Hanel ML , Hensey C .

057990/2/99/2 Wellcome Trust , 061410/GM/SP/JAT Wellcome Trust

             optic vesicle morphogenesis
             BMP signaling pathway
             positive regulation of apoptotic process

                coloboma

           MICROPHTHALMIA, ISOLATED, WITH COLOBOMA 6; MCOPCB6

Xla Wt + gdf6 (Fig.1.E)
Xla Wt + gdf6 MO (Fig.1.I,J)
Xla Wt + gdf6 MO (Fig.2.B,D,F,H)
Xla Wt + gdf6 MO (Fig.3.B,C,E,F)
Xla Wt + gdf6 MO (Fig.4.A,C,D,J)
Xla Wt + gdf6 MO (Fig.4.F,H,I,J)
Xla Wt + gdf6 MO (Fig.5.A-D, F)
Xla Wt + gdf6 MO (Fig.5.E,F)
Xla Wt + gdf6 MO

	Figure 1. Reduced eye size following GDF6 depletion. (A) In vitro translation of GDF6 mRNA is blocked by GDF6 MOrpholino (GDF6 MO) but is not affected by the standard control MOrpholino (STD MO). (B) Dorsal view of stage 41 tadpole injected with 10 ng GDF6 MO on the left side. (C) Lateral view of tadpoles showing (top to bottom) an unaffected tadpole; GDF6 MO injected sides with an eye 84% of normal contra lateral size and 66% of normal contra lateral size. (D) Graphical representation of the percent of embryos injected with GDF6 MO with the small eye phenotype at stage 41 compared with those injected with STD MO. (E) A severely ventralized embryo injected with 250 pg GDF6 mRNA at stage 41. The percent of ventralized embryos is shown. (F) An example of a partially rescued embryo injected with 250 pg GDF6 mRNA + 20 ng GDF6 MO. The percent of similarly ventralized embryos is shown. (G) A completely rescued embryo injected with 250 pg GDF6 mRNA + 20 ng GDF6 MO. (H) The untreated side of a stage 24 embryo immunostained with anti-phosphorylated-Smad1/5/8 showing staining within the retina and along the neural tube (arrow). Full embryo is shown in the inset. (I) The GDF6 MO (20 ng) injected side of the same embryo showing a loss of phosphorylated-Smad1/5/8 in the retina and neural tube. (J) Frontal view of the same stage 24 embryo showing the reduction in the intensity of the phosphorylated-Smad1/5/8 stain on the injected side (i). (K) Endogenous Smad1/5/8 phosphorylation in St.11.5–12.5 embryonic extracts, lane 2. No Smad phosphorylation is detected in 64 cell embryos lane 1.
	Figure 2. Loss of retinal differentiation and laminar structure in GDF6 depleted embryos. Histology of stage 41 embryos with small eyes following GDF6 MO injection (20 ng) compared with normal stage matched controls. DAPI staining of small eye (B, D, F, and H) compared with normal eye (A, C, E, and G) showing small eyes have a lack of laminar structure and appear disorganized. (A, B) Photoreceptors (p) are not stained in B compared with XAP-1 staining in A. (B) Cell bodies are visibly extending outward (white arrow) but do not stain for XAP-1. Red staining next to the lens is non-specific staining also detected in a proportion of negative controls. (C, D) 40.2D6 detects ganglion (g) and amacrine (a) cells within the normal retina, but these cells are not detected in the small eye. Photoreceptors are visible due to autofluorescence at increased exposure. (E, F) Small disorganized eyes show a loss of immunostaining for the cytoplasmic domain of NCAM (n). (G, H) Mitotic cells (m) detected using anti-phosphorylated-histoneH3 are few and found mainly in the ciliary marginal zone in normal stage 41 embryos. The small eye shows a few mitotic cells near the RPE layer.
	Figure 3. GDF6 knock down causes a loss of Pax6 expression as detected by in situ hybridization. Representative stage 20 embryos injected with 50 ng standard MO (A, D), 20 ng GDF6 MO (B, E), and 50 ng GDF6 MO (C, F). The injected sides are labelled i. Dorsal views show a loss of Pax6 stain within the two rhombomere bands in the hindbrain in the 20 ng GDF6 MO injected embryos (B), and an additional more severe loss of dorsal neural tube staining in the 50 ng GDF6 MO injected embryos (C), compared to the uninjected side (red arrows indicate rhombomeres) and standard MO injected embryos (A). Frontal views show a laterally reduced size of the Pax6 domain (E and F, injected side), compared to Pax6 staining in uninjected side (definition between forebrain and retinal stain indicated with white arrow) and standard MO injected embryo (D). (G, H) Dorsal and lateral views of stage 27 embryos injected with 25 ng of GDF6 MO show a reduced size of the Pax6 domain within the retina and a complete loss of stain within brain on the injected side. (I) Graph showing the percent stage 20 embryos injected at 20 ng and 50 ng of GDF6 MO and Standard MO showing a disruption of normal Pax6 stain.
	Figure 4. GDF6 knockdown disrupts neural differentiation. At neurulation stages, immunostaining for neural cell adhesion molecule (NCAM) is lost or reduced in intensity in the GDF6 MO injected side: A(i), C(i), D, F(i), H(i), I. (A) Stage 24 embryo injected with 50 ng GDF6 MO showing complete loss of NCAM stain along the neural tube on the injected side. Some patchy retina stain remains. This embryo is also curved and appears to be reduced in size on the injected side. (B) Normal NCAM stain in an embryo injected with 50 ng standard MO. (C) Stage 24 embryos injected with 20 ng GDF6 MO with NCAM stain lost along the posterior neural tube (top embryo, arrow marks posterior end of NCAM stain), or along the entire neural tube (bottom embryo). (D, E) Injected (D) and uninjected side (E) of bottom embryo in (C) showing patchy loss of NCAM stain in the retina of the injected side. (F) Dorsal view of stage 27 embryo injected with 50 ng GDF6 MO. The injected side (i) shows a loss of NCAM stain both along the neural tube and within the head and retina. In the head and retina small patches of intense NCAM staining remain. (G) Normal NCAM staining in a stage 27 embryo injected with 50 ng standard MO. (H, I) Full view and close up of a stage 27 embryo injected with 20 ng GDF6 MO. Neural tube NCAM staining is less intense on the injected side (top half of embryo). (J) Percent embryos with NCAM reduction on the injected side following GDF6 MO injection.
	Figure 5. Increased cell death during neurogenesis in GDF6 depleted embryos. (A) Stage 20 embryo injected with 20 ng GDF6 MO shows a patch of TUNEL at the anterior region of the injected side (i). (B) Stage 22 embryo injected with 20 ng GDF6 MO showing TUNEL positive cells near the midline at the anterior region of the injected side (i). (C) Stage 22 embryo injected with 50 ng GDF6 MO showing more extensive TUNEL staining which extends along the length of the injected side (i). There is an obvious reduction in physical size on the injected side. (D) Stage 27 embryo injected with 50 ng GDF6 MO showing dark concentrated TUNEL staining in the brain and retina on the injected side (i). This embryo is also curled towards the injected side. (E) Graphical representation of the percent embryos with increased TUNEL staining on the injected side at stage 20–24 and stage 27. (F, G) The GDF6 MO (25 ng) injected side (F) and uninjected side (G) of a stage 27 embryo, showing heavy TUNEL staining in the retina and brain.

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