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First published online 14 July 2004
doi: 10.1242/dev.01247

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Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina

¹ European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany
² Institute of Toxicology and Genetics, Research Center Karlsruhe, PO Box 3640, 76021 Karlsruhe, Germany

View larger version (135K): [in a new window]   Fig. 1. Expression of shh in the zebrafish retina. All panels show confocal sections through the retina, with anterior to the top. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ACs, amacrine cells. (A) Whole-mount detection of shh RNA in the retina at 46 hpf. Note expression in the GCL and proximal INL. (B) Expression of shh-GFP (green) and Zn5 (red). Note that shh-GFP and Zn5 are co-expressed in ganglion cells, and that shh-GFP is also expressed by a subset of amacrine cells. (C) High magnification of the retina shown in B. Note that GFP is also present in neurites that contribute to the inner plexiform layer (IPL). (D) Expression of the shh-GFP at 46 hpf (GFP single channel of the image shown in F). Note expression in the GCL, and proximal INL. (E) Isl1 single channel of the image shown in F. Note expression in the GCL and proximal INL, as well as in the distal INL. (F) Retina at 46 hpf, double labeled for Isl1 protein (red) and GFP (green) expressed by the shh-GFP line. (G) High magnification of the GCL double labeled for Isl1 (red) and shh-GFP (green) at 46 hpf. Note that some RGCs express only Isl1 (arrowhead), whereas some RGCs express both Isl1 and shh-GFP (arrow). (H) shh-GFP single channel of the image shown in G. (I) Isl1 single channel of the image shown in G.

View larger version (100K): [in a new window]   Fig. 2. In vivo time-lapse recording of shh-GFP expression in the retina. (A-F) Single frames taken from a film recording of shh-GFP expression in the retina, starting at 35 hpf and ending at 46 hpf. Note that the first amacrine cells expressing shh-GFP are already present at 35 hpf, and that the wave of shh-GFP expression in amacrine cells spreads rapidly following the wave in the GCL.

View larger version (116K): [in a new window]   Fig. 3. A wave of shh-GFP expression spreads through the GCL and INL independently of ath5 activity. All images are confocal sections of the retina, with anterior to the top. (A) Wild-type embryo double labeled for Zn5 protein (red) and shh-GFP expression (green) at 32 hpf. The expression of both markers has been initiated in the ventronasal sector. Note that some of the shh-GFP-expressing cells do not co-express Zn5, and are therefore amacrine cells (inset, arrowhead). (B) Wild-type embryo double labeled for Zn5 and shh-GFP at 57 hpf. Expression of both markers has spread through the retina. (C) ath5 mutant embryo double labeled for Zn5 (red) and shh-GFP expression (green) at 32 hpf. Note the absence of Zn5-expressing cells, while a small number of shh-GFP-expressing cells are already present (arrowheads). (D) ath5 mutant embryo double labeled for Zn5 and shh-GFP expression at 57 hpf. Note that shh-GFP expression has spread through both the GCL and the INL.

View larger version (73K): [in a new window]   Fig. 4. The differentiation of cell types found in the INL depends on shh signaling. All images are confocal sections of the retina, with anterior to the top. (A) Isl1 protein (red) in a wild-type retina at 64 hpf. Note expression in the GCL, as well as in three discrete domains in the INL: amacrine cells (AC) in the proximal INL, as well as bipolar cells (BC) and horizontal cells (HC) in the distal INL. (B) Isl1 protein in a shh–/– retina at 64 hpf. Note the overall reduction of Isl1-expressing cells, and that most of them are located in the proximal retina. Note also that these cells are disorganized, and that there is no sign of an INL or laminar organization. (C) Glutamine synthetase protein (red) in a wild-type retina at 72 hpf. Note expression in Mueller glia, which span all the retinal layers. (D) Glutamine synthetase protein in a shh–/– retina at 72 hpf. Note the complete absence of glutamine synthetase staining from the retina. (E) PKC protein (green) in a wild-type retina at 72 hpf. Note prominent expression in bipolar cells in the INL. (F) PKC protein in a shh–/– retina at 72 hpf. Note the complete absence of PKC staining from the retina. (G) GAD67 protein (green) in a wild-type retina at 72 hpf. Note prominent expression in amacrine cells of the INL, and in the inner plexiform layer. (H) GAD67 protein in a shh–/– retina at 72 hpf. Note the severe reduction of amacrine cells (arrowheads) and absence of inner plexiform layer.

