Rbpj direct regulation of Atoh7 transcription in the embryonic mouse retina.

In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.

Separate and coincident expression of Hes1 and Hes5 in the developing mouse eye.

BACKGROUND: Notch signaling is broadly required during embryogenesis, frequently activating the transcription of two basic helix-loop-helix transcription factors, Hes1 and Hes5. But, it remains unresolved when and where Hes1 and Hes5 act alone or together during development. Here, we analyzed a Hes5-green fluorescent protein (GFP) bacterial artificial chromosome (BAC) transgenic mouse, as a proxy for endogenous Hes5. We directly compared transgenic GFP expression with Hes1, and particular markers of embryonic lens and retina development. RESULTS: Hes5-GFP is dynamic within subsets of retinal and lens progenitor cells, and differentiating retinal ganglion neurons, in contrast to Hes1 found in all progenitor cells. In the adult retina, only Müller glia express Hes5-GFP. Finally, Hes5-GFP is up-regulated in Hes1 germline mutants, consistent with previous demonstration that Hes1 suppresses Hes5 transcription. CONCLUSIONS: Hes5-GFP BAC transgenic mice are useful for identifying Hes5-expressing cells. Although Hes5-GFP and Hes1 are coexpressed in particular developmental contexts, we also noted cohorts of lens or retinal cells expressing just one factor. The dynamic Hes5-GFP expression pattern, coupled with its derepressed expression in Hes1 mutants, suggests that this transgene contains the relevant cis-regulatory elements that regulate endogenous Hes5 in the mouse lens and retina. Developmental Dynamics 247:212-221, 2018. © 2017 Wiley Periodicals, Inc.

Cell autonomous and nonautonomous requirements for Delltalike1 during early mouse retinal neurogenesis.

BACKGROUND: In the vertebrate retina, six neuronal and one glial cell class are produced from a common progenitor pool. During neurogenesis, adjacent retinal cells use Notch signaling to maintain a pool of progenitors by blocking particular cells from differentiating prematurely. In mice there are multiple Notch pathway ligands and receptors, but the role(s) of each paralogue during retinal histogenesis remains only partially defined. RESULTS: Here we analyzed the cell autonomous and nonautonomous requirements for the Deltalike1(Dll1) ligand during prenatal retinogenesis. We used the α-Cre driver to simultaneously delete a Dll1 conditional allele and activate the Z/EG reporter, then quantified Dll1 mutant phenotypes within and outside of this α-Cre GFP-marked lineage. We found that Dll1 activity is required for Hes1 expression, both autonomously and nonautonomously, but were surprised that retinal ganglion cell differentiation is only blocked cell autonomously. Moreover, Dll1 does not act during cone photoreceptor neurogenesis. Finally, Dll1 mutant adult retinas contained small retinal rosettes and RGC patterning defects but were otherwise normal. CONCLUSIONS: Although Dll1 participates in bidirectional (cis + trans) Notch signaling to regulate Hes1 expression, it only acts cell autonomously (in cis) to interpret inhibitory signals from other cells that block RGC neurogenesis. Developmental Dynamics 245:631-640, 2016. © 2016 Wiley Periodicals, Inc.

Notch signaling differentially regulates Atoh7 and Neurog2 in the distal mouse retina.

Notch signaling regulates basic helix-loop-helix (bHLH) factors as an evolutionarily conserved module, but the tissue-specific mechanisms are incompletely elucidated. In the mouse retina, bHLH genes Atoh7 and Neurog2 have distinct functions, with Atoh7 regulating retinal competence and Neurog2 required for progression of neurogenesis. These transcription factors are extensively co-expressed, suggesting similar regulation. We directly compared Atoh7 and Neurog2 regulation at the earliest stages of retinal neurogenesis in a broad spectrum of Notch pathway mutants. Notch1 and Rbpj normally block Atoh7 and Neurog2 expression. However, the combined activities of Notch1, Notch3 and Rbpj regulate Neurog2 patterning in the distal retina. Downstream of the Notch complex, we found the Hes1 repressor mediates Atoh7 suppression, but Hes1, Hes3 and Hes5 do not regulate Neurog2 expression. We also tested Notch-mediated regulation of Jag1 and Pax6 in the distal retina, to establish the appropriate context for Neurog2 patterning. We found that Notch1;Notch3 and Rbpj block co-expression of Jag1 and Neurog2, while specifically stimulating Pax6 within an adjacent domain. Our data suggest that Notch signaling controls the overall tempo of retinogenesis, by integrating cell fate specification, the wave of neurogenesis and the developmental status of cells ahead of this wave.

