Increased neuron density in the midbrain of a foveate bird, pigeon, results from profound change in tissue morphogenesis.

The increase of brain neuron number in relation with brain size is currently considered to be the major evolutionary path to high cognitive power in amniotes. However, how changes in neuron density did contribute to the evolution of the information-processing capacity of the brain remains unanswered. High neuron densities are seen as the main reason why the fovea located at the visual center of the retina is responsible for sharp vision in birds and primates. The emergence of foveal vision is considered as a breakthrough innovation in visual system evolution. We found that neuron densities in the largest visual center of the midbrain - i.e., the optic tectum - are two to four times higher in modern birds with one or two foveae compared to birds deprived of this specialty. Interspecies comparisons enabled us to identify elements of a hitherto unknown developmental process set up by foveate birds for increasing neuron density in the upper layers of their optic tectum. The late progenitor cells that generate these neurons proliferate in a ventricular zone that can expand only radially. In this particular context, the number of cells in ontogenetic columns increases, thereby setting the conditions for higher cell densities in the upper layers once neurons did migrate.

A transient decrease in mitochondrial activity contributes to establish the ganglion cell fate in retina adapted for high acuity vision.

Although the plan of the retina is well conserved in vertebrates, there are considerable variations in cell type diversity and number, as well as in the organization and properties of the tissue. The high ratios of retinal ganglion cells (RGCs) to cones in primate fovea and bird retinas favor neural circuits essential for high visual acuity and color vision. The role that cell metabolism could play in cell fate decision during embryonic development of the nervous system is still largely unknown. Here, we describe how subtle changes of mitochondrial activity along the pathway converting uncommitted progenitors into newborn RGCs increase the recruitment of RGC-fated progenitors. ATOH7, a proneural protein dedicated to the production of RGCs in vertebrates, activates transcription of the Hes5.3 gene in pre-committed progenitors. The HES5.3 protein, in turn, regulates a transient decrease in mitochondrial activity via the retinoic acid signaling pathway few hours before cell commitment. This metabolic shift lengthens the progression of the ultimate cell cycle and is a necessary step for upregulating Atoh7 and promoting RGC differentiation.

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

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.

Conserved regulatory sequences in Atoh7 mediate non-conserved regulatory responses in retina ontogenesis.

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.

The basic domain of ATH5 mediates neuron-specific promoter activity during retina development.

In the developing retina, the gene encoding the beta3 subunit of the neuronal nicotinic receptor, a specific marker of retinal ganglion cells, is under the direct control of the atonal homolog 5 (ATH5) basic helix-loop-helix (bHLH) transcription factor. Although quite short (143 bp in length), the beta3 promoter has the remarkable capacity to discriminate between ATH5 and the other neuronal bHLH proteins expressed in the developing nervous system. We have identified three amino acids within the basic domain that confer specificity to the ATH5 protein. These residues do not mediate direct DNA binding but are required for interaction between ATH5 and chromatin-associated proteins during retina development. When misexpressed in neurons, the myogenic bHLH factor MyoD is also able to activate the beta3 gene. This, however, is achieved not by binding of the protein to the promoter but by dimerization of MyoD with a partner, a process that depends not on the basic domain but on the HLH domain. By sequestering an E-box-binding protein, MyoD relieves the active repression that blocks the beta3 promoter in most neurons. The mechanisms used by bHLH proteins to activate beta3 thus highlight how ATH5 is selected by the beta3 promoter and coordinates the derepression and transcriptional activation of the beta3 gene during the specification of retinal ganglion cells.

A positive feedback loop between ATOH7 and a Notch effector regulates cell-cycle progression and neurogenesis in the retina.

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.

Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating retinogenesis.

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.

A bHLH transcriptional network regulating the specification of retinal ganglion cells.

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.