bHLH-dependent and -independent modes ofAth5gene regulation during retinal development

Abstract

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 Xath5transgene, suggesting that additional mechanisms contribute to Xath5expression 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.

Conserved regulation of Math5 and Math1 revealed by Math5-GFP transgenes

“…1). The 5′ 2.1 Kb sequence is sufficient to drive GFP expression in the mouse retina, beginning at E11.5 (Hutcheson et al, 2005;Riesenberg et al, 2007 and Fig. 3A,4B).…”

“…In the upstream region of Math5, two highly conserved regions among the Xenopus, mouse and human Ath5 genes are shown (yellow boxes in Fig. 7A) (Brown et al, 2002;Hutcheson et al, 2005;Riesenberg et al, 2007). Within these two evolutionarily conserved regions are four E-boxes, bHLH consensus binding sites (blue boxes) (Murre et al, 1989;Hutcheson et al, 2005).…”

“…7A) (Brown et al, 2002;Hutcheson et al, 2005;Riesenberg et al, 2007). Within these two evolutionarily conserved regions are four E-boxes, bHLH consensus binding sites (blue boxes) (Murre et al, 1989;Hutcheson et al, 2005). For Math1, Helms et al (2000) demonstrated that two enhancers (A and B, located 3 Kb downstream) activate Math1 expression (green boxes in Figs.…”

“…However, the cis-regulatory elements regulating Math5 expression are not yet well defined. In Xenopus, Ath5 expression (Xath5) is regulated by upstream bHLH-dependent and independent elements (Hutcheson et al, 2005). Both proximal bHLH-specific binding sites and more distal cis-regulatory Xath5 sequences each independently drive transgenic expression of a GFP reporter in the Xenopus retina.…”

“…Both proximal bHLH-specific binding sites and more distal cis-regulatory Xath5 sequences each independently drive transgenic expression of a GFP reporter in the Xenopus retina. Importantly, a phylogenetically conserved 5′ distal element is required for retinal expression in the mouse retina and sufficient to drive retinal expression in the frog eye (Hutcheson et al, 2005;Riesenberg et al, 2007).…”