Notch pathway mutants do not equivalently perturb mouse embryonic retinal development.

In the vertebrate eye, Notch ligands, receptors, and ternary complex components determine the destiny of retinal progenitor cells in part by regulating Hes effector gene activity. There are multiple paralogues for nearly every node in this pathway, which results in numerous instances of redundancy and compensation during development. To dissect such complexity at the earliest stages of eye development, we used seven germline or conditional mutant mice and two spatiotemporally distinct Cre drivers. We perturbed the Notch ternary complex and multiple Hes genes to understand if Notch regulates optic stalk/nerve head development; and to test intracellular pathway components for their Notch-dependent versus -independent roles during retinal ganglion cell and cone photoreceptor competence and fate acquisition. We confirmed that disrupting Notch signaling universally blocks progenitor cell growth, but delineated specific pathway components that can act independently, such as sustained Hes1 expression in the optic stalk/nerve head. In retinal progenitor cells, we found that among the genes tested, they do not uniformly suppress retinal ganglion cell or cone differentiation; which is not due differences in developmental timing. We discovered that shifts in the earliest cell fates correlate with expression changes for the early photoreceptor factor Otx2, but not with Atoh7, a factor required for retinal ganglion cell formation. During photoreceptor genesis we also better defined multiple and simultaneous activities for Rbpj and Hes1 and identify redundant activities that occur downstream of Notch. Given its unique roles at the retina-optic stalk boundary and cone photoreceptor genesis, our data suggest Hes1 as a hub where Notch-dependent and -independent inputs converge.

Notch directs telencephalic development and controls neocortical neuron fate determination by regulating microRNA levels.

The central nervous system contains a myriad of different cell types produced from multipotent neural progenitors. Neural progenitors acquire distinct cell identities depending on their spatial position, but they are also influenced by temporal cues to give rise to different cell populations over time. For instance, the progenitors of the cerebral neocortex generate different populations of excitatory projection neurons following a well-known sequence. The Notch signaling pathway plays crucial roles during this process, but the molecular mechanisms by which Notch impacts progenitor fate decisions have not been fully resolved. Here, we show that Notch signaling is essential for neocortical and hippocampal morphogenesis, and for the development of the corpus callosum and choroid plexus. Our data also indicate that, in the neocortex, Notch controls projection neuron fate determination through the regulation of two microRNA clusters that include let-7, miR-99a/100 and miR-125b. Our findings collectively suggest that balanced Notch signaling is crucial for telencephalic development and that the interplay between Notch and miRNAs is essential for the control of neocortical progenitor behaviors and neuron cell fate decisions.

Not all Notch pathway mutations are equal in the embryonic mouse retina.

In the vertebrate retina, combinations of Notch ligands, receptors, and ternary complex components determine the destiny of retinal progenitor cells by regulating Hes effector gene activity. Owing to reiterated Notch signaling in numerous tissues throughout development, there are multiple vertebrate paralogues for nearly every node in this pathway. These Notch signaling components can act redundantly or in a compensatory fashion during development. To dissect the complexity of this pathway during retinal development, we used seven germline or conditional mutant mice and two spatiotemporally distinct Cre drivers. We perturbed the Notch ternary complex and multiple Hes genes with two overt goals in mind. First, we wished to determine if Notch signaling is required in the optic stalk/nerve head for Hes1 sustained expression and activity. Second, we aimed to test if Hes1, 3 and 5 genes are functionally redundant during early retinal histogenesis. With our allelic series, we found that disrupting Notch signaling consistently blocked mitotic growth and overproduced ganglion cells, but we also identified two significant branchpoints for this pathway. In the optic stalk/nerve head, sustained Hes1 is regulated independent of Notch signaling, whereas during photoreceptor genesis both Notch-dependent and -independent roles for Rbpj and Hes1 impact photoreceptor genesis in opposing manners.

Multiple roles for Pax2 in the embryonic mouse eye.

The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2GFP/+ knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.

The Atoh7 remote enhancer provides transcriptional robustness during retinal ganglion cell development.

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

Simultaneous Requirements for Hes1 in Retinal Neurogenesis and Optic Cup-Stalk Boundary Maintenance.

