DEV02493 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Bmp7 expression pattern during eye development. Bmp7 expression was determined using the activity of the LacZ transgene of the mutated allele (Bmp7^lacZ/Neo) in whole mounts (A,D,G,J) and frontal paraffin sections (B,C,E,F,H,I,K,L) of embryos at different developmental stages, as indicated in the figure. Bmp7 is expressed in the anteronasal half of the optic vesicle (A-C). In the early optic cup, Bmp7 localised to the optic stalk (E), nasal-ventral optic cup (E) prospective RPE (F) and surrounding extraocular mesenchyme (D-F). Additional expression is later observed in the developing hyaloid artery (G,H), perineurium of the optic nerve (J, K) and in scattered cells in the optic chiasm (L, broken line demarcates the ventricle). Abbreviations: aov, anterior optic vesicle; ch, chiasm; hv, hyaloid vessels; nvr, naso-ventral retina; on, optic nerve; os, optic stalk; rpe, retinal pigmented epithelium; vr, ventral retina. Scale bar: 200 μm in A; 300 μm in D; 250 μm in J; 125 μm in B,E,H,I; 60 μm C,F,K,L.

• Supplemental Figure 2 -

  Fig. S2. Segregation of the optic vesicle into neural retina and RPE is impaired in severely affected Bmp7-null embryos. The distribution of the prospective RPE (Otx2, A-D; Mitf, E-F) and neural retina (Chx10, I-M; Six3, N-Q) markers was determined by whole-mount in situ hybridisation in E10.5 wild-type (A, E, I, N) and Bmp7^-/- (C, G, L, P) embryos. Images in second and fourth columns are paraffin sections from the embryos shown in the first and third columns, respectively. Chx10 and Six3 expression is largely reduced in the mutants, whereas RPE markers are expanded into the neural retina domain. Scale bar: 60 μm; 150 μm in A,C; 230 μm in E,G,I,L,N,P.

• Supplemental Figure 3 -

  Fig. S3. Defects in the ventral optic cup are visible at early optic cup stages in the Bmp7 null microphthalmic embryos. E9.5 (A,B) and E10.5 (C-H) wild-type (A,C,E,G) or mutant (B,D,F,H) embryos were hybridised in toto with probes specific for Rx (A,B), a perspective neural retina marker; Pax6 (C,D), a distal optic cup determinant; Pax2 (E,F); and netrin 1 (G,), and then sectioned in the frontal plane with a vibratome. Sections in A,B were then immunostained with antibodies against PAX2. Initially, the optic vesicle of mutant embryos (B) presents a normal distribution of Pax2 and Rx when compared with a wild-type littermate (A). Similarly, the domain of Pax6 expression is normally limited to the distal optic cup, although the signal appears more uniformly distributed (C,D). By contrast, Pax2 and netrin 1 expression is reduced in the ventral optic cup (open arrows in F,H) of the mutants when compared with the wild type (yellow arrow in E,G), suggesting a reduction of invaginating/proliferating RF precursors. Abbreviations: lv, lens vesicle. Scale bar: 250 μm.

• Supplemental Figure 4 -

  Fig. S4. RGC axons defects in E14.5 Bmp7^-/- retinas are independent from abnormal cell differentiation or direct activity of BMP7 on RGC axon growth. Frontal cryostat sections from E14.5 wild-type (A) or mutant (B) embryos were immunostained with antibodies against islet 1 to recognise developing RGC. (C) The number of islet 1-positive cells/area was quantified in three different embryos. The density of RGC is similar in both wild-type and mutant embryos. (A,B) Retinal explants from E14.5 wild-type embryos were grown in collagen gel matrix in the presence (E) or absence (D) of soluble BMP7. After 48 hours, explants were fixed and immunostained with Tuj1 antibodies. The average growth area (a) and mean neurite length (l) were quantified in three different experiments (n) performed in quadruplicate. No statistically significant difference was observed between control and BMP7-treated explants (see boxes in D and E). Scale bar: 200 μm in A,B; 125 μm in D,E.