Heterochronic misexpression of Ascl1 in the Atoh7 retinal cell lineage blocks cell cycle exit.

Retinal neurons and glia arise from a common progenitor pool in a temporal order, with retinal ganglion cells (RGCs) appearing first, and Müller glia last. The transcription factors Atoh7/Math5 and Ascl1/Mash1 represent divergent bHLH clades, and exhibit distinct spatial and temporal retinal expression patterns, with little overlap during early development. Here, we tested the ability of Ascl1 to change the fate of cells in the Atoh7 lineage when misexpressed from the Atoh7 locus, using an Ascl1-IRES-DsRed2 knock-in allele. In Atoh7(Ascl1KI/+) and Atoh7(Ascl1KI/Ascl1KI) embryos, ectopic Ascl1 delayed cell cycle exit and differentiation, even in cells coexpressing Atoh7. The heterozygous retinas recovered, and eventually produced a normal complement of RGCs, while homozygous substitution of Ascl1 for Atoh7 did not promote postnatal retinal fates precociously, nor rescue Atoh7 mutant phenotypes. However, our analyses revealed two unexpected findings. First, ectopic Ascl1 disrupted cell cycle progression within the marked Atoh7 lineage, but also nonautonomously in other retinal cells. Second, the size of the Atoh7 retinal lineage was unaffected, supporting the idea of a compensatory shift of the non-proliferative cohort to maintain lineage size. Overall, we conclude that Ascl1 acts dominantly to block cell cycle exit, but is incapable of redirecting the fates of early RPCs.

Rbpj cell autonomous regulation of retinal ganglion cell and cone photoreceptor fates in the mouse retina.

Vertebrate retinal progenitor cells (RPCs) are pluripotent, but pass through competence states that progressively restrict their developmental potential (Cepko et al., 1996; Livesey and Cepko, 2001; Cayouette et al., 2006). In the rodent eye, seven retinal cell classes differentiate in overlapping waves, with RGCs, cone photoreceptors, horizontals, and amacrines forming predominantly before birth, and rod photoreceptors, bipolars, and Müller glia differentiating postnatally. Both intrinsic and extrinsic factors regulate each retinal cell type (for review, see Livesey and Cepko, 2001). Here, we conditionally deleted the transcription factor Rbpj, a critical integrator of multiple Notch signals (Jarriault et al., 1995; Honjo, 1996; Kato et al., 1997; Han et al., 2002), during prenatal mouse retinal neurogenesis. Removal of Rbpj caused reduced proliferation, premature neuronal differentiation, apoptosis, and profound mispatterning. To determine the cell autonomous requirements for Rbpj during RGC and cone formation, we marked Cre-generated retinal lineages with GFP expression, which showed that Rbpj autonomously promotes RPC mitotic activity, and suppresses RGC and cone fates. In addition, the progressive loss of Rbpj-/- RPCs resulted in a diminished progenitor pool available for rod photoreceptor formation. This circumstance, along with the overproduction of Rbpj-/- cones, revealed that photoreceptor development is under homeostatic regulation. Finally, to understand how the Notch pathway regulates the simultaneous formation of multiple cell types, we compared the RGC and cone phenotypes of Rbpj to Notch1 (Jadhav et al., 2006b; Yaron et al., 2006), Notch3, and Hes1 mutants. We found particular combinations of Notch pathway genes regulate the development of each retinal cell type.

Pax6 regulation of Math5 during mouse retinal neurogenesis.

Activation of the bHLH factor Math5 (Atoh7) is an initiating event for mammalian retinal neurogenesis, as it is critically required for retinal ganglion cell formation. However, the cis-regulatory elements and trans-acting factors that control Math5 expression are largely unknown. Using a combination of transgenic mice and bioinformatics, we identified a phylogenetically conserved regulatory element that is required to activate Math5 transcription during early retinal neurogenesis. This element drives retinal expression in vivo, in a cross-species transgenic assay. Previously, Pax6 was shown to be necessary for the initiation of Math5 mRNA expression. We extend this finding by showing that the Math5 retinal enhancer also requires Pax6 for its activation, via Pax6 binding to a highly conserved binding site. In addition, our data reveal that other retinal factors are required for accurate regulation of Math5 by Pax6.

