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First published online 24 January 2007
doi: 10.1242/dev.02780

Development 134, 845-855 (2007)
Published by The Company of Biologists 2007
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Expanded expression of Sonic Hedgehog in Astyanax cavefish: multiple consequences on forebrain development and evolution

Arnaud Menuet1,*, Alessandro Alunni2,*, Jean-Stéphane Joly2, William R. Jeffery3 and Sylvie Rétaux1,{dagger}

1 CNRS-UPR2197 DEPSN, Institut Fessard, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France.
2 MSNC INRA Group, Institut Fessard, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France.
3 Department of Biology, University of Maryland, MD 20742, USA.


Figure 1
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  		Fig. 1. Enlarged Shh expression is maintained during cavefish development. Brain distribution of Shh mRNA in surface fish (left) and cavefish (right) embryos at indicated stages. Transverse sections through the hypothalamus (E,F), or in toto ventral (A-D) and lateral views (G,H) show expansion of Shh signal in cavefish at all stages. In all panels and following figures, anterior is right and dorsal is up. In G,H, the eyes were removed to facilitate observation. In this and subsequent figures, note that pigment cells (asterisks) are only present in surface fish embryos, most of them corresponding to the retina pigmented epithelium. Scale bar: 50 µm. ddi, dorsal diencephalon; fp, floor plate; hyp, hypothalamus; po, preoptic region; SP, subpallium; tel, telencephalon; zli, zona limitans.

 

Figure 2
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  		Fig. 2. Differential modulation of Nkx and Dlx2 expression domains in cavefish embryos. Dlx2 and Nkx gene expression in surface fish (left) and cavefish embryos (right) at indicated stages. (A-D) Dlx2 and Nkx2.2 on in toto lateral views. (E-H) Nkx2.1b in toto (E,F) and on horizontal sections (G,H, orientation indicated in E). The arrow in F and arrowheads in G,H point to larger expression in cavefish SP. (I,J) Nkx2.1a distribution in toto and in transverse sections through the hypothalamus (insets in I and J). Scale bar: 50 µm. hyp, hypothalamus; p3, prosomere 3 (ventral thalamus); po, preoptic region; pal, pallium; SP, subpallium; tel, telencephalon.

 

Figure 3
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  		Fig. 3. Increased proliferation in the preoptic region and hypothalamus of cavefish embryos is Shh-dependent. (A-H) PCNA immunoreactivity in transverse sections (surface fish, left; cavefish, right) at 60 hpf (A-D) and 5 days (E-H, high magnification) through the preoptic region (A,B,E,F) and the hypothalamus (C,D,G,H). Note that proliferation in the dorsal diencephalon and mesencephalon is identical in both populations. (I,J) Cavefish embryos were treated with 20 µmol/l cyclopamine from 15 to 24 hpf, allowed to develop until 48 hpf, and proliferation was assessed with anti-phosphohistone H3 immunostaining (see Fig. S1 in the supplementary material for pictures). The results are expressed as the density of phospho-H3-positive cells (number of cells per surface area in arbitrary units). See methods for details. * indicates a significant effect (P<0.05, n=4 embryos; Student's t-test) when compared with the ethanol-treated controls. Scale bars: 50 µm. ddi, diencephalon; hyp, hypothalamus; mes, mesencephalon; po, preoptic region.

 

Figure 4
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  		Fig. 4. Phylogenetic analysis and expression domains of Lhx6 and Lhx7 in Astyanax. (A) Lhx6 and Lhx7 sequences from different species were aligned and a phylogenetic tree was constructed. (B,C) Forty-eight hpf in toto lateral views of Lhx6 (B) and Lhx7 (C) expression patterns (purple) in cavefish brains that have been double-hybridised with Shh (red). (D,E) At 36 hpf, in toto lateral (D) and ventral (E, high power) views of cavefish brains double-hybridised for Lhx6 (purple) and Lhx7 (red). Scale bars: 50 µm. fp, floor plate; hyp, hypothalamus; pit, pituitary; po, preoptic region; SP, subpallium; zli, zona limitans.