The bHLH transcription factor Hes1 is a key downstream effector for the Notch signaling pathway. During embryogenesis neural progenitors express low levels of Hes1 in an oscillating pattern, whereas glial brain boundary regions (e.g., isthmus) have high, sustained Hes1 levels that suppress neuronal fates. Here, we show that in the embryonic mouse retina, the optic nerve head and stalk express high Hes1, with the ONH constituting a boundary between the neural retina and glial cells that ultimately line the optic stalk. Using two Cre drivers with distinct spatiotemporal expression we conditionally inactivated Hes1, to delineate the requirements for this transcriptional repressor during retinal neurogenesis versus patterning of the optic cup and stalk. Throughout retinal neurogenesis, Hes1 maintains proliferation and blocks retinal ganglion cell formation, but surprisingly we found it also promotes cone photoreceptor genesis. In the postnatal eye, Hes1 inactivation with Rax-Cre resulted in increased bipolar neurons and a mispositioning of Müller glia. Our results indicate that Notch pathway regulation of cone genesis is more complex than previously assumed, and reveal a novel role for Hes1 in maintaining the optic cup-stalk boundary.SIGNIFICANCE STATEMENT The bHLH repressor Hes1 regulates the timing of neurogenesis, rate of progenitor cell division, gliogenesis, and maintains tissue compartment boundaries. This study expands current eye development models by showing Notch-independent roles for Hes1 in the developing optic nerve head (ONH). Defects in ONH formation result in optic nerve coloboma; our work now inserts Hes1 into the genetic hierarchy regulating optic fissure closure. Given that Hes1 acts analogously in the ONH as the brain isthmus, it prompts future investigation of the ONH as a signaling factor center, or local organizer. Embryonic development of the ONH region has been poorly studied, which is surprising given it is where the pan-ocular disease glaucoma is widely believed to inflict damage on RGC axons.

Jagged1 protein processing in the developing mammalian lens.

Notch signaling regulates a multitude of cellular processes. During ocular lens development this pathway is required for lens progenitor growth, differentiation and maintenance of the transition zone. After ligand-receptor binding, the receptor proteins are processed, first by ADAM proteases, then by γ-secretase cleavage. This results in the release of a Notch intracellular domain (N-ICD), which is recruited into a nuclear transcription factor complex that activates Notch target genes. Previous in vitro studies showed that the Delta-like and Jagged ligand proteins can also be cleaved by the γ-secretase complex, but it remains unknown whether such processing occurs during in vivo vertebrate development. Here we show that mouse and human lens progenitor cells endogenously express multiple Jagged1 protein isoforms, including a Jagged1 intracellular domain. We also found that pharmacologic blockage of γ-secretase activity in vitro resulted in an accumulation of Jagged1 polypeptide intermediates. Finally, overexpression of an epitope-tagged Jagged1 intracellular domain displayed nuclear localization and induced the upregulation of endogenous JAG1 mRNA expression. These findings support the idea that along with its classical role as a Notch pathway ligand, Jagged1 is regulated post-translationally, to produce multiple active protein isoforms.

Eye organogenesis: A hierarchical view of ocular development.

This chapter provides an overview of the early developmental origins of six ocular tissues: the cornea, lens, ciliary body, iris, neural retina, and retina pigment epithelium. Many of these tissue types are concurrently specified and undergo a complex set of morphogenetic movements that facilitate their structural interconnection. Within the context of vertebrate eye organogenesis, we also discuss the genetic hierarchies of transcription factors and signaling pathways that regulate growth, patterning, cell type specification and differentiation.

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens.

BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

Presenilin gene function and Notch signaling feedback regulation in the developing mouse lens.

