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

Spectrum of Mutations in NDP Resulting in Ocular Disease; a Systematic Review

Aims and Rationale: The inner retina is supplied by three intraretinal capillary plexi whereas the outer retina is supplied by the choroidal circulation: NDP is essential for normal intraretinal vascularisation. Pathogenic variants in NDP (Xp11.3) may result in either a severe retinal phenotype associated with hearing loss (Norrie Disease) or a moderate retinal phenotype (Familial Exudative Vitreoretinopathy, FEVR). However, little is known about whether the nature or location of the NDP variant is predictive of severity. In this systematic review we summarise all reported NDP variants and draw conclusions about whether the nature of the NDP variant is predictive of the severity of the resulting ocular pathology and associated hearing loss and intellectual disability. Findings: 201 different variants in the NDP gene have been reported as disease-causing. The pathological phenotype that may result from a disease-causing NDP variant is quite diverse but generally comprises a consistent cluster of features (retinal hypovascularisation, exudation, persistent foetal vasculature, tractional/exudative retinal detachment, intellectual disability and hearing loss) that vary predictably with severity. Previous reviews have found no clear pattern in the nature of NDP mutations that cause either FEVR or Norrie disease, with the exception that mutations affecting cysteine residues have been associated with Norrie Disease and that visual loss amongst patients with Norrie disease tends to be more severe if the NDP mutation results in an early termination of translation as opposed to a missense related amino acid change. A key limitation of previous reviews has been variability in the case definition of Norrie disease and FEVR amongst authors. We thus reclassified patients into two groups based only on the severity of their retinal disease. Of the reported pathogenic variants that have been described in more than one patient, we found that any given variant caused an equivalent severity of retinopathy each time it was reported with very few exceptions. We therefore conclude that specific NDP mutations generally result in a consistent retinal phenotype each time they arise. Reports by different authors of the same variant causing either FEVR or Norrie disease conflict primarily due to variability in the authors' respective case definitions rather than true differences in disease severity

Multiple GF-1 binding sites flank the erythroid specific transcription unit of the human carbonic anhydrase I gene

AbstractSix potential GF-1 sites which bind an erythroid factor are present in the 5' and 3' regions flanking the erythroid-speciflc transcription unit of the human carbonic anhydrase 1 (HCAI) gene. When two of these sites are placed upstream of a minimal eukaryotic promoter they confer upregulated expression in erythroid over non-erythroid cells. The presence of the erythroid factor in TPA-treated HEL cells in which the level of HCAI transcript has greatly decreased and in non-HCAI-expressing K562 cells suggests that in these cases the presence of the factor is not sufficient for HCAI expression

Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution

Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies

Generation of 3D retinal tissue from human pluripotent stem cells using a directed small molecule-based serum-free microwell platform

Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by replacing lost photoreceptors and/or retinal pigment epithelium. Development of protocols to generate retinal tissue from human pluripotent stem cells (hPSC), reliably and at scale, can provide a platform to generate photoreceptors for cell therapy and to model retinal disease in vitro. Here, we describe an improved differentiation platform to generate retinal organoids from hPSC at scale and free from time-consuming manual microdissection steps. The scale up was achieved using an agarose mould platform enabling generation of uniform self-assembled 3D spheres from dissociated hPSC in microwells. Subsequent retinal differentiation was efficiently achieved via a stepwise differentiation protocol using a number of small molecules. To facilitate clinical translation, xeno-free approaches were developed by substituting Matrigel™ and foetal bovine serum with recombinant laminin and human platelet lysate, respectively. Generated retinal organoids exhibited important features reminiscent of retinal tissue including correct site-specific localisation of proteins involved in phototransduction

Systemic gene therapy rescues retinal dysfunction and hearing loss in a model of Norrie disease

Deafness affects 5% of the world's population, yet there is a lack of treatments to prevent hearing loss due to genetic causes. Norrie disease is a recessive X-linked disorder, caused by NDP gene mutation. It manifests as blindness at birth and progressive sensorineural hearing loss, leading to debilitating dual sensory deprivation. To develop a gene therapy, we used a Norrie disease mouse model (Ndptm1Wbrg ), which recapitulates abnormal retinal vascularisation and progressive hearing loss. We delivered human NDP cDNA by intravenous injection of adeno-associated viral vector (AAV)9 at neonatal, juvenile and young adult pathological stages and investigated its therapeutic effects on the retina and cochlea. Neonatal treatment prevented the death of the sensory cochlear hair cells and rescued cochlear disease biomarkers as demonstrated by RNAseq and physiological measurements of auditory function. Retinal vascularisation and electroretinograms were restored to normal by neonatal treatment. Delivery of NDP gene therapy after the onset of the degenerative inner ear disease also ameliorated the cochlear pathology, supporting the feasibility of a clinical treatment for progressive hearing loss in people with Norrie disease

Systemic gene therapy rescues retinal dysfunction and hearing loss in a model of Norrie disease

Deafness affects 5% of the world's population, yet there is a lack of treatments to prevent hearing loss due to genetic causes. Norrie disease is a recessive X‐linked disorder, caused by NDP gene mutation. It manifests as blindness at birth and progressive sensorineural hearing loss, leading to debilitating dual sensory deprivation. To develop a gene therapy, we used a Norrie disease mouse model (Ndp$^{tm1Wbrg}$), which recapitulates abnormal retinal vascularisation and progressive hearing loss. We delivered human NDP cDNA by intravenous injection of adeno‐associated viral vector (AAV)9 at neonatal, juvenile and young adult pathological stages and investigated its therapeutic effects on the retina and cochlea. Neonatal treatment prevented the death of the sensory cochlear hair cells and rescued cochlear disease biomarkers as demonstrated by RNAseq and physiological measurements of auditory function. Retinal vascularisation and electroretinograms were restored to normal by neonatal treatment. Delivery of NDP gene therapy after the onset of the degenerative inner ear disease also ameliorated the cochlear pathology, supporting the feasibility of a clinical treatment for progressive hearing loss in people with Norrie disease

Isolation and Comparative Transcriptome Analysis of Human Fetal and iPSC-Derived Cone Photoreceptor Cells.

Loss of cone photoreceptors, crucial for daylight vision, has the greatest impact on sight in retinal degeneration. Transplantation of stem cell-derived L/M-opsin cones, which form 90% of the human cone population, could provide a feasible therapy to restore vision. However, transcriptomic similarities between fetal and stem cell-derived cones remain to be defined, in addition to development of cone cell purification strategies. Here, we report an analysis of the human L/M-opsin cone photoreceptor transcriptome using an AAV2/9.pR2.1:GFP reporter. This led to the identification of a cone-enriched gene signature, which we used to demonstrate similar gene expression between fetal and stem cell-derived cones. We then defined a cluster of differentiation marker combination that, when used for cell sorting, significantly enriches for cone photoreceptors from the fetal retina and stem cell-derived retinal organoids, respectively. These data may facilitate more efficient isolation of human stem cell-derived cones for use in clinical transplantation studies

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease