Sbt1 Is Required Downstream of Proneural bHLH Factors for Neurogenesis in the Developing Retina

Abstract

Purpose:: Proneural basic helix-loop helix (bHLH) transcription factors are key regulators of retinal neurogenesis, and they function by activating the expression of target genes that execute a program of neuronal differentiation within progenitors. In a previous screen for proneural target genes we identified a novel gene called sbt1 (shared bHLH target 1). The purpose of this study is to investigate the expression and function of sbt1 and determine whether it plays a role in regulating retinal neurogenesis.

Methods:: We isolated full-length clones for sbt1 by library screening, and analyzed expression during retinal development by in situ hybridization. To assess subcellular localization the protein was epitope-tagged and expressed in Xenopus embryos for immunostaining. Protein regions required for subcellular localization were mapped by generating deletion constructs. To assess the role of sbt1 in retinal neurogenesis we injected either sbt1 RNA or antisense morpholino oligonucleotides together with RNA for GFP into one blastomere at the 16-32 cell stage and assessed effects on retinal cell fates at stage 41.

Results:: sbt1 is conserved across vertebrate species and encodes a protein that shows no homology to any previously described protein. sbt1 is transiently expressed in late proliferating or early differentiating cells in both the Xenopus and mouse retina. The protein localizes to both the plasma membrane and the nucleus in Xenopus animal caps, and the N-terminal region is required for membrane localization. Inhibition of SBT1 translation in Xenopus retinal progenitors prevented retinal neuron differentiation, and progenitors either adopted Müller glial fates or remained as progenitors. Conversely, overexpression of either mouse or Xenopus sbt1 promoted differentiation of early born retinal neurons, and also enhanced the ability of proneural bHLH factors to promote neurogenesis.

Conclusions:: Sbt1 is expressed in retinal progenitors as they initiate neuronal differentiation, and appears to function as a conserved component of the neuronal differentiation program downstream of proneural bHLH factors during retinal development.

Keywords: retinal development • differentiation • gene/expression