Nadean L. Brown

Genome-wide association identifies ATOH7 as a major gene determining human optic disc size

Optic nerve assessment is important for many blinding diseases, with cup-to-disc ratio (CDR) assessments commonly used in both diagnosis and progression monitoring of glaucoma patients. Optic disc, cup, rim area and CDR measurements all show substantial variation between human populations and high heritability estimates within populations. To identify loci underlying these quantitative traits, we performed a genome-wide association study in two Australian twin cohorts and identified rs3858145, P = 6.2 × 10−10, near the ATOH7 gene as associated with the mean disc area. ATOH7 is known from studies in model organisms to play a key role in retinal ganglion cell formation. The association with rs3858145 was replicated in a cohort of UK twins, with a meta-analysis of the combined data yielding P = 3.4 × 10−10. Imputation further increased the evidence for association for several SNPs in and around ATOH7 (P = 1.3 × 10−10 to 4.3 × 10−11, top SNP rs1900004). The meta-analysis also provided suggestive evidence for association for the cup area at rs690037, P = 1.5 × 10−7, in the gene RFTN1. Direct sequencing of ATOH7 in 12 patients with optic nerve hypoplasia, one of the leading causes of blindness in children, revealed two novel non-synonymous mutations (Arg65Gly, Ala47Thr) which were not found in 90 unrelated controls (combined Fishers exact P = 0.0136). Furthermore, the Arg65Gly variant was found to have very low frequency (0.00066) in an additional set of 672 controls.

Notch signaling regulates growth and differentiation in the mammalian lens.

The Notch signal transduction pathway regulates the decision to proliferate versus differentiate. Although there are a myriad of mouse models for the Notch pathway, surprisingly little is known about how these genes regulate early eye development, particularly in the anterior lens. We employed both gain-of-function and loss-of-function approaches to determine the role of Notch signaling in lens development. Here we analyzed mice containing conditional deletion of the Notch effector Rbpj or overexpression of the activated Notch1 intracellular domain during lens formation. We demonstrate distinct functions for Notch signaling in progenitor cell growth, fiber cell differentiation and maintenance of the transition zone. In particular, Notch signaling controls the timing of primary fiber cell differentiation and is essential for secondary fiber cell differentiation. Either gain or loss of Notch signaling leads to formation of a dysgenic lens, which in loss-of-function mice undergoes a profound postnatal degeneration. Our data suggest both Cyclin D1 and Cyclin D2, and the p27(Kip1) cyclin-dependent kinase inhibitor act downstream of Notch signaling, and define multiple critical functions for this pathway during lens development.

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.

Neurog2 controls the leading edge of neurogenesis in the mammalian retina.

In the mammalian retina, neuronal differentiation begins in the dorso-central optic cup and sweeps peripherally and ventrally. While certain extrinsic factors have been implicated, little is known about the intrinsic factors that direct this process. In this study, we evaluate the expression and function of proneural bHLH transcription factors during the onset of mouse retinal neurogenesis. Dorso-central retinal progenitor cells that give rise to the first postmitotic neurons express Neurog2/Ngn2 and Atoh7/Math5. In the absence of Neurog2, the spread of neurogenesis stalls, along with Atoh7 expression and RGC differentiation. However, neurogenesis is eventually restored, and at birth Neurog2 mutant retinas are reduced in size, with only a slight increase in the retinal ganglion cell population. We find that the re-establishment of neurogenesis coincides with the onset of Ascl1 expression, and that Ascl1 can rescue the early arrest of neural development in the absence of Neurog2. Together, this study supports the hypothesis that the intrinsic factors Neurog2 and Ascl1 regulate the temporal progression of retinal neurogenesis by directing overlapping waves of neuron formation.

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. genesis 47:175–187, 2009.

