DEV028308 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Scheme of Pax6 locus and the morphological and molecular phenotype of Pax6^del/del embryonic eyes. Scheme of Pax6^flox (A) and the resulting Pax6^del (B) allele following Cre activity. loxP, black arrowheads; FRT, red arrowheads; exons, filled boxes. (C) Eye development is morphologically arrested at the optic-vesicle stages as shown for the Pax6^del/del E15.5 embryos. In the Pax6^del/del optic rudiment, the VC1.1 epitope is detected by antibody labeling (C-E, red) in a mostly non-overlapping pattern with the Crx transcripts, detected on the same section by fluorescent in-situ hybridization (D,E, green). E is a higher magnification of the boxed region in D. Abbreviations: ov, optic vesicle; se, surface ectoderm. Arrow points to the direction of the surface ectoderm. Scale bar: 100 µm in; 50 µm in E.

• Supplemental Figure S2 -

  Fig. S2. Expression of Atoh4 and Crx in the Pax6^flox/flox;α-Cre;Z/AP mutants demarcates the two populations of Pax6-deficient RPCs. In the normal E15 OC, Atoh4 (A) and Crx (B) are expressed in the neuroblast layer or the prospective photoreceptor layer, respectively, and their expression extends to the non-neuronal progenitors in the peripheral OC. In the Pax6^flox/flox;a-Cre;Z/AP eyes, Atoh4 expression is abolished from the region of Cre activity (C). The domain of Cre activity was determined on adjacent sections by detection of human alkaline phosphatase (hAP) activity expressed from the Z/AP transgene (E). Thus, the region of Atoh4 loss overlaps with the Pax6 deficient area (compare C with E). In contrast with the complete loss of Atoh4 in the hAP⁺ region, Crx upregulation in the Pax6^flox/flox;a-Cre;Z/AP mutants is detected only in a peripheral region of the Pax6-deficient area (D). Thus, in the Pax6^flox/flox;a-Cre;Z/AP mutants, the Crx⁺Atoh4^- cells populate region 1, whereas the Crx^-Atoh4^- cells delineate region 2 (D). Abbreviations: nnp, non-neuronal progenitors; nbl, neuroblast layer. Scale bar: 100 µm

• Supplemental Figure S3 -

  Fig. S3. Loss of Pax6 in the Pax6^flox/flox;Chx10-Cre RPCs results in differentiation of the mutant cells to amacrine interneurons. The recombination pattern mediated by Chx10-Cre (A) and α-Cre (D) was monitored by detection of human alkaline phosphatase (hAP) protein expressed from the Z/AP transgene, and this reporter transgene was employed to trace the recombinant cells in the Pax6^flox/flox;Chx10-Cre;Z/AP (B,C) or Pax6^flox/flox;α-Cre;Z/AP (E) postnatal (P15 or P20) retinas. In the normal Chx10-Cre;Z/AP retina, patches of cells expressed hAP (A). In some regions, hAP was detected in all retinal layers overlapping with recoverin, which is expressed in photoreceptors, and syntaxin, which is expressed in amacrine cells and their axonal projections in the inner nuclear layer (F), thus corresponding with the activity of Cre in the multipotent RPCs that give rise to all cell types (A, white arrowheads). In other regions, the recombination was restricted to the cells in the ganglion and inner cell layers (A, green arrowhead), indicating a second stage of Chx10-Cre activity after cell specification is initiated. In the somatic retinal mutants, the laminated structure was lost in regions located in the central or peripheral retina (Pax6^flox/flox;Chx10-Cre;Z/AP) or only in the peripheral retina (Pax6^flox/flox;α-Cre;Z/AP); in these regions, the hAP was co-expressed with the amacrine-specific marker syntaxin (compare B with G and E with J) but not with the photoreceptor marker recoverin (compare C with H). Abbreviations: co, cornea; inl, inner nuclear layer; le, lens; oc, optic cup; prl, photoreceptor layer; rpe, retinal pigmented epithelium. Scale bar: 50 µm