for the development of the eye (Chu et al., 2006) 1.5 Comparing Hedgehog Orthologs 1.5.1 Patched The vertebrate homologs of Ptc, Ptc1 and Ptc2 appear to display redundancy in terms of their functions (Goodrich et al., 1997) Loss of Ptc1 in mice mimics the ectopic activation of the Hh pathway resulting in embryonic lethality However, the loss of Ptc2 in mice (Nieuwenhuis et al., 2006) and zebrafish (Koudijs et al., 2005) does not result in lethality and mutant embryos not exhibit any phenotype similar to ectopic Hh activation This is probably due to compensation by Ptc1 which strongly suggests that Ptc1 is the ortholog of Drosophila Ptc How Ptc inhibits the activity of Smo remains unknown although sterols may regulate the inhibitory activity of Ptc1 on Smo (Bijlsma et al., 2006; Chen et al., 2002b; Frank-Kamenetsky et al., 2002; Stanton and Peng, 2010) Ptc has been shown to play a role in limiting or increasing the concentration of these small molecules (Callejo et al., 2008) 1.5.2 Smoothened The likelihood that Smo functions similarly in both invertebrates and vertebrates is possible (van den Heuvel and Ingham, 1996) given that there is only a single copy of the gene across the species (Akiyama et al., 1997; Zhang et al., 2001) However, structural analysis revealed disparity which may result in different working mechanisms between flies and mammals (Varjosalo et al., 2006) In the absence of Hh, Drosophila Smo (dSmo) remains unphosphorylated and this targets the protein for endocytosis and degradation by lysosomes When Hh binds to its receptor Ptc, dSmo gets hyperphosphorylated at its C-terminal tail and creates a conformational change which leads to the activation of the pathway although the exact mechanism in the signal transduction remains unknown (Denef et al., 2000; 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for 5mins before use) Hybe B 10 Blocking Reagent 11 in situ Staining buffer 12 Growth Medium 13 Trypsin 14 Freezing Medium 25ml Formamide, 12.5ml 20X SSC, 0.5ml Triton X-100 Adjust pH to 5.5 with HCl 1.25g Blocking reagent (Roche), 50ml PBT Adjust pH to 7.5 with NaOH 4ml of 1M Tris pH9.5, 2ml of 1M MgCl2, 1ml of 4M NaCl, 400µl of 10% Triton X-100 Top up to 40ml with water 10% FBS and 1% PenicillinStreptomycin (10000U-10mg/ml) added to 500ml of DMEM 0.125% Trypsin/Versene FBS and 10% DMSO Usage Zebrafish fixative Antibody insitu hybridization Mammalian cell culture 131 15 PBST 16 10X Running buffer 17 Commassie Blue staining solution 18 Destaining solution 19 IP Buffer 20 Low salt IP Buffer 0.1% (v/v) Tween20 in PBS 250mM TrisHCl, 2M Glycine and 1% SDS diluted in PBS 50% methanol, 10% acetic acid and 0.05% Coomassie brilliant blue R-250 (Bio-rad) diluted water Filter to obtain homogenous solution 10% methanol and 5% acetic acid diluted in water 1% Triton X-100, 0.5% NP-40, 150mM NaCl, 10mM Tris pH7.4, 1mM EDTA pH8.0, 1mM EGTA pH8.0, 0.2mM sodium orthovanadate and 0.2mM PMSF 21 Low melting agarose (LMA) same components as IP Buffer without detergent and only 15mM NaCl 1.5% agar and % sucrose diluted in water 22 30% Surcose SDS-PAGE, Protein analysis Co-IP 30% Surcose diluted in PBS Cryo-sectioning 132 ... kinesin-like protein of 1363 amino acids was assembled Like Drosophila Cos2 and other members of the kinesin heavy chain (KHC) superfamily of proteins, the N-terminal and C-terminal regions of. .. about the mechanism of the pathway The aim of this study focuses on two proteins, Cos2 /Kif7 and Iguana /Dzip1 Drosophila Cos2 plays an important role in Hh signaling, regulating the activity of the. .. the vertebrate homolog of the Cos2 was not identified The aim of the first part of this thesis was to identify a cos2 gene in the zebrafish genome and elucidate its function in Hh signaling The