Characterization of gli2 2b genes in zebrafish hindbrain development 3

Results Fig. 3-14 gli2b MO blocks zebrafish gli2b mRNA splicing. genomic sequence used for designing splicing (A) Partial gli2b morpholino. Sequence complementary to morpholino is underlined in red. Exons (gli2b cDNA:1125-1314, 1315-1425, 1426-1560 nt) are in cyan and introns are in black. Arrows indicate primers showed in B. (B) Diagram shows the strategy of using splice MO (Red bar) for blocking the normal splice site and organization of predicted mRNA. The blue and green arrows represent the primers for PCR used to test whether the splice MO affects formation of gli2b mRNA. The sequences for primers F1, F2, R2 are indicated in A; R1 is indicated in Fig. 3-2. (C) RT-PCR results. Control RNA (lane C) was extracted from gli2bMMS morphants; morphant RNA (lane M) was extracted from gli2bSPL. Primers used are indicated in B. EF1α primers were used for internal control of mRNA quality. 118 Results 3.4.2 Classification of phenotypes of gli2b morphants Both the gli2bATG and gli2bSPL morphants showed similar morphological changes. Compared with wild type or control MO injected embryos, gli2b morphants demonstrated dosage-dependent phenotypes (Table 3-1). The mild phenotype (Type I) of gli2b morphants consisted of the enlarged hindbrain ventricle and smaller brain and eyes by 48 hpf (Fig. 3-15C, D). The severe phenotype (Type II) of gli2b morphants demonstrated also abnormality of the trunk and in ~50% cases conversed otoliths at 48 hpf (Fig. 3-15E, F). Interestingly, after injection of a high dose (>2 pmol) of gli2b MO, a defect of the convergence-extension movement could be observed. By the end of gastrulation these morphants showed a phenotype similar to that of mutants with affected BMP signaling (Fig. 3-15 H, I), indicating that the early function of Gli2b might be related to dorso-ventral (D-V) patterning. This early function may be due to maternal expression of this gene and not Hh related. Therefore, to circumvent the early defect and investigate the function of gli2b in neural development, we typically injected embryos with the almost 10 times lower dose of morpholino (0.3 pmol). 119 Results Table 3-2 Phenotypes obtained after injection of gli2b morpholino oligonucleotides in zebrafish embryos. Mortality Looks Mild Strong gli2bAtg MO Injected injected(pmol) number >24hpf(Num/percentage) normal phenotype phenotype 0.15 100 12(12%) 28(28%) 54(54%) 6(6%) 0.3 100 15(15%) 13(13%) 49(49%) 23(23%) 0.6 50 7(14%) 5(10%) 17(34%) 21(42%) 100 35(35%) - 11(11%) 54(54%) gli2bMMA 50 3(6%) 45(94%) 2(4%) - gli2bSpl MO Injected Mortality Looks Mild Strong injected(pmol) number >24hpf(Num/percentage) normal phenotype phenotype 0.15 100 8(8%) 46(46%) 43(43%) 3(3%) 0.3 100 7(7%) 18(18%) 61(61%) 14(14%) 0.6 100 13(13%) 7(7%) 52(52%) 28(28%) 100 18(18%) - 33(33%) 49(49%) gli2bMMS 50 2(4%) 48(96%) - - 120 Results A B C D E F G H Fig. 3-15 Classification of Gli2b morphants. I (A-F) Gli2b morphant with mild phenotype (Type I) and severe phenotype (Type II) at 30 hpf and 48 hpf. The mild phenotype (C, D) of Gli2b morphant is characterized by an enlarged hindbrain ventricle but relatively normal trunk. The severe phenotype (E, F) of Gli2b morphant shows not only enlarged hindbrain ventricle, but also curled trunk. (H, I) gli2b morphants in high doses (dose I, pmol; dose II, 1.5 pmol) showed convergence-extension defects (arrows) in different levels compared with control (G). 121 Results 3.4.3. Comparison of the phenotypes of gli2 and gli2b morphants Both the gli2b and gli2 morphants by 48 hpf were characterized by the similarly enlarged 4th ventricle and smaller brain and eyes (Fig.3-16B, C). Further analysis of the Gli2b morphant under the differential interference contrast (DIC) microscope showed the intact horizontal myoseptum in contrast to Gli2 morphants, which lacked this structure (Fig.3-16E,F; 45/52 embryos). Even when injected with the much higher doses of MO (1 - pmol), the gli2b morphants acquired the curl-down tail phenotype with much more severe abnormality of the brain, but preserved the horizontal myoseptum. gli2b is not expressed in the adaxial slow muscle cells, thus in muscle development gli2b probably plays a minor role if any. In contrast, both gli2 and gli2b show similar late expression in the hindbrain and its deficiency in morphants is in line with expression of these genes in this part of brain. ctrl/brain A gli2b MO/brain B ctrl/trunk D gli2 MO/brain C gli2b MO/trunk E gli2 MO/trunk F Fig. 3-16 Comparative analysis of Gli2b and Gli2 morphants. Both Gli2b and Gli2 morphants show similar abnormality in the hindbrain but the trunk is affected differently. Gli2b (B) and Gli2 morphants (C) have enlarged fourth ventricle (arrowhead). In the trunk of Gli2b morphant (E) the horizontal myoseptum (arrow) is normal (D), while in Gli2 morphant the horizontal myoseptum is abnormal and somites U-shaped (F). 