QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00515

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dakubo, G. D. Articles by Wallace, V. A. Search for Related Content PubMed PubMed Citation Articles by Dakubo, G. D. Articles by Wallace, V. A.

Retinal ganglion cell-derived sonic hedgehog signaling is required for optic disc and stalk neuroepithelial cell development

¹ Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada
² Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
³ Eye Institute, Center for Neuromuscular Disease, and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada

View larger version (126K): [in a new window]   Fig. 1. Expression analysis suggests that Shh from RGCs signals to cells in the retinal neuroblast, optic disc and optic stalk. (A,E) Sections through the optic vesicle of an E9 embryo hybridized with Shh (A) and Ptch (E) riboprobes demonstrate the upregulation of Ptch expression in the neuroepitheliun and cephalic mesenchyme adjacent to the Shh-expressing cells. (B,F) Frontal sections through the developing eye of an E11 embryo reveals a graded Ptch expression (F) in the ventral forebrain, with high levels in the anterior hypothalamic neuroepithelium (ahn) and low levels in the optic stalk (os), consistent with an established morphogen gradient of Shh (B) from the ventral midline. Ptch (arrows in F) expression in the diencephalic neuroepithelium is due to Shh from the zona limitans intrathalamica (not shown). The period from E12 to E14 is when most neuroepithelial cells of the optic stalk transform into astrocyte progenitor cells (Kuwabara, 1975), and this period is coincident with the rapid RGC differentiation and expression of Shh (C,D), as well as uniform Ptch (G,H) expression in the optic nerve. (I-P) The response of optic disc astrocyte precursor cells (odap) to RGC-derived Shh signaling. As RGCs differentiate in the central retina and express Shh (I), optic disc astrocyte precursor cells and retinal neuroblasts in close proximity to the Shh-expressing cells respond by upregulating the Hh target gene, Gli (J). It is likely that the Gli-expressing cells at the disc are the same cells that express Pax2 (K) and netrin 1 (Ntn1; L). Although RGCs continue to express Shh (M) into late embryogenesis and the underlying neuroblasts respond to this by expressing Gli (N), the Pax2-(O) and Pdgfra- (P) expressing retinal astrocyte precursor cells migrating into the retina (arrowheads in N,O,P), and those at the optic disc (arrows in N,O,P), downregulate their Hh responsiveness. ov, optic vesicle; tv, telencephalic vesicle; oc, optic cup; os, optic stalk; ahn, anterior hypothalamic neuroepithelium; odap, optic disc astocyte precursor cells.

View larger version (87K): [in a new window]   Fig. 2. Ihh signaling is not required for Hh target gene expression in the retina and optic nerve. (A-E) Gli and (F) Pax2 transcripts detected by in situ hybridization in sections through the developing eye and optic nerve of E14 wild-type (A,B) and Ihh-/- (D-F) embryos, and E15 ThyCreShhn/c embryos (C). (A,B,D-F) Horizontal sections. (C) Frontal section. Compared with wild type (A,B), Gli expression is specifically lost in the peri-ocular mesenchyme of Ihh-/- mice (D,E) and in the retinal neuroblast layer of ThyCreShhn/c embryos (C). Arrowheads (A,C,D) indicate Gli-expressing cells adjacent to the retinal pigment epithelium. Astrocyte precursor cells in the optic nerve and at the optic disc develop normally in Ihh-/--mutant embryos, as indicated by normal Pax2 expression (F).

View larger version (67K): [in a new window]   Fig. 3. Craniofacial phenotype and Hh target gene expression in the retina of wild-type and ThyCreShhn/c mice. (A,D) Photographs of E17 embryos showing that ThyCreShhn/c embryos (D) develop well separated bilateral eyes but, compared with wild type (A), have hypoplastic craniofacial structures and failed eyelid closure. (B,C,E,F) Expression of Hh target genes in retinal neuroblasts of E17 wild-type (B,C) and ThyCreShhn/c embryos (E,F) reveals a marked downregulation of Gli (E) and Ptch (F) expression in the retina of ThyCreShhn/c embryos in comparison with wild-type littermates (B,C). Arrows (E,F) point to retinal neuroblasts responding to Shh signaling from a few RGCs that escaped Cre-mediated recombination of the Shh allele in this part of the retina.

View larger version (91K): [in a new window]   Fig. 8. Dorsal ventral patterning and optic fissure closure are normal in ThyCreShhn/c embryos. (A,B,D,E) Sagittal sections through the eyes of E13 wild-type (A,B) and ThyCreShhn/c (D,E) embryos hybridized with Vax2 (A,D) and Pax6 (B,E) riboprobes. Note the absence of coloboma (D,E), and the normal expression of Vax2 (D) in the ventral retina, and Pax6 (E) in the retina, lens, and corneal epithelium of ThyCreShhn/c embryos in comparison with wild-type littermates. (C,F) Collagen type IV immunostaining reveals the presence of normal vascular tunics in the posterior chamber of E17 ThyCreShhn/c eyes (compare hyaloid vessels in C and F). ac, anterior chamber; l, lens; hv, hyaloid vessels; cb, ciliary body.

