Highly Conserved Sequences Mediate the Dynamic Interplay of Basic Helix-Loop-Helix Proteins Regulating Retinogenesis

Abstract

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.Retina development in vertebrates relies on regulatory proteins, most of which are widely expressed in the developing nervous system. However, the expression of the basic helixloop-helix protein atonal homolog 5 (ATH5) 3 appears to be restricted to retina ontogenesis. ATH5 activates neurogenesis and is required for the production of retinal ganglion cells (RGCs) (1-4). It is transiently expressed during the period of development when RGCs are produced and underlies the pathway leading to the conversion of proliferating progenitors into newborn RGCs (5, 6). Retinotopic differences in the timing of RGC production may reflect the wave-like expression of ATH5 (5, 7). In zebrafish, a signal from the optic stalk appears to induce the first patch of ATH5-expressing cells, and the continued spread of ATH5 expression beyond that patch may require a cascade of cell-to-cell interactions (7,8). It has been suggested that Sonic hedgehog derived from newborn RGCs drives a self-propagating wave of ATH5 expression and RGC production across the zebrafish retina (9, 10). In contrast, related experiments in zebrafish and chick highlight the importance of intrinsic factors for triggering ATH5 expression and neurogenesis (11-13).The presence of consensus E-box binding sites in the highly conserved upstream sequence of the ATH5 gene suggests that bHLH transcription factors are directly involved in the regulation of ATH5. This idea is supported by experiments showing the selective binding of neuronal bHLH proteins to the upstream sequence of ATH5 and the simultaneous changes in the expression level of ATH5 in response to several of these proteins (5, 6, 14 -16). These findings indicate a requirement for different combinations of bHLH proteins in regulating the different phases of ATH5 expression...

Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6

“…We cannot exclude that a number of bHLH might compete to occupy the conserved Six6 E-box and control different phases of Six6 expression, as reported for Ath5 regulation in RGCs (Hernandez et al, 2007). Nevertheless, our results support a highly specific role of the NeuroD-E47 heterodimer in the binding and transactivation of the E-box in the Six6 enhancer.…”

“…Development 137 (14) Different subfamilies of bHLH proteins can form heterodimers and recognize the same canonical E-box sequence (Hernandez et al, 2007;Powell and Jarman, 2008). We cannot exclude that a number of bHLH might compete to occupy the conserved Six6 E-box and control different phases of Six6 expression, as reported for Ath5 regulation in RGCs (Hernandez et al, 2007).…”

Delayed neurogenesis with respect to eye growth shapes the pigeon retina for high visual acuity

“…Despite the fact that Hes5.3 expression is detected in the whole subset of Atoh7-expressing progenitor cells, only 25-30% of them upregulate Atoh7 and enter this lineage in chick between E3 and E5 (Chiodini et al, 2013;Matter-Sadzinski et al, 2005). This low efficiency of RGC production at early stages is attributed to the negative effect of HES1 on Atoh7 cis-acting regulatory elements (Hernandez et al, 2007), hence keeping transcription of the Atoh7 gene at a low level in progenitor cells. Thus, we could expect that, at the onset of Atoh7 expression in pigeon, a larger subset of cells express Atoh7 at high levels.…”

“…HES1 is expressed in the developing retina and it represses Atoh7 and Ngn2 expression (Hernandez et al, 2007;Lee et al, 2005;Matter-Sadzinski et al, 2005). We examined whether prolonged expression of HES1 could account for delaying neuronal differentiation in pigeon.…”

Conserved regulatory sequences inAtoh7mediate non-conserved regulatory responses in retina ontogenesis

“…Whereas site occupancy and functional analyses have shown that the Ngn2 (Neurog2) and Atoh7 basic helix-loop-helix (bHLH) proteins are required for transcription of the chicken Atoh7 gene (Hernandez et al, 2007;Matter-Sadzinski et al, 2001;Matter-Sadzinski et al, 2005;Skowronska-Krawczyk et al, 2004), the late onset of Ngn2 expression and the continued expression of the mutated Atoh7 gene in suggested that these factors are not involved in the regulation of Atoh7 in mouse (Brown et al, 1998;Brown et al, 2001;Hutcheson et al, 2005;Wang et al, 2001). The differences between mouse and chicken in the regulation of Atoh7 are used in the present study to compare the function of evolutionarily conserved regulatory sequences that include several bHLH binding sites.…”