122 Results 3.4.4 Gli2b morphant showed cell proliferation defects in the hindbrain In the 30 hpf hindbrain, the radial astrocytes (RA) stained with zrf-1 antibody appeared as seven double segmental clusters that form curtains along rhombomere boundaries (Trevarrow et al, 1990) (Fig. 3-17A) (for discussion of characteristics of cell types, see Morest and Silver, 2003). These clusters were reduced in Gli2b morphants (Fig. 3-17B; 10/10 embryos). Further increase of gli2b MO dose led to decrease of zrf-1 staining at this stage, but a gross morphology of morphants remains relatively normal (not shown). Perhaps, elimination of RAs could be due to apoptosis in the hindbrain triggered by the knockdown of Gli2b . However, TUNEL staining of Gli2b morphants failed to detect increase of apoptosis (data not shown). Another possibility is that cell proliferation slowed down. Consistent with this explanation, the number of mitotic cells detected by the anti-phospho-histone H3 (P-H3) antibody was reduced in the hindbrain both dorsally in the ventricular zone (VZ) and ventrally in association with RGCs (Fig. 3-17C, D). Our analysis of the number and distribution of proliferating cells in the consecutive sections of the hindbrain of Gli2b morphants and controls (n=3, sections/embryo=15) demonstrated moderate reduction of cell proliferation in the ventricular zone and more significant reduction in the ventral hindbrain (Fig. 3-17E). Since the RAs are the neural progenitors, these results indicated that Gli2b could be necessary in the progenitor cells, including the neural progenitors in the hindbrain. 123 Results Ventricle RA Fig. 3-17 gli2b and cell proliferation. (A, B) Whole mount immunostaining of radial astrocytes in 30 hpf control (A) and Gli2b morphant (B) by zrf-1 antibody followed by POD staining. (C, D) Cross sections of 30 hpf control (C) and Gli2b morphant (D) at the hindbrain level stained by anti-P-H3 (purple) and zrf-1 (green) antibody. (E) Chart shows the average number of proliferating cells in the ventricular zone of 4th ventricle and the ventral region associated with processes of RA cells in continuous sections of hindbrain of Gli2b morphant and wild type embryos. 124 Results 3.4.5 zath3 expression was affected in Gli2/Gli2b morphants Since expression of gli2b takes place in the CNS (Ke et al, 2005) and Gli2b knockdown caused abnormality of cell proliferation and differentiation, we decided to check markers of early neurodifferentiation. zath3 (neurod4) is involved in early neurogenesis downstream of ngn1 (Bae et al, 2003; Park et al, 2003; Wang et al, 2003). zath3 detects two main groups of cells. First, the looped clusters in the hindbrain consist of cell groups extended along the D-V axis; these are associated with curtains of RAs on both sides of rhombomeric boundaries (DV clusters) (Fig. 3-18A). Second, the ventral (V) clusters in a central part of rhombomeres 2, 3, and (Fig. 3-18C). In the single Gli2 and Gli2b morphants, the dorsal extent of zath3 expression in the DV clusters is shifted more ventrally (Fig. 3-18B, E), while the V clusters diminished (Fig. 3-18D, E; 10/10 embryos each). In the double Gli2/Gli2b morphants, zath3 expression in the DV clusters is strongly reduced; however, some zath3 transcripts were still detected ventrally in rhombomeres and (Fig. 3-18F; 10/10 embryos). Thus it seems that in both positions Gli2b acts in parallel with Gli2, but at different D-V levels zath3-positive cells demonstrate different dependence on Gli2 proteins. To get better understanding of this phenomenon, we checked expression of zath3 in two mutants affecting Hh pathway. In yot-/-, where in result of mutation the dominant repressor form of Gli2 (Gli2DR) appeared (Karlstrom et al, 1999), the expression of zath3 in the DV clusters was similar to that in control. In contrast, the V clusters in rhombomeres and disappeared and the ectopic domain of zath3 expression in rhombomere appeared (Fig. 3-18G). In smu-/- mutant deficient in the receptor of Hh signaling, Smoothened (Chen et al, 2001; Varga et al, 2001), the zath3-positive DV clusters were intact while all V clusters disappeared (Fig. 3-18I). 125 Results Similar result was obtained after Gli2b knockdown of Gli2b on a yot-/- background (Fig. 3-18H; 10/10 embryos). Hence, it seems that Hh-dependent Gli2 activating role is required for expression of zath3 only in the ventral hindbrain probably containing more differentiated neurons. Here some other factors may also be involved in addition to the two Gli2s. 