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doi: 10.1242/10.1242/dev.00305

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Hedgehog signalling maintains the optic stalk-retinal interface through the regulation of Vax gene activity

Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (22K): [in a new window]   Fig. 1. Phylogenic relationship of the Vax gene family. (A) Lineage tree of vertebrate Vax genes using Emx genes as an `out group'. The out group branch separates Vax1 and Vax2 subgroups, both of which contain putative orthologues from different Classes of vertebrates, suggesting that the ancestral Vax gene was duplicated prior to the separation of the vertebrate Classes. Amino acid sequences from GenBank database entries were used. (B) Vax protein structure. Emx and Vax proteins share more than 60% amino acid sequence in the homeodomain (Hallonet et al., 1999), and additionally Vax proteins share nearly invariant homeodomains and have two Vax gene-specific conserved domains, VCDa and VCDb.

View larger version (101K): [in a new window]   Fig. 2. Zebrafish Vax genes are expressed in the optic stalk and the preoptic area. Expression patterns of vax1 and vax2 genes (indicated bottom right) in the anterior neural keels (A,D) and brains (B,C,E-L) of wild-type embryos at various stages (indicated bottom left). Dorsal is to the top in the frontal views (A-F,I). Anterior is to the left in the dorsal views (G,J) and in the lateral views (H,K,L). Eyes are removed to observe the preoptic area in L. Arrowheads indicate optic recess (H,L). Abbreviations: cf, choroid fissure; e, eye; hyp, hypothalamus; mb, midbrain; nr, nasal retina; poa, preoptic area; or, optic recess; os, optic stalk; re, retina; t, telencephalon; tr, temporal retina.

View larger version (81K): [in a new window]   Fig. 3. Abrogation of Vax1 and Vax2 function disrupts eye development. Stages indicated bottom left. The morpholino oligonucleotides (MOs) injected are indicated top right. (A-H) Lateral views of left eyes of living embryos with anterior to the left. Bars indicate the choroid fissure, which fails to close in the MO-injected embryos. (I-T) Toluidine blue-stained transverse sections at the level of the choroid fissure or optic nerve head. S and T show higher magnifications of K and R, respectively. Arrows indicate disorganization of cells at the choroid fissure (O,S) and the arrowhead indicates reduced retinal pigment epithelium in the ventral retina (N). (U-X) Dorsal views of Vax (U,V) and rx1 (W,X) expression with anterior to the left (compare U and V with Fig. 2G,J). Arrowheads indicate that the retinal marker rx1 is extended into the optic stalk region in the MO-injected embryos. (Y,Z) Confocal projection of DiI-labeled optic nerve with Z' showing a higher magnification of the midline of Z. Abbreviations: cf, choroid fissure; 1, lens; nr, neural retina; oc, optic chiasm; on, optic nerve; ot, optic tectum; pr, photoreceptor layer; rg, retinal ganglion cells; rpe, retinal pigment epithelium.

View larger version (121K): [in a new window]   Fig. 4. Medial optic vesicle cells are viable but fail to constrict to form an optic nerve in vax1/vax2 morphants. (A) Schematic representation of the experiment (left) and dorsal view of an embryo with fluorescein uncaged in both left and right optic stalks (right). (B-G) Confocal images of living eyes, optic nerves and preoptic area of wild-type (B-D) and vax1-morpholino oligonucleotide (MO)/vax2-MO (E-G) embryos at the stages indicated bottom left. Arrowbars indicate the width of the optic stalk or nerve territory. Abbreviations: di, diencephalon; on, optic nerve; r, retina.

View larger version (121K): [in a new window]   Fig. 5. Hedgehog signals acting through Smoothened induce Vax gene expression. Anterior is to the left in the lateral views (A-H,J,K) and anterior is to the top in the dorsal views (I,L) of 24 hpf brains showing expression of genes (indicated bottom right) in wild-type (WT) (A-C,I), shh-injected wild-type (WT+shh) (D-F,L) embryos, smoothened-/- (smu) (G,H), and shh-injected smoothened-/- (smu+shh) (J,K) mutant embryos. In shh-injected wild-type embryos, expression of the ventral retinal gene, vax2, expands dorsally (E), whereas expression of the dorsal retinal gene, tbx5, is abolished (F). Abbreviations: cf, choroid fissure; dr, dorsal retina; nr, nasal retina; tr, temporal retina.

View larger version (114K): [in a new window]   Fig. 6. Hedgehog signals act downstream of the Nodal signalling pathway to induce Vax gene expression. Lateral views of gene expression (indicated bottom right) in 24-hour stage cyc-/- (cyc) (A,B), oep-/- (oep) (C,D), shh-injected oep-/- (oep+shh) (E,F) embryos. Abbreviations: r, retina; t, telencephalon.

View larger version (93K): [in a new window]   Fig. 7. Lack of Nodal signaling activity leads to Fgf receptor-dependent induction of Vax gene expression. (A-J) Lateral views of brains of maternal and zygotic oep-/- (MZoep) (A-E), and shh-injected maternal and zygotic oep-/- (MZoep+shh) (F-J) mutant embryos analysed for expression of various genes (indicated bottom right). (K-M) atv-injected sibling embryos from smu+/- x smu+/- crosses showing expression of Vax genes and pax2.1 expression. (N-O) Dorsal views of Vax expression in embryos treated with the Fgf receptor inhibitor SU5402. (P-Q) Lateral views showing absence of Vax expression in atv-injected embryos treated with SU5402. Abbreviations: mb, midbrain; os, optic stalk; poa, preoptic area; r, retina; t, telencephalon.

View larger version (77K): [in a new window]   Fig. 8. Belladonna regulates Vax gene expression in the preoptic area. Anterior is to the left in the dorsal views of heads of noi-/- (noi) (A,B), shh-injected noi-/- (noi+shh) (C,D), bel-/- (bel) (E,F), and shh-injected bel-/- (bel+shh) (G,H) embryos. Note that the expression of Vax genes is reduced in the preoptic area but not in the optic stalk and ventral retina of bel-/- embryos (E,F, compare with Fig. 2G,J). Abbreviations: os, optic stalk; poa, preoptic area; r, retina.

View larger version (33K): [in a new window]   Fig. 9. Schematic indicating genetic pathways involved in the regulation of Vax genes and pax2.1 expression and patterning of the early eye primordium. Cyc/Nodal signals from axial tissues promote medial fates in the anterior neural plate (including optic stalks and preoptic area), in part through the regulation of Hh expression. Hh proteins promote both pax2.1 and Vax gene expression in parallel. Within the forebrain, Bel and Fgf activity is required for Hh to promote Vax expression. We currently do not know the epistatic relationship between Bel and Fgf signals. It is also probable that Fgf signals act more widely to pattern optic stalks as well as the preoptic area. Both pax2.1 and Vax genes are required for closure of the choroid fissure, whereas Vax genes have a more prominent role than pax2.1 in limiting retinal differentiation to the eye cup. See text for further details.

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