Neuronal Bmal1 regulates retinal angiogenesis and neovascularization in mice.

Circadian clocks in the mammalian retina regulate a diverse range of retinal functions that allow the retina to adapt to the light-dark cycle. Emerging evidence suggests a link between the circadian clock and retinopathies though the causality has not been established. Here we report that clock genes are expressed in the mouse embryonic retina, and the embryonic retina requires light cues to maintain robust circadian expression of the core clock gene, Bmal1. Deletion of Bmal1 and Per2 from the retinal neurons results in retinal angiogenic defects similar to when animals are maintained under constant light conditions. Using two different models to assess pathological neovascularization, we show that neuronal Bmal1 deletion reduces neovascularization with reduced vascular leakage, suggesting that a dysregulated circadian clock primarily drives neovascularization. Chromatin immunoprecipitation sequencing analysis suggests that semaphorin signaling is the dominant pathway regulated by Bmal1. Our data indicate that therapeutic silencing of the retinal clock could be a common approach for the treatment of certain retinopathies like diabetic retinopathy and retinopathy of prematurity.

Histopathological assessments reveal retinal vascular changes, inflammation, and gliosis in patients with lethal COVID-19.

PURPOSE: The purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. METHODS: Eyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for Epon embedding and immunocytochemistry. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the controls. OCT-based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes; however, the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggests that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Histopathological assessments reveal retinal vascular changes, inflammation and gliosis in patients with lethal COVID-19.

PURPOSE: To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. DESIGN: Comparative analysis of human eyes. SUBJECTS: Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed. METHODS: Globes were evaluated ex-vivo with macroscopic, SLO and OCT imaging. Macula and peripheral regions were processed for epon-embedding and immunocytochemistry. MAIN OUTCOME MEASURES: Retinal thickness and histopathology, detection of SARS-CoV-2 Spike protein, changes in vascular density, gliosis, and degree of inflammation. RESULTS: Fundus analysis shows hemorrhagic spots and increased vitreous debris in several of the COVID-19 eyes compared to the control. OCT based measurements indicated an increased trend in retinal thickness in the COVID-19 eyes, however the difference was not statistically significant. Histology of the retina showed presence of hemorrhages and central cystoid degeneration in several of the donors. Whole mount analysis of the retina labeled with markers showed changes in retinal microvasculature, increased inflammation, and gliosis in the COVID-19 eyes compared to the controls. The choroidal vasculature displayed localized changes in density and signs of increased inflammation in the COVID-19 samples. CONCLUSIONS: In situ analysis of the retinal tissue suggested that there are severe subclinical abnormalities that could be detected in the COVID-19 eyes. This study provides a rationale for evaluating the ocular physiology of patients that have recovered from COVID-19 infections to further understand the long-term effects caused by this virus.

Thyroid Activating Enzyme, Deiodinase II Is Required for Photoreceptor Function in the Mouse Model of Retinopathy of Prematurity.

Purpose: Retinopathy of prematurity (ROP) is a severe complication of premature infants, leading to vision loss when untreated. Presently, the molecular mechanisms underlying ROP are still far from being clearly understood. This study sought to investigate whether thyroid hormone (TH) signaling contributes to the neuropathology of ROP using the mouse model of ROP to evaluate longitudinal photoreceptor function. Methods: Animals were exposed to hyperoxia from P7 to P12 to induce retinopathy, thereafter the animals were returned to room air (normoxia). The thyroid-activating enzyme type 2 deiodinases (Dio2) knockout (KO) mice and the littermate controls that were exposed to hyperoxia or maintained in room air and were then analyzed. The retinal function was evaluated using electroretinograms (ERGs) at three and seven weeks followed by histologic assessments with neuronal markers to detect cellular changes in the retina. Rhodopsin protein levels were measured to validate the results obtained from the immunofluorescence analyses. Results: In the ROP group, the photoreceptor ERG responses are considerably lower both in the control and the Dio2 KO animals at P23 compared to the non-ROP group. In agreement with the ERG responses, loss of Dio2 results in mislocalized cone nuclei, and abnormal rod bipolar cell dendrites extending into the outer nuclear layer. The retinal function is compromised in the adult Dio2 KO animals, although the cellular changes are less severe. Despite the reduction in scotopic a-wave amplitudes, rhodopsin levels are similar in the adult mice, across all genotypes irrespective of exposure to hyperoxia. Conclusions: Using the mouse model of ROP, we show that loss of Dio2 exacerbates the effects of hyperoxia-induced retinal deficits that persist in the adults. Our data suggest that aberrant Dio2/TH signaling is an important factor in the pathophysiology of the visual dysfunction observed in the oxygen-induced retinopathy model of ROP.

The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.

A single pool of multipotent retinal progenitor cells give rise to the diverse cell types within the mammalian retina. Such cellular diversity is due to precise control of various cellular processes like cell specification, proliferation, differentiation, and maturation. Circadian clock genes can control the expression of key regulators of cell cycle progression and therefore can synchronize the cell cycle state of a heterogeneous population of cells. Here we show that the protein encoded by the circadian clock gene brain and muscle arnt-like protein-1 (Bmal1) is expressed in the embryonic retina and is required to regulate the timing of cell cycle exit. Accordingly, loss of Bmal1 during retinal neurogenesis results in increased S-phase entry and delayed cell cycle exit. Disruption in cell cycle kinetics affects the timely generation of the appropriate neuronal population thus leading to an overall decrease in the number of retinal ganglion cells, amacrine cells, and an increase in the number of the late-born type II cone bipolar cells as well as the Müller glia. Additionally, the mislocalized Müller cells are observed in the photoreceptor layer in the Bmal1 conditional mutants. These changes affect the functional integrity of the visual circuitry as we report a significant delay in visual evoked potential implicit time in the retina-specific Bmal1 null animals. Our results demonstrate that Bmal1 is required to maintain the balance between the neural and glial cells in the embryonic retina by coordinating the timing of cell cycle entry and exit. Thus, Bmal1 plays an essential role during retinal neurogenesis affecting both development and function of the mature retina.-Sawant, O. B., Jidigam, V. K., Fuller, R. D., Zucaro, O. F., Kpegba, C., Yu, M., Peachey, N. S., Rao, S. The circadian clock gene Bmal1 is required to control the timing of retinal neurogenesis and lamination of Müller glia in the mouse retina.