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Ichthyol. 1: 31-34. 202 [...]... role of gli2/ gli2b in development of the zebrafish brain, especially the hindbrain Our results indicated that Gli2b plays a more important role in regulating Hh signaling in zebrafish hindbrain than Gli2 Also, our results indicated that Gli2 dominant repressor was not sufficient to interfere with all possible functions of Gli2b in yot-/-; in particular, Gli2b might have an essential activator role in. .. view of netrin1a expression in the hindbrain of 30 hpf controls and Gli2b morphants (B, D) In Gli2b morphants netrin1a expression in the ventral hindbrain was up-regulated and expanded laterally (E) In yot-/-, netrin1a expression was less affected in r4 However, its expression in the dorsal hindbrain was less affected After injection of Gli2b MO into yot-/-, a faint netrin1a expression remained only in. .. decrease in early gliogenesis comparing to that of neurogenesis Thus, Gli2b in the hindbrain may act preferentially in regulating early differentiation of non-neuronal cell lineages 132 Results Fig 3- 20 Expression of early neurodifferentiation markers in the hindbrain of Gli2b morphant at 30 hpf (A, B) Expression of notch1a in Gli2b morphant (A) and control (B) (C, D) Expression of notch1b in Gli2b morphant... mutant resulted in a severe reduction of netrin1a transcripts in the hindbrain (Fig 3- 25F; 10/10 embryos) These results indicated that, first, the netrin1a patterning is regulated by the Hh signaling involving Gli2b, and, second, knockdown of Gli2b in yot-/- blocks multiple events of the Hh-dependent gliogenesis in the hindbrain 146 Results Fig 3- 25 Gli2/ Gli2b regulate expression of netrin1a (A) Lateral... Expression of ngn1 in Gli2b morphant (E) and control (F) (G, H) Expression of gfap in Gli2b morphant (G) and control (H) The relative positions of expression domains are indicated in left side of the panel: midline, blue bar; intermediate medio-lateral cluster, green bar; lateral loops; red bar 133 Results 3. 4.8 Inhibition of gli2b did not affect the segmentation of hindbrain Since loss -of- function of Gli2b...Results Fig 3- 18 Gli2/ Gli2b regulate expression of zath3 zath3 expression in hindbrain in 30 hpf in controls (A,C,E,G,I) or Gli2b MO injected embryos (B, D, F, H,J) White broken lines indicate the r4 (A,C) control; (B,D) Gli2b morphant; (E) Gli2 morphant; (F) Gli2/ Gli2b morphant; (G) yot-/- mutant; (H) yot-/mutant/Gli2b morphant; (I) smu-/- mutant; (J) smu-/- mutant/Gli2b morphant Black lines indicate... expression In Gli2b morphant, nkx2.2 expression only slightly expanded (Fig .3- 22A, B; 5/5 embryos) In Gli2 morphant, nkx2.2 expression was similar to that in Gli2b morphant (data now shown) These results indicated a minor role of both gli2 and gli2b in regulating expression of nkx2.2 in the ventral hindbrain Due to duplication of gli2 in zebrafish, the function of Gli2b might be redundant with Gli2 Thus,... hindbrain the residual activity of some other Gli could be responsible for maintaining nkx2.2 expression in r4 It was proposed that r4 plays an important role of the signaling center within a hindbrain (Maves et al, 2002) Hence it could be of interest to study a role of Gli2b in 137 Results this rhombomere The expression of nkx2.2 was completely lost in the hindbrain after injection of Gli2b MO into... Control embryos of ET3 line and ET3 Gli2b morphants (H) 4 dpf ET3 controls, hindbrain (F) and posterior trunk (G) GFP expression in the ET3 Gli2b morphant, hindbrain (I) and posterior trunk (J) 142 Results 3. 5 .3 Gli2b and neuronal differentiation The early changes in organization of neural precursors prompted us to explore a role of Gli2b in neurodevelopment Islet1 is an early marker of neurodifferentiation... (Fig 3- 24F) In contrast, their number was reduced in Gli2b morphants (Fig 3- 24D; 6/10 embryos) and even more so after injection of Gli2b MO into yot-/- mutant (Fig 3- 24H; 8/10 embryos) demonstrating, first, a redundant role of Gli2b in this cell lineage and, second, incomplete suppression of Gli2b function in the dorsal neural tube of yot-/- mutants 144 Results Fig 3- 24 Gli2/ Gli2b and differentiation of . These results indicated a minor role of both gli2 and gli2b in regulating expression of nkx2.2 in the ventral hindbrain. Due to duplication of gli2 in zebrafish, the function of Gli2b might be. gli2b show similar late expression in the hindbrain and its deficiency in morphants is in line with expression of these genes in this part of brain. ctrl/brain gli2b MO/brain gli2. processes of RA cells in continuous sections of hindbrain of Gli2b morphant and wild type embryos. Ventricle RA Results 125 3. 4.5 zath3 expression was affected in Gli2/ Gli2b morphants Since