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First published online 10 January 2007
doi: 10.1242/dev.02756

Development 134, 735-746 (2007)
Published by The Company of Biologists 2007
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The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum

Molly K. Nyholm1, Shan-Fu Wu2, Richard I. Dorsky2 and Yevgenya Grinblat1,*

1 Departments of Zoology and Anatomy, University of Wisconsin, Madison, WI, 53706, USA.
2 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA.


Figure 1
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  		Fig. 1. Zic2a and zic5 are linked and co-expressed in presumptive dorsal brain. (A) Genomic arrangement of zic2a and zic5. Coding regions are shown as gray boxes, non-coding transcribed regions as white boxes, and introns as lines. (B-G) Embryos stained by ISH for zic2a or zic5 (purple), and dlx3 (orange). (B) At midgastrula stages, zic2a is expressed throughout neurectoderm, whereas dlx3 marks the adjacent non-neural ectoderm. (C) By late gastrula stages, zic2a is restricted to the lateral neural plate and forms a sharp border with dlx3. (D,E) During somitogenesis, zic2a and zic5 are similarly expressed in the dorsal neural tube, ventral diencephalon and optic stalks. (F,G) At 24 hpf, zic2a and zic5 are coexpressed in the optic stalk, telencephalon and dorsal diencephalon, and weakly in the tectum. All views are lateral, anterior to the left, except in B,C where anterior (a) is at the top. Arrowheads mark anterior and posterior borders of presumptive midbrain. d, dorsal; m, midbrain; h, hindbrain; os, optic stalk; tel, telencephalon.

 

Figure 2
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  		Fig. 2. Transient transgenic analysis of zic2a-zic5 genomic regions. (A) Conserved DNA regions (thick lines) at the zic2a-zic5 locus. P2 and P5, each approximately 300 nt, are 70% identical with Fugu. IG (840 nt) is 49% identical to Fugu, and D5 (535 nt) is 60% identical to Fugu. Eight consensus Tcf/Lef-binding sites were found in IG and six in D5 (asterisks). (B) Transgenic constructs tested in transient assays. (C,D) Dorsal midbrain and hindbrain expression of Gfp in representative zic5D5:gfp and zic2aD5:gfp transient transgenics. Insets are dorsal views of the embryos shown. Asterisks mark mid-hindbrain organizer. (E-G) gfp RNA in representative transient transgenics. In embryos injected with 25 pg of zic2aD5:gfp (E) or zic2aD5{Delta}IG:gfp (F), expression was concentrated in dorsal midbrain and hindbrain. Embryos injected with 25, 50 or 70pg of zic2a:gfp (G) did not show enhanced expression. All views are lateral, except for insets.

 

Figure 3
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  		Fig. 3. Stable zic2aD5:gfp expression recapitulates endogenous midbrain and posterior hindbrain expression. Gfp expression in Tg(zic2aD5:gfp) embryos detected by ISH (A,D,G,H), or by fluorescence (B,E). (C,F,I) Endogenous zic2a expression detected by ISH. (A-C) gfp RNA at the 11- to 12-somite stage (A) and Gfp protein at 14-somites (B) are restricted to dorsal midbrain and posterior hindbrain. Endogenous zic2a is transcribed in the dorsal diencephalon, midbrain and hindbrain at 11-somites (C). (F-H) Transgenic (D,E) and endogenous (F) expression overlap in the midbrain and posterior hindbrain at the 20-somite stage. At 24 hpf, transgenic expression (G,H) overlaps endogenous expression (I) in the dorsal midbrain. All views are lateral, anterior to the left, except H,I which are dorsal views. Arrowheads mark anterior and posterior limits of tectum. m, midbrain; h, hindbrain.

 

Figure 4
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  		Fig. 4. Tcf/Lefs are required for zic gene expression. (A) The method used to disrupt Tcf/Lef signaling at controlled times. (B) Temporal correlation between Gfp{Delta}Tcf induction (by fluorescence) and zic2a RNA (by ISH). (C-L) Representative embryos after ISH. Stage of heat-shock and recovery time are indicated at upper right. Zic2a expression is normal in {Delta}Tcf-negative heat-shocked controls (C,E), and reduced or absent in {Delta}Tcf-positive siblings (D,F). Zic5 expression was normal in {Delta}Tcf-negative (G) embryos, and absent in {Delta}Tcf-positive embryos (H). Zic2aD5:gfp expression was normal in {Delta}Tcf-negative embryos (I) and absent in {Delta}Tcf-positive embryos (J). Wnt3a expression was normal in heat-shocked controls (K) and in {Delta}Tcf-positive siblings (L). All views are lateral, anterior to the left. Arrowheads mark anterior and posterior tectal borders. (M,N) Gfp ChIP analysis of heat-shocked Tg(hs:gfp{Delta}tcf) and Tg(hs:gfp) embryos. Genotype of embryos and PCR templates are shown above, as follows: GFP Ab, chromatin after anti-Gfp IP; input, total chromatin before IP; no Ab, no-antibody ChIP; mock, no chromatin. Regions amplified are labeled next to the corresponding bands. (M) Promoters of known Wnt targets, ngn1 and nacre. An upstream region of ngn1, lacking functional Tcf/Lef-binding sites (lane 5). (N) IG and D5 regions of zic2a-zic5 and zic5 intron.

