Ubiquitin-mediated proteasome degradation regulates optic fissure fusion

Warlen Pereira Piedade,S Veith,J K Famulski

doi:10.1242/bio.044974

Abstract

ABSTRACTOptic fissure fusion is a critical event during retinal development. Failure of fusion leads to coloboma, a potentially blinding congenital disorder. Pax2a is an essential regulator of optic fissure fusion and the target of numerous morphogenetic pathways. In our current study, we examined the negative regulator of pax2a expression, Nz2, and the mechanism modulating Nlz2 activity during optic fissure fusion. Upregulation of Nlz2 in zebrafish embryos resulted in downregulation of pax2a expression and fissure fusion failure. Conversely, upregulation of pax2a expression also led to fissure fusion failure suggesting Pax2 levels require modulation to ensure proper fusion. Interestingly, we discovered Nlz2 is a target of the E3 ubiquitin ligase Siah. We show that zebrafish siah1 expression is regulated by Hedgehog signaling and that Siah1 can directly target Nlz2 for proteasomal degradation, in turn regulating the levels of pax2a mRNA. Finally, we show that both activation and inhibition of Siah activity leads to failure of optic fissure fusion dependent on ubiquitin-mediated proteasomal degradation of Nlz2. In conclusion, we outline a novel, proteasome-mediated degradation regulatory pathway involved in optic fissure fusion.

Highlights

Coloboma is a congenital blindness disorder occurring approximately once in every 2077 live births in the US (Nakamura et al, 2011)
Nlz2 is a negative regulator of pax2a gene expression To confirm Nlz2 is a negative regulator of pax2a expression we injected Nlz2 mRNA into single-cell zebrafish embryos and subsequently analyzed pax2a expression
Whole-mount in situ hybridization (WISH) analysis of Nlz2 mRNA injections confirmed a dose-dependent reduction of pax2a expression (Fig. 1A) which was validated by qPCR (Fig. 1B)

Summary

Introduction

Coloboma is a congenital blindness disorder occurring approximately once in every 2077 live births in the US (Nakamura et al, 2011). We sought to determine whether Nlz2-dependent modulation of pax2a expression levels would have consequences on optic fissure fusion. Optic fissure fusion in embryos injected with Pax2a, Nlz2 or both mRNAs was analyzed by examining the degradation of basement membrane (BM) as indicated by laminin immunohistochemical (IHC) staining (Fig. 1C).

Results

Conclusion