In the developing retina, the production of ganglion cells is dependent on the proneural proteins NGN2 and ATH5, whose activities define stages along the pathway converting progenitors into newborn neurons. Crossregulatory interactions between NGN2, ATH5 and HES1 maintain the uncommitted status of ATH5-expressing cells during progenitor patterning, and later on regulate the transition from competence to cell fate commitment. Prior to exiting the cell cycle, a subset of progenitors is selected from the pool of ATH5-expressing cells to go through a crucial step in the acquisition of a definitive retinal ganglion cell fate. The selected cells are those in which the upregulation of NGN2, the downregulation of HES1 and the autostimulation of ATH5 are coordinated with the progression of progenitors through the last cell cycle. This coordinated pattern initiates the transcription of ganglion cell-specific traits and determines the size of the ganglion cell population.

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

Expression of the neuronal non‐alpha nicotinic acetylcholine receptor (n alpha nAChR) gene is transiently stimulated in the chick optic tectum between embryonic days 7 and 16 with a peak value reached around embryonic day 12. This stimulation takes place at the time when optic nerve axons are invading this region of the brain and proceeds along a rostral to caudal gradient. Transcripts of the n alpha nAChR gene are localized in the superficial layers of the tectum at the time when cells in these layers are forming synapses with retina axons. The transient expression of n alpha nAChR gene does not take place in the optic tectum of ‘eyeless’ embryos. The results of our study suggest that the neuronal n alpha nAChR gene may play a role in neurogenesis of retino‐tectal connections.

The HES proteins are known Notch effectors and have long been recognized as important in inhibiting neuronal differentiation. However, the roles that they play in the specification of neuronal fate remain largely unknown. Here, we show that in the differentiating retinal epithelium, the proneural protein ATOH7 (ATH5) is required for the activation of the transcription of the Hes5.3 gene before the penultimate mitosis of progenitor cells. We further show that the HES5.3 protein slows down the cell-cycle progression of Atoh7-expressing cells, thereby establishing conditions for Atoh7 to reach a high level of expression in S phase and induce neuronal differentiation prior to the ultimate mitosis. Our study uncovers how a proneural protein recruits a protein known to be a component of the Notch signaling pathway in order to regulate the transition between an initial phase of selection among uncommitted progenitors and a later phase committing the selected progenitors to neuronal differentiation.

A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the a7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which C7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that c7 regulatory elements acquire their neuronal specificity in the course of embryogenesis.

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...

We have previously shown that transcription of the beta 3 nicotinic receptor gene within the chick CNS is regulated by a promoter 143 base pairs (bp) in length. Here, we demonstrate that in the developing visual system this promoter is active in a subset of retinal cells, the majority of which are ganglion cells. Because the beta 3 promoter is activated very early during retina development, it can provide a marker of ganglion cell induction and differentiation. Transfection of neuroretina explants enabled us to detect activity of the beta 3 promoter in premigratory cells localized on the ventricular side of the retina. Double-labeling experiments showed that activation of the beta 3 promoter takes place before the last S-phase, suggesting that this particular phenotypic trait is determined when precursor cells are still proliferating. The beta 3 phenotype is induced in about one-tenth of the total pool of retinal progenitor cells and is stable upon changing the cellular environment. Our study suggests that at the very early stages of retina neurogenesis, some lineage restrictions have already occurred in the population of retinal progenitor cells.

The macula and fovea located at the optical centre of the retina make primate visual perception unique among mammals. Our current understanding of retina ontogenesis is primarily based on animal models having no macula and no fovea. However, the pigeon retina and the human macula share a number of structural and functional properties that justify introducing the former as a new model system for retina development. Comparative transcriptome analysis of pigeon and chicken retinas at different embryonic stages reveals that the genetic programmes underlying cell differentiation are postponed in the pigeon until the end of the period of cell proliferation. We show that the late onset of neurogenesis has a profound effect on the developmental patterning of the pigeon retina, which is at odds with the current models of retina development. The uncoupling of tissue growth and neurogenesis is shown to result from the fact that the pigeon retinal epithelium is inhibitory to cell differentiation. The sum of these developmental features allows the pigeon to build a retina that displays the structural and functional traits typical of primate macula and fovea.