 

Figure 5
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  		Fig. 5. Ectopic activation of the canonical Wnt pathway induces zic gene transcription. (A-C) Embryos were injected with hs:ßCat-gfp DNA, heat shocked at shield-stage, and assayed for zic2a (purple) and gfp (orange) at late-gastrula. (A) ßCat-gfp is expressed in the medial neural plate; the zic2a pattern is normal (no ectopic expression; n=10, two experiments). (B,C) ßCat-gfp is expressed in lateral ectoderm; zic2a is ectopically expressed (arrows; n=8, 8 ectopic, two experiments). (D) Control embryo expressing hs:gfp in lateral ectoderm showing no ectopic zic2a expression (n=18, 0 ectopic, one experiment). a, anterior. (E-G) Zic2aD5:gfp embryos injected with GR-Lef RNA and assayed for gfp RNA at late-gastrula (purple). (E) GR-Lef-injected embryo showing weak ectopic gfp (arrow). (F,G) GR-Lef-injected embryos treated with DEX (F) or DEX and CHX (G). (H) Uninjected zic2aD5:gfp control treated with DEX. (I) Summary of GR-Lef overexpression experiments, showing the proportion of embryos expressing gfp for each treatment condition. The number of embryos scored for each condition is listed along the x-axis.

 

Figure 6
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  		Fig. 6. Multiple Tcf/Lefs are required for zic gene expression. Embryos injected with the MOs shown at the lower left of each panel and stained for zic2aD5:gfp (A,C,E,G,I) or zic2a (B,D,F,H,J) at 18 hpf. (C-D) Strongly affected tcf3 morphants had anterior trunctions and reduced zic2aD5:gfp (asterisks) and zic2a domains. (E-J) lef1 morphants showed a strong reduction in zic2aD5:gfp (E) and a mild reduction in zic2a (F). tcf7 morphants showed a mild reduction in zic2aD5:gfp (G), but normal zic2a (H). lef1+tcf7 morphants showed a strong reduction in zic2aD5:gfp (I) and mild reduction in zic2a (J). All views are lateral, anterior to the left. Arrowheads indicate the midbrain.

 

Figure 7
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  		Fig. 7. MO knockdown of zic2a and zic5 disrupts dorsal midbrain formation. (A) MO-binding sites in the zic2a-zic5 locus. Coding regions are shown as gray boxes, non-coding transcribed regions as white boxes, and introns as lines. MO-binding sites are designated with red lines. (B-J) Embryos injected with indicated MOs were assayed for expression of wnt1 or wnt3a at 24-25 hpf. Error bars in B show s.e.m. (C,D) Embryo injected with conMO (8 ng) showing normal wnt1 expression. (E,F) Severely affected embryo injected with 2MOC (2 ng) showing disorganized wnt1 expression in dorsal midbrain and hindbrain. (G,H) Mildly affected embryo injected with 2MOA+2MOB (6.6 ng). (I,J) Embryos co-injected with 2MOC and 5MOC (1 ng each) showing severe defects in wnt1 expression. C,E,G,I are lateral views; D,F,H,J are dorsal views of the embryos shown to the left; embryos are positioned with anterior to the left. Arrowheads indicate the midbrain.

 

Figure 8
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  		Fig. 8. Knockdown of zic2a and zic5 disrupts midbrain morphology, but not DV pattern. Embryos were injected with conMO (8 ng), or 2MOC and 5MOC combined (1 ng each), and stained for DV marker expression by ISH. (A-D) Marker expression in 19-somite embryos. wnt3a marks dorsal midline and adjacent cells in midbrains of conMO-injected embryos (A) and in zic morphants (B). pax7 marks alar midbrain in controls (C) and morphants (D). (E-T) Marker expression in 24-hpf embryos. wnt3a is expressed in dorsal tectum of conMO-injected embryos (E-G). (H-J) wnt3a domain in zic morphants. (K-M) pax7 in the alar tectum of controls. (N-P) pax7 in zic morphants is reduced, but correctly positioned. (Q,R) hlx1 marks basal plate midbrain of controls (Q) and zic morphants (R). (S,T) shh in ventral midbrain of controls and zic morphants. A-D,E,H,K,N,Q-T are lateral views, anterior to the left; F,I,L,O are dorsal views of embryos at left; G,J,M,P are midbrain cross-sections at positions indicated by lines in images to left. Arrowheads indicate the midbrain.

 

Figure 9
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  		Fig. 9. Cell proliferation is reduced in the dorsal midbrain of zic morphants. (A) A comparison of average mitotic index in control (blue) and 2MOC+5MOC (pink) morphant midbrains. Mitotic index (y-axis) was calculated at nine DV positions (x-axis) at the 19-somite stage. Error bars show s.e.m. Asterisks mark statistically significant reductions in mitotic index. (B-D) PH3 (green) staining in representative confocal z-sections through midbrain dorsal position 3 in control (B) and zic MO-injected (C,D) embryos. (E-G) A merge of PH3 and nuclear ToPro3 (red) staining in embryos shown above. (H-M) Confocal z-stacks of 19-somite conMO (H-J) and 2MOC+5MOC-injected (K-M) embryos immunostained for Pax7 (red) and PH3 (green). H,K are stacks of dorsal-most Pax7 domain; I,L are stacks of ventral Pax7 domain; J,M are stacks of ventral midbrain.

 

Figure 10
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  		Fig. 10. A proposed role for zic genes in the developing midbrain. Expression of zic2a and zic5 is activated through canonical Wnt signaling in the dorsal midbrain. Activation of zic tectal expression is mediated through Tcf/Lefs, including Lef1 and Tcf7 acting through an enhancer in the downstream zic5 (D5) region. Zics subsequently control proliferation in the alar plate.

 

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© The Company of Biologists Ltd 2007