Presenilins (Psen1 and Psen2 in mice) are polytopic transmembrane proteins that act in the γ-secretase complex to make intra-membrane cleavages of their substrates, including the well-studied Notch receptors. Such processing releases the Notch intracellular domain, allowing it to physically relocate from the cell membrane to the nucleus where it acts in a transcriptional activating complex to regulate downstream genes in the signal-receiving cell. Previous studies of Notch pathway mutants for Jagged1, Notch2, and Rbpj demonstrated that canonical signaling is a necessary component of normal mouse lens development. However, the central role of Psens within the γ-secretase complex has never been explored in any developing eye tissue or cell type. By directly comparing Psen single and double mutant phenotypes during mouse lens development, we found a stronger requirement for Psen1, although both genes are needed for progenitor cell growth and to prevent apoptosis. We also uncovered a novel genetic interaction between Psen1 and Jagged1. By quantifying protein and mRNA levels of key Notch pathway genes in Psen1/2 or Jagged1 mutant lenses, we identified multiple points in the overall signaling cascade where feedback regulation can occur. Our data are consistent with the loss of particular genes indirectly influencing the transcription level of another. However, we conclude that regulating Notch2 protein levels is particularly important during normal signaling, supporting the importance of post-translational regulatory mechanisms in this tissue.

Requirements for Neurogenin2 during mouse postnatal retinal neurogenesis.

During embryonic retinal development, the bHLH factor Neurog2 regulates the temporal progression of neurogenesis, but no role has been assigned for this gene in the postnatal retina. Using Neurog2 conditional mutants, we found that Neurog2 is necessary for the development of an early, embryonic cohort of rod photoreceptors, but also required by both a subset of cone bipolar subtypes, and rod bipolars. Using transcriptomics, we identified a subset of downregulated genes in P2 Neurog2 mutants, which act during rod differentiation, outer segment morphogenesis or visual processing. We also uncovered defects in neuronal cell culling, which suggests that the rod and bipolar cell phenotypes may arise via more complex mechanisms rather than a simple cell fate shift. However, given an overall phenotypic resemblance between Neurog2 and Blimp1 mutants, we explored the relationship between these two factors. We found that Blimp1 is downregulated between E12-birth in Neurog2 mutants, which probably reflects a dependence on Neurog2 in embryonic progenitor cells. Overall, we conclude that the Neurog2 gene is expressed and active prior to birth, but also exerts an influence on postnatal retinal neuron differentiation.

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.

All in the family: proneural bHLH genes and neuronal diversity.

Proneural basic Helix-Loop-Helix (bHLH) proteins are required for neuronal determination and the differentiation of most neural precursor cells. These transcription factors are expressed in vastly divergent organisms, ranging from sponges to primates. Here, we review proneural bHLH gene evolution and function in the Drosophila and vertebrate nervous systems, arguing that the Drosophila gene atonal provides a useful platform for understanding proneural gene structure and regulation. We also discuss how functional equivalency experiments using distinct proneural genes can reveal how proneural gene duplication and divergence are interwoven with neuronal complexity.

Integral bHLH factor regulation of cell cycle exit and RGC differentiation.

BACKGROUND: In the developing mouse embryo, the bHLH transcription factor Neurog2 is transiently expressed by retinal progenitor cells and required for the initial wave of neurogenesis. Remarkably, another bHLH factor, Ascl1, normally not present in the embryonic Neurog2 retinal lineage, can rescue the temporal phenotypes of Neurog2 mutants. RESULTS: Here we show that Neurog2 simultaneously promotes terminal cell cycle exit and retinal ganglion cell differentiation, using mitotic window labeling and integrating these results with retinal marker quantifications. We also analyzed the transcriptomes of E12.5 GFP-expressing cells from Neurog2GFP/+ , Neurog2GFP/GFP , and Neurog2Ascl1KI/GFP eyes, and validated the most significantly affected genes using qPCR assays. CONCLUSIONS: Our data support the hypothesis that Neurog2 acts at the top of a retinal bHLH transcription factor hierarchy. The combined expression levels of these downstream factors are sufficiently induced by ectopic Ascl1 to restore RGC genesis, highlighting the robustness of this gene network during retinal ganglion cell neurogenesis. Developmental Dynamics 247:965-975, 2018. © 2018 Wiley Periodicals, Inc.

The dynamics of native Atoh7 protein expression during mouse retinal histogenesis, revealed with a new antibody.

The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.

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.

Regulation of Brn3b by DLX1 and DLX2 is required for retinal ganglion cell differentiation in the vertebrate retina.

Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.

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.