bHLH-dependent and -independent modes of Ath5 gene regulation during retinal development

In a wide range of vertebrate species, the bHLH transcription factor Ath5 is tightly associated with both the initiation of neurogenesis in the retina and the genesis of retinal ganglion cells. Here, we describe at least two modes of regulating the expression of Ath5 during retinal development. We have found that a proximal cis-regulatory region of the Xenopus Ath5 gene (Xath5) is highly conserved across vertebrate species and is sufficient to drive retinal-specific reporter gene expression in transgenic Xenopus embryos. Xath5 proximal transgene expression depended upon two highly conserved bHLH factor binding sites (E-boxes) as well as bHLH factor activity in vivo. However, we found that bHLH activity was not required for expression of a longer Xath5 transgene, suggesting that additional mechanisms contribute to Xath5 expression in vivo. Consistent with this, we showed that a more distal fragment that does not include the conserved proximal region is sufficient to promote transgene expression in the developing retina. In mouse, we found that a longer fragment of the cis-regulatory region of either the mouse or Xenopus Ath5 gene was necessary for transgene expression, and that expression of a mouse Math5 (Atoh7) transgene was not dependent upon autoregulation. Thus, despite extensive conservation in the proximal region, the importance of these elements may be species dependent.