Characterization of a transient TCF/LEF-responsive progenitor population in the embryonic mouse retina.

PURPOSE: High mobility group (HMG) transcription factors of the T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) family are a class of intrinsic regulators that are dynamically expressed in the embryonic mouse retina. Activation of TCF/LEFs is a hallmark of the Wnt/beta-catenin pathway; however, the requirement for Wnt/beta-catenin and noncanonical Wnt signaling during mammalian retinal development remains unclear. The goal of the study was to characterize more fully a TCF/LEF-responsive retinal progenitor population in the mouse embryo and to correlate this with Wnt/beta-catenin signaling. METHODS: TCF/LEF activation was analyzed in the TOPgal (TCF optimal promoter) reporter mouse at embryonic ages and compared to Axin2 mRNA expression, an endogenous readout of Wnt/beta-catenin signaling. Reporter expression was also examined in embryos with a retina-specific deletion of the beta-catenin gene (Ctnnb1), using Six3-Cre transgenic mice. Finally, the extent to which TOPgal cells coexpress cell cycle proteins, basic helix-loop-helix (bHLH) transcription factors, and other retinal cell markers was tested by double immunohistochemistry. RESULTS: TOPgal reporter activation occurred transiently in a subpopulation of embryonic retinal progenitor cells. Axin2 was not expressed in the central retina, and TOPgal reporter expression persisted in the absence of beta-catenin. Although a proportion of TOPgal-labeled cells were proliferative, most coexpressed the cyclin-dependent kinase inhibitor p27/Kip1. CONCLUSIONS: TOPgal cells give rise to the four earliest cell types: ganglion, amacrine, horizontal, and photoreceptor. TCF/LEF activation in the central retina does not correlate with Wnt/beta-catenin signaling, pointing to an alternate role for this transcription factor family during retinal development.

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

Retinal ganglion cell genesis requires the proneural bHLH transcription factor Math5 (Atoh7), but little is known about the regulatory elements that control its expression. Here, we investigate Math5 gene regulation using transgenic mice. These mice express GFP in the prenatal retina, live-labeling RGC axon migration and innervation of the brain. Unexpectedly, these Math5-GFP transgenes are also found in Math1 expression domains throughout the nervous system, intriguing since Math5 and Math1 normally exhibit nonoverlapping expression. Furthermore, Math5-GFP and Math1 are regulated similarly, by both Pax6 and Math1 itself, in the lower rhombic lip and dorsal spinal cord. We also show that Pax6 binds to particular Math5 and Math1 regulatory sequences in vitro. Together these data suggest that these atonal semi-orthologues may share conserved regulatory elements that are normally silent in the Math5 gene.

Math5 is required for both early retinal neuron differentiation and cell cycle progression.

CNS progenitors choose a fate, exit mitosis and differentiate. Basic helix-loop-helix (bHLH) transcription factors are key regulators of neurogenesis, but their molecular mechanisms remain unclear. In the mouse retina, removal of the bHLH factor Math5 (Atoh7) causes the loss of retinal ganglion cells (RGCs) and appearance of excess cone photoreceptors. Here, we show a simultaneous requirement for Math5 in retinal neuron formation and cell cycle progression. At embryonic day E11.5, Math5-/- cells are unable to assume the earliest fates, particularly that of an RGC, and instead adopt the last fate as Müller glia. Concurrently, the loss of Math5 causes mitotically active retinal progenitors to undergo aberrant cell cycles. The drastic fate shift of Math5-/- cells correlates with age-specific alterations in p27/Kip1 expression and an inability to become fully postmitotic. Finally, Math5 normally suppresses NeuroD1 within Math5-expressing cells and inhibits Ngn2 expression and cone photoreceptor genesis within separate cell populations. Thus, Math5 orchestrates neurogenesis in multiple ways, regulating both intrinsic and extrinsic processes.