Biology subjects

Collery, R. F.

Publications and source records attributed to Collery, R. F..

4 recordsLinked to original sources

bioRxiv2024

The USH3A causative gene clarin1 functions in Muller glia to maintain retinal photoreceptors

Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular balance issues. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafish clrn1. Retina of clrn1 mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina in clrn1 mutants, including actin-based structures of the Muller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Muller glia or photoreceptor cells. Muller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type and clrn1 mutant animals. However, rod- or cone-specific Clrn1 re-expression did not rescue the extent of cell death. Taken together, our findings underscore three crucial insights. First, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Muller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Muller glia suggests a structural support mechanism, possibly through direct interactions between Muller glia and photoreceptors mediated in part by Clrn1 protein. AUTHOR SUMMARYMutations in USH-associated genes profoundly impact patients, affecting auditory, visual, and vestibular function. While the basis of inner ear defects is reasonably well understood for USH and auditory devices can improve hearing, the mechanisms underlying photoreceptor loss are unknown, and there are no approved treatments for vision deficits. In USH3A, the affected gene, clarin1 (clrn1), is predominantly expressed in Muller glia. The role of Muller glia in maintaining photoreceptor health and contributions to USH3 pathology is understudied, in part as Clrn1 mutant mice - the traditional experimental model used to study retinal diseases - do not phenocopy the photoreceptor loss of USH3 patients. In the present study, we developed a zebrafish model of USH3A that displays many features of the human disease. Our research shows that the loss of Clrn1 affects actin-based structures of the outer retina, including those of photoreceptor cells and Muller glia. Importantly, we demonstrate that the expression of Clrn1 in Muller glia, but not rods and cones, alleviated light-induced damage in clrn1 mutant zebrafish. We also highlight that the dosage of Clrn1 in Muller glia is critical for maintaining proper photoreceptor function. These findings demonstrate the key contribution of Muller glia to USH pathology and can guide strategies for gene-replacement therapies.

cell biology↗

bioRxiv2021

Ablation of mpeg+ macrophages exacerbates mfrp-related hyperopia

PURPOSEProper refractive development of the eye, termed emmetropization, is critical for focused vision and impacted by both genetic determinants and several visual environment factors. Improper emmetropization caused by genetic variants can lead to congenital hyperopia, which is characterized by small eyes and relatively short ocular axial length. To date variants in only four genes have been firmly associated with human hyperopia, one of which is MFRP. Zebrafish mfrp mutants also have hyperopia and similar to reports in mice, exhibit increased macrophage recruitment to the retina. The goal of this research was to examine the effects of macrophage ablation on emmetropization and mfrp-related hyperopia. METHODSWe utilized a chemically inducible, cell-specific ablation system to deplete macrophages in both wild-type and mfrp mutant zebrafish. Spectral-domain optical coherence tomography (SD-OCT) was used to measure components of the eye and determine relative refractive state. Histology, immunohistochemistry, and transmission electron microscopy was used to further study the eyes. RESULTSWhile macrophage ablation does not cause significant changes to the relative refractive state of wild-type zebrafish, macrophage ablation in mfrp mutants significantly exacerbates their hyperopic phenotype. CONCLUSIONSGenetic inactivation of mfrp leads to hyperopia as well as abnormal accumulation of macrophages in the retina. Ablation of the mpeg1-positive macrophage population exacerbates the hyperopia, suggesting that macrophages are recruited in an effort help preserve emmetropization and ameliorate hyperopia.

genetics↗

bioRxiv2019

notum1, acting downstream of pitx2, is essential for proper eye and craniofacial development.

Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant developmental disorder characterized by ocular anterior chamber anomalies with an increased risk of glaucoma and systemic defects. Mutations in the transcription factor PITX2 were the first identified genetic cause of ARS. Despite the developmental importance of PITX2 and its role in ARS, the pathways downstream of PITX2 have yet to be fully characterized. Comparative transcriptome analyses involving pitx2-enriched cell populations isolated via fluorescence activated cell sorting of tissues expressing (Tg(-2.6pitx2-CE4:GFP)) reporter in wild-type and pitx2M64* mutant zebrafish embryos identified the highly down-regulated target notum1b, an ortholog of human NOTUM encoding a secreted carboxylesterase that cleaves a necessary palmitoleate moiety from WNT proteins. Further experiments confirmed a decrease in notum1b and identified down-regulation of another NOTUM ortholog, notum1a, in the developing mutant eye. CRISPR-generated permanent double knockout zebrafish lines of notum1b and notum1a, notum1-/-, displayed defects in craniofacial and ocular development, including corneal defects, small lenses, increased sizes of the anterior and posterior chambers, and anomalies in teeth development. Analysis of head transcriptome of notum1-/- zebrafish in comparison to wild-type predicted an up-regulation of the WNT pathway. We present NOTUM/notum1 as an important factor in ocular and craniofacial development and a novel downstream member of the PITX2/pitx2 pathway.

genetics↗

bioRxiv2018

Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error

Mutations in LRP2, a transmembrane receptor, cause ocular enlargement and high-myopia. LRP2 is expressed by the RPE and eye ciliary epithelia, binding many extracellular ligands, including Bmp4 and Shh. Signaling mediated by LRP2 is very context-dependent, and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled, sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia (proper refraction). Using zebrafish, we demonstrate that BACE sheddase-dependent LRP2 cleavage produces a soluble domain that binds BMP4, inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand-binding receptor forms available: a soluble BMP trap, and a membrane-bound RPE signaling facilitator. By modulating LRP2 cleavage, cells can fine-tune and coordinate multiple signaling pathways, as well as growth and turnover of the extracellular matrix, control of which is important to maintain proper eye size. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth.\n\nAuthor SummaryFor proper focusing and normal vision, the axial length of the eye needs to match the refractive power of the lens. This is achieved by fine-tuning multiple signaling pathways to regulate the shape of the eye primarily by remodeling of the sclera, the outermost layer of the eye. This process is termed emmetropization. Emmetropization cues are initiated by visual input, but how signals are transduced from the photoreceptors across the retinal pigment epithelium to the sclera is incompletely understood. Here we show that cleavage of Lrp2, a large receptor expressed on RPE cells in the eye, alters BMP signaling, which contributes to proper eye size control. Dysregulation of BMP signaling by a) absence of Lrp2 in mutant zebrafish or b) overexpression of BMP antagonists from the RPE both cause eye enlargement and myopia. Understanding how regulated cleavage of Lrp2 affects paracrine signaling provides critical insight to emmetropization, raising the possibility for development of therapeutic agents to combat the epidemic incidence of refractive error.

molecular biology↗