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BACKGROUND: RERE is a highly conserved transcriptional co-regulator that is associated with a human neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH, OMIM: 616975). RESULTS: We show that the zebrafish rerea mutant (babyface) robustly recapitulates optic fissure closure defects resulting from loss of RERE function, as observed in humans. These defects result from expansion of proximal retinal optic stalk (OS) and reduced expression of some of the ventral retinal fate genes due to deregulated protein signaling. Using zebrafish and cell-based assays, we determined that NEDBEH-associated human RERE variants function as hypomorphs in their ability to repress shh signaling and some exhibit abnormal nuclear localization. Inhibiting shh signaling by the protein inhibitor HPI-1 rescues coloboma, confirming our observation that coloboma in rerea mutants is indeed due to deregulation of shh signaling. CONCLUSIONS: Zebrafish rerea mutants exhibit OS and optic fissure closure defects. The optic fissure closure defect was rescued by an shh signaling inhibitor, suggesting that this defect could arise due to deregulated shh signaling.

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Sending an axon out of the eye and into the target brain nuclei is the defining feature of retinal ganglion cells (RGCs). The literature on RGC axon pathfinding is vast, but it focuses mostly on decision making events such as midline crossing at the optic chiasm or retinotopic mapping at the target nuclei. In comparison, the exit of RGC axons out of the eye is much less explored. The first checkpoint on the RGC axons' path is the optic cup - optic stalk junction (OC-OS). OC-OS development and the exit of the RGC pioneer axons out of the eye are coordinated spatially and temporally. By the time the optic nerve head domain is specified, the optic fissure margins are in contact and the fusion process is ongoing, the first RGCs are born in its proximity and send pioneer axons in the optic stalk. RGC differentiation continues in centrifugal waves. Later born RGC axons fasciculate with the more mature axons. Growth cones at the end of the axons respond to guidance cues to adopt a centripetal direction, maintain nerve fiber layer restriction and to leave the optic cup. Although there is extensive information on OC-OS development, we still have important unanswered questions regarding its contribution to the exit of the RGC axons out of the eye. We are still to distinguish the morphogens of the OC-OS from the axon guidance molecules which are expressed in the same place at the same time. The early RGC transcription programs responsible for axon emergence and pathfinding are also unknown. This review summarizes the molecular mechanisms for early RGC axon guidance by contextualizing mouse knock-out studies on OC-OS development with the recent transcriptomic studies on developing RGCs in an attempt to contribute to the understanding of human optic nerve developmental anomalies. The published data summarized here suggests that the developing optic nerve head provides a physical channel (the closing optic fissure) as well as molecular guidance cues for the pioneer RGC axons to exit the eye.

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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.

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Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.

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Cell cycle-related kinase (CCRK) is a conserved regulator of ciliogenesis whose loss in mice leads to a wide range of developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, and microphthalmia. Here, we investigate the role of CCRK in mouse eye development. Ccrk mutants show dramatic patterning defects, with an expansion of the optic stalk domain into the optic cup, as well as an expansion of the retinal pigment epithelium (RPE) into neural retina (NR) territory. In addition, Ccrk mutants display a shortened optic stalk. These defects are associated with bimodal changes in Hedgehog (Hh) pathway activity within the eye, including the loss of proximal, high level responses but a gain in distal, low level responses. We simultaneously removed the Hh activator GLI2 in Ccrk mutants (Ccrk-/-;Gli2-/-), which resulted in rescue of optic cup patterning and exacerbation of optic stalk length defects. Next, we disrupted the Hh pathway antagonist GLI3 in mutants lacking CCRK (Ccrk-/-;Gli3-/-), which lead to even greater expansion of the RPE markers into the NR domain and a complete loss of NR specification within the optic cup. These results indicate that CCRK functions in eye development by both positively and negatively regulating the Hh pathway, and they reveal distinct requirements for Hh signaling in patterning and morphogenesis of the eyes.

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Astrocytes represent the earliest glial population in the embryonic optic nerve, contributing critically to retinal angiogenesis and formation of brain-retinal-barrier. Despite of many developmental and clinical implications of astrocytes, answers to some of the most fundamental questions of this unique type of glial cells remain elusive. This review provides an overview of the current knowledge about the origination, proliferation, and differentiation of astrocytes, their journey from the optic nerve toward the neuroretina, and their involvement in physiological and pathological development of the visual system.

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Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previously, we have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. We studied the roles of the integrin complexes in these glial developmental processes during OS/eye development. The common beta subunit ßPS and two alpha subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of ßPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. Our results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development.

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BACKGROUND: Heparan sulfate proteoglycans (HSPG) are important for embryonic development by means of the regulation of gradient formation and signaling of multiple growth factors and morphogens. Previous studies have shown that Bmp/Shh/Fgf signaling are required for the regionalization of the optic vesicle (OV) and for the closure of the optic fissure (OF), the disturbance of which underlie ocular anomalies such as microphthalmia, coloboma, and optic nerve hypoplasia. RESULTS: To study HSPG-dependent coordination of these signaling pathways during mammalian visual system development, we have generated a series of OV-specific mutations in the heparan sulfate (HS) N-sulfotransferase genes (Ndst1 and Ndst2) and HS O-sulfotransferase genes (Hs2st, Hs6st1, and Hs6st2) in mice. Of interest, the resulting HS undersulfation still allowed for normal retinal neurogenesis and optic fissure closure, but led to defective optic disc and stalk development. The adult mutant animals further developed optic nerve aplasia/hypoplasia and displayed retinal degeneration. We observed that MAPK/ERK signaling was down-regulated in Ndst mutants, and consistent with this, HS-related optic nerve morphogenesis defects in mutant mice could partially be rescued by constitutive Kras activation. CONCLUSIONS: These results suggest that HSPGs, depending on their HS sulfation pattern, regulate multiple signaling pathways in optic disc and stalk morphogenesis.

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An improved and efficient protocol was developed based on the TaKaRa RNAiso Plus Kit (Code: D9108A) for isolating good-quality total RNA from the optic stalk of mud crab, Scylla paramamosain. The protocol was based on the Trizol method with modifications. The carapace overlapping the optic stalk was retained with RNA in regular protocol. In order to remove the abundant deposition correlative with the carapace which makes the isolation of RNA particularly difficult, 5M potassium acetate solution (pH = 6.0) was added before the precipitation of RNA, and the temperature of RNA deposition was also decreased to -70ºC to ensure the stabilization of RNA. Good-quality total RNA from the optic stalk of S. paramamosain could be easily isolated with this modified protocol and three conventional methods were also employed to confirm the quality of RNA. This improved method would be helpful in facilitating molecular research of crabs involving RNA from the optic stalk.

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