View larger version (66K): [in a new window]   Fig. 5. The formation of plexiform layers and the differentiation of photoreceptors depends on shh signaling. All images are confocal sections of the retina, with anterior to the top. (A) F-actin staining in a wild-type retina at 50 hpf. Note the prominent staining of the inner and outer plexiform layers. (B) F-actin staining in a shh–/– retina at 50 hpf. Note the complete absence of plexiform layer formation. (C) Zpr1 protein in a wild-type retina at 72 hpf. Note the staining in cones of the ONL. (D) Zpr1 protein in a shh–/– retina at 72 hpf. Note the severely reduced number of cells expressing Zpr1, which are present in a ventronasal patch. (E) Zpr3 protein in a wild-type retina at 72 hpf. Note the staining in rods of the ONL. (F) Zpr3 protein in a shh–/– embryo at 72 hpf. Note the reduced cell number, as observed with Zpr1 (E).

View larger version (85K): [in a new window]   Fig. 6. Non-autonomous rescue of Mueller glia at 72 hpf by wild-type cells transplanted into shh mutant embryos. Wild-type cells are labeled with biotin (blue) and they also carry the shh-GFP transgene (green). Mueller glia are detected with an antibody recognizing Glutamine Synthetase (GS, red). (A) Mueller glia are rescued in the immediate vicinity of shh-expressing wild-type cells. The arrowheads point to shh mutant cells that express GS. Arrows indicate wild-type donor cells that express GS. (B) Higher magnification of the image shown in A. (B'-B''') Single channels of the image shown in B. Arrowheads point to mutant cells that are rescued. (C) Another example showing non-autonomous rescue of mutant cells (arrowheads). Note only two shh-GFP-expressing wild-type cells are present in the INL in the lower portion of the retina, and that these cells rescue nearby mutant cells. (D) Higher magnification of the image shown in C. (E) An example of a small clone of wild-type cells expressing shh-GFP in the neural retina that leads to local rescue of Mueller glia (arrowheads). (F) A slightly larger clone of wild-type cells expressing shh-GFP that leads to local rescue of Mueller glia (arrowheads). (G) An example of failure to rescue by wild-type cells expressing shh-GFP present in the pigmented retina (RPE, arrowheads). (Inset) Enlarged view of framed area.

View larger version (90K): [in a new window]   Fig. 7. Non-autonomous rescue of photoreceptors at 72 hpf by wild-type cells transplanted into shh mutant embryos. Wild-type cells are labeled with biotin (blue) and they also carry the shh-GFP transgene (green). Photoreceptors are detected with an antibody recognizing Zpr1 (red). (A) Photoreceptors are rescued when they are found in the region of the retina containing wild-type shh-expressing cells in the neural retina. The arrow indicates shh mutant cells that are rescued. (B) Higher magnification of the image shown in A. Arrowheads point to non-autonomously rescued cells. (C) Another example of the rescue of photoreceptors. (D) Higher magnification of the image shown in C. Arrowheads point to non-autonomously rescued cells. (E) A further example of rescue. Note that there is a gap in the rescued photoreceptors in exactly the same region as where there are no shh-expressing cells in the neural retina (arrow). (F) Example of failure to rescue photoreceptors when wild-type cells are located only in the RPE (arrowheads). (Inset) Enlarged view of framed area.

View larger version (65K): [in a new window]   Fig. 8. Non-autonomous rescue of lamination at 50 hpf by wild-type cells transplanted into shh mutant embryos. Wild-type cells are unlabeled, but they carry the shh-GFP transgene (green). The inner plexiform layer is detected with phalloidin staining (red). (A) An example of a very small group of wild-type shh-expressing cells that lead to elevated levels of F-actin in adjacent cells (arrowhead) in the position where the IPL would normally form. (B) Higher magnification of the image in A. (C) Two groups of wild-type shh- expressing cells lead to local rescue of the IPL (arrowheads). (D) Higher magnification of the image in C. (E-G). Local rescue of the IPL by groups of wild-type cells expressing shh. In G, wild-type cells are labeled in blue, but rescue is only observed close to shh-GFP-expressing wild-type cells (arrowhead). (H) Large clones (blue) fail to rescue lamination if they do not contain shh-GFP expressing cells (green). (I) Wild-type cells expressing shh-GFP in the RPE fail to rescue lamination. (I') The GFP single channel of the image shown in I, showing shh-GFP-expressing cells in the RPE (arrowhead, inset).