The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

Background: Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp ( homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.Results: Our data show that the level of BMP4 signaling is critical for the regulation of distinct Tbx2, Tbx3, Tbx5 and Vax2 gene expression domains along the dorso-ventral axis of the mouse optic cup. BMP4 signaling gradients were manipulated in whole mouse embryo cultures during optic cup development, by implantation of beads soaked in BMP4, or the BMP antagonist Noggin, to provide a local signaling source. Tbx2, Tbx3 and Tbx5, showed a differential response to alterations in the level of BMP4 along the entire dorso-ventral axis of the optic cup, suggesting that BMP4 acts across a distance. Increased levels of BMP4 caused expansion of Tbx2 and Tbx3, but not Tbx5, into the ventral retina and repression of the ventral marker Vax2. Conversely, Noggin abolished Tbx5 expression but only shifted Tbx2 expression dorsally. Increased levels of BMP4 signaling caused decreased proliferation, reduced retinal volume and altered the shape of the optic cup.Conclusion: Our findings suggest the existence of a dorsal-high, ventral-low BMP4 signaling gradient across which distinct domains of Tbx2, Tbx3, Tbx5 and Vax2 transcription factor gene expression are set up. Furthermore we show that the correct level of BMP4 signaling is critical for normal growth of the mammalian embryonic eye

Pattern Onset ERGs and VEPs Produced by Patterns Arising From Light Increment and Decrement

PURPOSE: Our aim was to elaborate how on and off signals contribute to pattern ERGs and pattern visual evoked potentials (VEPs) by using pedestal patterns arising from incremental and decremental onset stimulation. METHODS: Pattern onset/offset ERGs and VEPs were produced by black and white checks of 60' side length and 88% spatial contrast appearing in a 16° field for 200 ms from white (110 cd/m2), black (7 cd/m2), and gray (48 cd/m2) backgrounds and disappeared for 1000 ms. Twenty healthy subjects participated in the study (median age 19.5, range, 5-31 years), 10 of whom also underwent pattern onset/offset ERG recordings to the same stimuli (median age 25.7, range, 22-31 years). VEPs were recorded from an occipital array referred to Fz. Pattern electroretinograms (PERGs) were recorded from "Dawson-Trick-Litzkow" (DTL) plus corneal electrodes referred to ipsilateral outer canthi. RESULTS: There was high correlation within subjects of the VEP waveform produced by patterns arising from light increment and decrement (group mean correlation coefficient of PVEPs to check appearance from black versus white: 87%). An average of increment and decrement PERGs simulated the onset PERG from a gray background. This waveform is akin to standard International Society for Clinical Electrophysiology of Vision (ISCEV) clinical PERGs to reversing checks. CONCLUSIONS: In healthy individuals, the early components of the pattern onset/offset VEP waveforms are comparable to light increment and decrement pedestal stimulation. Pattern onset/offset ERGs to pedestal stimulation may be used to probe simultaneous recording of ERGs with VEPs in order to obtain an assessment of retinal ganglion cell and optic pathway function in patients with less stable fixation

Genes and pathways in optic fissure closure

Embryonic development of the vertebrate eye begins with the formation of an optic vesicle which folds inwards to form a double-layered optic cup with a fissure on the ventral surface, known as the optic fissure. Closure of the optic fissure is essential for subsequent growth and development of the eye. A defect in this process can leave a gap in the iris, retina or optic nerve, known as a coloboma, which can lead to severe visual impairment. This review brings together current information about genes and pathways regulating fissure closure from human coloboma patients and animal models. It focuses especially on current understanding of the morphological changes and processes of epithelial remodelling occurring at the fissure margins

A molecular and cellular analysis of human embryonic optic fissure closure related to the eye malformation coloboma

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC), and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup

Missed case of Axenfeld-Rieger syndrome: a case report

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

NRL^{-/-} gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl‐deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S‐cone‐like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL‐deficient embryonic stem cell (ESC) line (NRL^{−/−}), and differentiated it into retinal organoids. Retinal organoids self‐organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL−/− OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL^{−/−} OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S‐OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC‐derived organoid system that NRL is required to define rod identity, and that in its absence S‐cone‐like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases

Advancing practice for back pain through stratified care (STarT Back)

Background Low back pain (LBP) is common, however research comparing the effectiveness of different treatments over the last two decades conclude either no or small differences in the average effects of different treatments. One suggestion to explain this is that patients are not all the same and important subgroups exist that might require different treatment approaches. Stratified care for LBP involves identifying subgroups of patients and then delivering appropriate matched treatments. Research has shown that stratified care for LBP in primary care can improve clinical outcomes, reduce costs and increase the efficiency of health-care delivery in the UK. The challenge now is to replicate and evaluate this approach in other countries health care systems and to support services to implement it in routine clinical care. Results The STarT Back approach to stratified care has been tested in the National Health Service, within the UK, it reduces unnecessary overtreatment in patients who have a good prognosis (those at low risk) yet increases the likelihood of appropriate healthcare and associated improved outcomes for those who are at risk of persistent disabling pain. The approach is cost-effective in the UK healthcare setting and has been recommended in recent guidelines and implemented as part of new LBP clinical pathways of care. This approach has subsequently generated international interest, a replication study is currently underway in Denmark, however, some lessons have already been learnt. There are potential obstacles to implementing stratified care in low-and-middle-income settings and in other high-income settings outside of the UK, however, implementation science literature can inform the development of innovations and efforts to support implementation of stratified care. Conclusions The STarT Back approach to stratified care for LBP is a promising method to advance practice that has demonstrated clinical and cost effectiveness in the UK. Over time, further evidence for both the effectiveness and the adaptations needed to test and implement the STarT Back stratified care approach in other countries is needed

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-derived Retinal Organoids and Fetal Retinae

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were FAC-sorted using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. This article is protected by copyright. All rights reserved

Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application. Stem Cells 2015;33:2469-2482