Jagged 1 is necessary for normal mouse lens formation

In mammals, two spatially and temporally distinct waves of fiber cell differentiation are crucial steps for normal lens development. In between these phases, an anterior growth zone forms in which progenitor cells migrate circumferentially, terminally exit the cell cycle and initiate differentiation at the lens equator. Much remains unknown about the molecular pathways orchestrating these processes. Previously, the Notch signal transduction pathway was shown to be critical for anterior lens progenitor cell growth and differentiation. However, the ligand or ligand(s) that direct these events are unknown. Using conditional gene targeting, we show that Jagged1 is required for lens fiber cell genesis, particularly that of secondary fiber cells. In the absence of Jagged1, the anterior growth and equatorial transition zones fail to develop fully, with only a handful of differentiated fiber cells present at birth. Adult Jagged1 conditional mutants completely lack lenses, along with severe anterior chamber deformities. Our data support the hypothesis that Jagged1-Notch signaling conveys a lateral inductive signal, which is indispensable for lens progenitor cell proliferation and differentiation.

Evolution of arthropod visual systems: Development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus linnaeus, 1758 (Chelicerata, Xiphosura)

Despite ongoing interest into the architecture, biochemistry, and physiology of the visual systems of the xiphosuran Limulus polyphemus, their ontogenetic aspects have received little attention. Thus, we explored the development of the lateral eyes and associated neuropils in late embryos and larvae of these animals. The first external evidence of the lateral eyes was the appearance of white pigment spots—guanophores associated with the rudimentary photoreceptors—on the dorsolateral side of the late embryos, suggesting that these embryos can perceive light. The first brown pigment emerges in the eyes during the last (third) embryonic molt to the trilobite stage. However, ommatidia develop from this field of pigment toward the end of the larval trilobite stage so that the young larvae at hatching do not have object recognition. Double staining with the proliferation marker bromodeoxyuridine (BrdU) and an antibody against L. polyphemus myosin III, which is concentrated in photoreceptors of this species, confirmed previous reports that, in the trilobite larvae, new cellular material is added to the eye field from an anteriorly located proliferation zone. Pulse–chase experiments indicated that these new cells differentiate into new ommatidia. Examining larval eyes labeled for opsin showed that the new ommatidia become organized into irregular rows that give the eye field a triangular appearance. Within the eye field, the ommatidia are arranged in an imperfect hexagonal array. Myosin III immunoreactivity in trilobite larvae also revealed the architecture of the central visual pathways associated with the median eye complex and the lateral eyes. Double labeling with myosin III and BrdU showed that neurogenesis persists in the larval brain and suggested that new neurons of both the lamina and the medulla originate from a single common proliferation zone. These data are compared with eye development in Drosophila melanogaster and are discussed with regard to new ideas on eye evolution in the Euarthropoda. Developmental Dynamics 235:2641–2655, 2006.

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.

bHLH-dependent and -independent modes of Ath5 gene regulation during retinal development

In a wide range of vertebrate species, the bHLH transcription factor Ath5 is tightly associated with both the initiation of neurogenesis in the retina and the genesis of retinal ganglion cells. Here, we describe at least two modes of regulating the expression of Ath5 during retinal development. We have found that a proximal cis-regulatory region of the Xenopus Ath5 gene (Xath5) is highly conserved across vertebrate species and is sufficient to drive retinal-specific reporter gene expression in transgenic Xenopus embryos. Xath5 proximal transgene expression depended upon two highly conserved bHLH factor binding sites (E-boxes) as well as bHLH factor activity in vivo. However, we found that bHLH activity was not required for expression of a longer Xath5 transgene, suggesting that additional mechanisms contribute to Xath5 expression in vivo. Consistent with this, we showed that a more distal fragment that does not include the conserved proximal region is sufficient to promote transgene expression in the developing retina. In mouse, we found that a longer fragment of the cis-regulatory region of either the mouse or Xenopus Ath5 gene was necessary for transgene expression, and that expression of a mouse Math5 (Atoh7) transgene was not dependent upon autoregulation. Thus, despite extensive conservation in the proximal region, the importance of these elements may be species dependent.

Conditional ablation of the Notch2 receptor in the ocular lens.

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.