View larger version (54K): [in a new window]   Fig. 4. The optic nerves of ThyCreShhn/c embryos are hypocellular and covered by a thick layer of melanotic cells. (A,B,F,G) RNA in situ hybridization for Ntn1 (A,F) and Pax2 (B,G) expression in sections through the eye and optic nerve of E16 wild-type (A,B) and ThyCreShhn/c (F,G) embryos. Note the expression of Ntn1 and Pax2 (arrows in F,G) in only proximal optic stalks of ThyCreShhn/c mice. Although RGC axons invaded the optic nerves of ThyCreShhn/c mice, as evidenced by anti-neurofilament-3A10 immunostaining (compare C and H), neuroepithelial cells in the nerve failed to differentiate as glial cells, resulting in the hypocellularity of the optic nerves (compare D,E with I,J). (E,J) High magnifications of the boxed areas in D and I. Dashed lines (E,J) outline the optic nerves, and arrowheads (I) indicate nuclei of some cells in optic nerves of ThyCreShhn/c mice.

View larger version (123K): [in a new window]   Fig. 5. Normal optic vesicle patterning and abnormal gene expression in the optic nerves of ThyCreShhn/c mice. (A-C,E-G) Pax2 expression detected by in situ hybridization in sections through the developing optic vesicle of E10 wild-type (A-C) and ThyCreShhn/c (E-G) embryos. Pax2 expression and optic vesicle morphology appear normal in ThyCreShhn/c when compared to wild-type littermates. Note that wild-type (C) and ThyCreShhn/c (G) embryo sections were not exactly through the same frontal plane. By E12, wild-type (D) optic nerves contained Ntn1-expressing astrocyte lineage cells, but the neuroepithelial cells in the optic stalk of ThyCreShhn/c embryos (H) were pigmented. Arrow indicates proximal optic stack with normal Ntn1 expression. (I-N) Cross sections through the optic nerve of E13 wild-type (I-K) and ThyCreShhn/c (L-N) embryos analyzed for Pax2 (I,L), Pax6 (J,M) and Mitf (K,N) expression. Compared with wild-type, almost all the neuroepithelial cells in the optic nerves of ThyCreShhn/c mice were pigmented and expressed Pax6 (M) and Mitf (N), but not Pax2 (L), and the pigmented cells were already separating to the periphery of the axons. (I-N) dorsal, top; ventral, bottom.

View larger version (82K): [in a new window]   Fig. 6. RGC axon guidance defects in ThyCreShhn/c mice. (A,D) Hematoxylin and Eosin staining of sections through the optic disc of E12 wild-type (A) and ThyCreShhn/c (D) embryos. Note that in the wild type, the eosinophilic RGC axons are separated from the potential subretinal space (black asterisk in A) by the optic disc astrocyte precursor cells (white arrows in A), and that these cells are missing in ThyCreShhn/c mutants (D) thereby exposing the subretinal space (black asterisk in D) to invasion by RGC axons (white asterisk in D). Anti-neurofilament-associated protein immunostaining (B,C,E,F) of E17 retinal sections of wild-type (B) and ThyCreShhn/c embryos (C,E,F) reveals the retinal axon guidance defects of ThyCreShhn/c embryos. Compared with wild type (B), ThyCreShhn/c embryos display axon coiling in the subretinal space at the optic disc (white asterisks in C and D), and axon misrouting into the peripheral retina (open white asterisks in E,F). Note the severe axon misguidance in (F) that creates what looks like multiple optic discs (white arrows in F).

View larger version (136K): [in a new window]   Fig. 7. Loss of optic disc astrocyte precursor cells in ThyCreShhn/c mice is not caused by a global defect in proliferation. Ntn1 (A,D), Pax2 (B,E) and Pdgfra (C,F) transcript expression detected by in situ hybridization in sections through the optic disc of wild-type (A-C) and ThyCreShhn/c (D-F) embryos, at E12 (A,D) and E17 (B,C,E,F). Compared with wild-type mice (A-C), there was no Ntn1 (D), Pax2 (E) or Pdgfra (F) expression in optic discs of ThyCreShhn/c-mutant mice. Pax2 (G,H) and Pdgfra (I,J) expression is comparable between E18 wild-type (G,I) and cyclin D1-/- (H,J) mice.

View larger version (143K): [in a new window]   Fig. 9. Normal development and expression of ventral markers in the hypothalamus of ThyCreShhn/c embryos. (A-F) Frontal sections through the hypothalamus hybridized with Shhexon2 (A,D), Nkx2-1 (B,E) and Ntn1 (C,F) riboprobes reveal comparable expression of functional Shh (A,D), Nkx2-1 (B,E) and Ntn1 (C,F) in wild-type (A-C) and ThyCreShhn/c (D-F) mice. ah, anterior hypothalamus; v3, third ventricle.

View larger version (80K): [in a new window]   Fig. 10. Modulation of the Pax2-positive astrocyte precursor cell population at the optic disc by Shh signaling. Optic cups of E12 (A-C) and E14 (D-H) C57BL/6 embryos were cultured for 48 hours, untreated (A,E) or treated with recombinant Shh-N (B,D,F,H) or the anti-Shh antibody 5E1 (C,G), and sectioned for in situ hybridization for Pax2 (A-G) and Gli (H) expression. Compared with control (A), the size of the Pax2-positive cell population at the optic disc was markedly increased by Shh-N treatment (B), whereas 5E1 treatment (C) resulted in an almost complete loss of these cells. (D,H) The kinetics of Hh responsiveness by optic disc cells is maintained in vitro, as the Pax2-positive cells (arrow in D) of the E14 Shh-N treated explants downregulate Gli (arrow in H) expression by the second day in culture. Consistent with this observation, the size of the Pax2-positive cell population at the optic disc did not differ much between controls (E) and Shh-N treated (F) cultures, but was much reduced in the 5E1 (G) treated explants at E14.