Nlz2 regulates optic fissure closure by inhibiting pax2a gene expression. (A) WISH for pax2a in 24 hpf embryos injected with varying amounts of Nlz2 mRNA. OF-associated pax2a signal is decreased in response to increasing Nlz2 mRNA. (B) qPCR results at 24 hpf for pax2a expression±s.d. *P<0.05 compared to uninjected, ^$P<0.05 compared to Nlz2 50 pg. (C) 72 hpf Tg[rx3:GFP] (green) embryos injected with Pax2a, Nlz2 or Pax2+Nlz2 mRNA stained for laminin (red) and DAPI (blue). Arrowheads indicate persistence of laminin signal. Scale bar: 50 µm. (D) Proportion of embryos with failure of fusion (1) or completed fusion (0)±s.d. Region of analysis is outlined by dashed lines. *P<0.05 compared to uninjected, one-way ANOVA; P<0.0001. Dashed boxes outline region of inset.

Siah targets Nlz2 for proteasomal degradation. (A) Conservation of Nlz2 ‘degron’ motif sequence. (B) Western blot analysis of nlz2-FLAG protein stability in response to siah1-myc, siah1ΔR-myc or siah1-myc+MG132. Actin was used as a loading control. * indicates potential ubiquitination products. Nlz2-FLAG band intensity quantification is shown. (C) Co-immunoprecipitation of nlz2-FLAG co-transfected with HA-ubiquitin, siah1-myc or siah1ΔR-myc probed for FLAG (green), MYC (red) and HA (B/W). * indicates potential ubiquitination. (D) Upper panels: endogenous Siah activity reporter assay expression in eyes of 24 hpf GFP-NxN or GFP-VSP mRNA-injected embryos, ±12.5 µM MG132. mCherry mRNA was co-injected for normalization. Scale bar: 50 µm. Lower panel: quantification of normalized GFP fluorescence intensity in the eye±s.d. *P<0.05 compared to GFP-NxN, ^#P<0.05 compared to GFP-VSP, one-way ANOVA; P<0.0001.

siah1, siah2l and nlz2 gene expression during eye morphogenesis. (A) WISH for siah1, siah2l and nlz2 between 24 and 48 hpf. (B) Two-color fluorescent WISH (FWISH) for nlz2, siah1, siah2 and pax2a. Arrowheads indicate co-localization. (C) siah1 WISH after treatment with RA (AGN194310), BMP (DMH1), hedgehog (cyclopamie) inhibitors, hedgehog agonist (purmorphamine) or in smo^hi1640Tg embryos at 24 hpf. (D) qPCR results for siah1 and pax2a expression±s.d. *P<0.05 compared to respective controls.

Siah activity indirectly regulates pax2a expression. (A) WISH for pax2a after injection of Siah1, Siah1ΔR, Siah2l or Siah2lΔR±MG132 at 24 hpf. (B) qPCR results for pax2a expression at 24 hpf. *P<0.05 compared to uninjected. (C) WISH for pax2a gene expression upon co-injection of Siah1+Nlz2 or Nlz2^nxn. (D) qPCR results for pax2a expression at 24 hpf. *P<0.05 compared to uninjected, ^@P<0.05 compared to Nlz2, ^#P<0.05 compared to Siah1, ^$P<0.05 compared to Siah1+Nlz2. (E) Tg[rx3:GFP] (green) embryos injected with siah1, siah2l, siah1ΔR or siah2lΔR±100 pg Nlz2 mRNA stained for laminin (red) and DAPI (blue) at 72 hpf. Arrowheads indicate persistence of laminin. Scale bar: 50 µm. (F) Proportion of embryos with failure of fusion (1) or completed fusion (0). Region of analysis is outlined by dashed lines. *P<0.05 compared to uninjected, ^#P<0.05 compared to Siah1, ^$P<0.05 compared to Siah1+Nlz2. One-way ANOVA; P<0.0001. Dashed boxes indicate region of inset.