 

Figure 5
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  		Fig. 5. Lhx7 is expressed earlier in the subpalium, and as a larger domain in the preoptic region of cavefish embryos. Surface fish (left) and cavefish embryos (right) were hybridised for Lhx7 at indicated stages (inset in H, 60 hpf). In each panel, insets show transverse (A,B, orientation given by black line in A) or horizontal sections (C-H, orientation given in C) at the level of the SP or po. In A,B, arrows points to the fewer than three cells reproducibly found in the surface fish subpallium, to be compared with the already large expression in cavefish at this stage. In G,H, arrowheads point to Lhx7 cells in the lateral subpallium. pit, pituitary; po, preoptic region; SP, subpallium; Vl area ventralis lateralis. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Lhx6 expression is enlarged in cavefish, especially in columns of cells extending from the SP. (A-H) Comparison of Lhx6 pattern between surface fish (left) and cavefish embryos (right) at indicated stages on lateral (A,B) and ventral (C-F) in toto views. G and H show frontal sections through the forebrain of 72 hpf embryos, where arrows point to anteroventral located Lhx6 cells. In A and B, an inset shows a transverse section at SP level (orientation given in A). Scale bar: 50 µm.

 

Figure 7
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  		Fig. 7. Characterisation of Lhx6-positive columns of cells in the telencephalon. (A-D) Frontal sections through the telencephalon of 48 hpf surface fish (left) and cavefish embryos (right) showing Lhx6 and corresponding GAD65 expression in the putative migratory stream. Arrows point to the increased Lhx6 or GAD65 component in cavefish. (E,F) Horizontal sections showing that Lhx6 (E) and Nkx2.1 (F) patterns are indistinguishable at 60 hpf in the columns of cells. (G-I) DiI labelling of the olfactory projection onto the olfactory bulbs of a 7-day-old cavefish, in dorsal views, where anterior is up. G (bright field), H (fluorescence) and I (combined) photomicrographs indicate that the olfactory bulbs are located in the anterolateral part of Astyanax telencephalon, i.e. where the Lhx6- and GAD65-positive cells terminate their migration. (J-M) Hemi-sections through the telencephalon of surface (J,K) or cavefish (L,M) embryos, to illustrate Lhx6 pattern with regard to Pax6 and Lhx9. In L,M, dashed lines delineate the brain and the adjacent olfactory epithelium (oe), which also expresses Lhx9. (N) Schema, in which gene expression is indicated on the left, with corresponding anatomical regions on the right. dSP, dorsal part of subpallium; hyp, hypothalamus; n, nostril; ob, olfactory bulb; po, preoptic region; SP, subpallium; tel, telencephalon. Scale bars: 50 µm.

 

Figure 8
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  		Fig. 8. Forebrain neuronal patterning differences observed in cavefish are Shh-dependent. Cavefish embryos were treated between 15 and 24 hpf with 0.1% ethanol (control), 20 µmol/l or 200 µmol/l cyclopamine and compared to surface fish embryos. In toto lateral views (A-U) and ventral views (insets in V-X) of 48 hpf brains hybridised with the indicated probes. Note the dose-dependence and regionspecificity of the effects. For example, the pituitary Lhx7 or the dorsal hypothalamic Lhx6 signals are not affected by cyclopamine. Scale bar: 50 µm. hyp, hypothalamus; mes, mesencephalon; p3, prosomere 3 (ventral thalamus); pit, pituitary; po, preoptic region; SP, subpallium; zli, zona limitans.

 

Figure 9
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  		Fig. 9. A summary of gene expression differences in the developing forebrain of cavefish and surface fish embryos. Schematic representations of lateral views of embryonic brains. The expression patterns of Shh (A), Nkx2.1a (B), Nkx2.1b (C), Lhx6 (D) and Lhx7 (E) are drawn in grey, blue, green, yellow and pink, respectively. For each gene, the dark and light colour schematises the enlarged expression domains in cavefish versus surface fish, respectively. In B to E, the Shh expression pattern, which is larger in cavefish, is also shown in grey. For each gene, the region-specific changes in expression observed in cavefish relative to surface fish embryos or after cyclopamine treatment in cavefish are indicated. The effect on Lhx6 in the hypothalamus is probably mediated through the increase of Nkx2.1a in this region, whereas the effects on Lhx6 and Lhx7 in the po and telencephalon are probably mediated through the increase of Nkx2.1b in the respective regions.

 

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© The Company of Biologists Ltd 2007