Sox2 is required for olfactory pit formation and olfactory neurogenesis through BMP restriction and Hes5 upregulation.

The transcription factor Sox2 is necessary to maintain pluripotency of embryonic stem cells, and to regulate neural development. Neurogenesis in the vertebrate olfactory epithelium persists from embryonic stages through adulthood. The role Sox2 plays for the development of the olfactory epithelium and neurogenesis within has, however, not been determined. Here, by analysing Sox2 conditional knockout mouse embryos and chick embryos deprived of Sox2 in the olfactory epithelium using CRISPR-Cas9, we show that Sox2 activity is crucial for the induction of the neural progenitor gene Hes5 and for subsequent differentiation of the neuronal lineage. Our results also suggest that Sox2 activity promotes the neurogenic domain in the nasal epithelium by restricting Bmp4 expression. The Sox2-deficient olfactory epithelium displays diminished cell cycle progression and proliferation, a dramatic increase in apoptosis and finally olfactory pit atrophy. Moreover, chromatin immunoprecipitation data show that Sox2 directly binds to the Hes5 promoter in both the PNS and CNS. Taken together, our results indicate that Sox2 is essential to establish, maintain and expand the neuronal progenitor pool by suppressing Bmp4 and upregulating Hes5 expression.

Apical constriction and epithelial invagination are regulated by BMP activity.

Epithelial invagination is a morphological process in which flat cell sheets transform into three-dimensional structures through bending of the tissue. It is accompanied by apical constriction, in which the apical cell surface is reduced in relation to the basal cell surface. Although much is known about the intra-cellular molecular machinery driving apical constriction and epithelial invagination, information of how extra-cellular signals affect these processes remains insufficient. In this study we have established several in vivo assays of placodal invagination to explore whether the external signal BMP regulates processes connected to epithelial invagination. By inhibiting BMP activity in prospective cranial placodes, we provide evidence that BMP signals are required for RhoA and F-actin rearrangements, apical constriction, cell elongation and epithelial invagination. The failure of placode invagination after BMP inhibition appears to be a direct consequence of disrupted apical accumulation of RhoA and F-actin, rather than changes in cell death or proliferation. In addition, our results show that epithelial invagination and acquisition of placode-specific identities are two distinct and separable developmental processes. In summary, our results provide evidence that BMP signals promote epithelial invagination by acting upstream of the intracellular molecular machinery that drives apical constriction and cell elongation.

Neural retina identity is specified by lens-derived BMP signals.

The eye has served as a classical model to study cell specification and tissue induction for over a century. Nevertheless, the molecular mechanisms that regulate the induction and maintenance of eye-field cells, and the specification of neural retina cells are poorly understood. Moreover, within the developing anterior forebrain, how prospective eye and telencephalic cells are differentially specified is not well defined. In the present study, we have analyzed these issues by manipulating signaling pathways in intact chick embryo and explant assays. Our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character, but from neural tube/optic vesicle stages, BMP signals from the lens are crucial for the maintenance of eye-field character, inhibition of dorsal telencephalic cell identity and specification of neural retina cells. Subsequently, our results provide evidence that a Rax2-positive eye-field state is not sufficient for the progress to a neural retina identity, but requires BMP signals. In addition, our results argue against any essential role of Wnt or FGF signals during the specification of neural retina cells, but provide evidence that Wnt signals together with BMP activity are sufficient to induce cells of retinal pigment epithelial character. We conclude that BMP activity emanating from the lens ectoderm maintains eye-field identity, inhibits telencephalic character and induces neural retina cells. Our findings link the requirement of the lens ectoderm for neural retina specification with the molecular mechanism by which cells in the forebrain become specified as neural retina by BMP activity.

Development of cranial placodes: insights from studies in chick.

This review focuses on how research, using chick as a model system, has contributed to our knowledge regarding the development of cranial placodes. This review highlights when and how molecular signaling events regulate early specification of placodal progenitor cells, as well as the development of individual placodes including morphological movements. In addition, we briefly describe various techniques used in chick that are important for studies in cell and developmental biology.

BMP-induced L-Maf regulates subsequent BMP-independent differentiation of primary lens fibre cells.

Bone morphogenetic protein (BMP) signals are essential for lens development. However, the temporal requirement of BMP activity during early events of lens development has remained elusive. To investigate this question, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that BMP activity is both required and sufficient to induce L-Maf expression, whereas the onset of δ-crystallin and initial elongation of primary lens fibre cells are BMP-independent. Moreover, before lens placode formation and L-Maf onset, but not after, prospective lens placodal cells can switch to an olfactory placodal fate in response to decreased BMP activity. In addition, L-Maf is sufficient to up-regulate δ-crystallin independent of BMP signals. Taken together, these results show that before L-Maf induction BMP activity is required for lens specification, whereas after L-Maf up-regulation, the early differentiation of primary lens fibre cells occurs independent of BMP signals.