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Hocking, J. C.

Publications and source records attributed to Hocking, J. C..

6 recordsLinked to original sources

A Müller glia-retinal pigment epithelium apical network surrounding cone outer segments in fish and mouse

Homeostasis of the vertebrate retina is supported by two non-neuronal cell types, Muller glia and the retinal pigment epithelium (RPE). Their apical domains delineate the subretinal space, where photoreceptor outer segments reside. While the individual interactions between the supporting cells and photoreceptors are well studied, Muller glia and RPE are presumed to remain physically separate in a healthy retina. Here, we describe a revised model of the subretinal space architecture based on detailed FIB-SEM and confocal microscopy imaging of the zebrafish and mouse outer retina. Long apical processes extend from both Muller glia and the RPE, interacting with one another while also encircling cone outer segments. These contact regions, at the interface of the three cell types are highly elaborated in fish and, although sparser, are still present in mouse. The growth and overlap of Muller glial and RPE apical processes occurs subsequent to the emergence of the outer segment in both species, and coincides with the onset of visual function in mouse. Although enriched at cone sites, this specialized interaction does not appear to depend solely on cone localization in either species, pointing to a general hallmark of the vertebrate subretinal space. Together, our work provides major insights into the architecture of the subretinal space and the interactions between Muller glia, RPE and photoreceptors, while demonstrating conservation across species.

cell biology↗

The BMP ligand Gdf6a Regulates Development of the Zebrafish Craniofacial Skeleton in a Pharyngeal Arch-Specific Manner

Craniofacial development involves the concerted action of several different tissue types and cellular processes that must be intricately regulated. Here, we describe the role of the BMP ligand Growth and Differentiation Factor 6a (gdf6a) in the development of the zebrafish craniofacial skeleton. Larval gdf6a mutant zebrafish have malformations in the midline of the craniofacial skeleton that correlate with the expression of gdf6a in the embryonic pharyngeal arches. We show that Gdf6a has arch-specific roles in craniofacial morphogenesis; Gdf6a promotes chondrogenesis and alignment of midline craniofacial elements in the posterior pharyngeal arches (arches 2 and 3) and regulates morphogenesis within the mandibular symphysis of pharyngeal arch 1. We demonstrate that Gdf6a regulates craniofacial development through activation of canonical BMP signaling, likely acting cooperatively with additional BMP ligands. Taken together, this work elucidates how Gdf6a/BMP signaling directs development of the craniofacial skeleton, specifically along the ventral midline. Summary StatementGdf6a regulates the development of the midline structures in the zebrafish craniofacial skeleton in an arch-specific manner via canonical BMP signaling.

developmental biology↗

PIKfyve is an essential component of the endolysosomal pathway within photoreceptors and the retinal pigment epithelium

Phosphoinositides (PIs) are a family of seven low abundance membrane lipids, each with distinct signaling functions. The phosphoinositide kinase PIKfyve generates phosphoinositide-3,5-bisphosphate (PI(3,5)P2) and PI5P. Emerging evidence implicates PIKfyve in key cellular processes, including autophagy, phagocytosis, endosomal trafficking, lysosomal maintenance, and melanosome formation. Complete loss of PIKfyve function is embryonic lethal in model organisms. In humans, heterozygous mutations in PIKFYVE are associated with Fleck corneal dystrophy and congenital cataracts. In this study, we investigate the role of PIKfyve in photoreceptors and the adjacent retinal pigment epithelium (RPE), host to dynamic endolysosomal pathways required for enduring the high oxidative stress environment, transporting metabolites and phototransduction components, and the breakdown of outer segment discs. To assess PIKfyve function in the retina and RPE in our zebrafish model, we employed CRISPR/Cas9-mediated gene editing and pharmacological inhibition using the specific PIKfyve inhibitor apilimod. Loss of PIKfyve activity leads to RPE expansion characterized by the accumulation of LC3- and LAMP1-positive vacuoles, along with defects in phagosome degradation and minor changes to melanosome biogenesis. Photoreceptors deprived of PIKfyve function develop a single large vacuole in the inner segment, while the OS remains largely intact over the timespan analyzed. Electroretinography (ERG) recordings revealed complete visual impairment in pikfyve crispant larvae, and significantly reduced visual function in larvae treated with apilimod post embryogenesis. These findings highlight the critical role of PIKfyve in the development and homeostasis of the RPE and retina.

cell biology↗

Polysialic acid is a versatile marker for retinal Müller glia in common vertebrate model organisms and systems

Muller glia are retinal support cells that play crucial roles in tissue structure, waste management, and repair. A challenge for the field has been to find Muller glia markers that detect non-reactive cells or work well in non-mammalian models. We introduce two novel markers for identifying reactive and non-reactive Muller glia in vertebrate retinas: a modified enzyme lectin (GFP-EndoNDM) and a monoclonal antibody (mAb735). These markers recognize polysialic acid (polySia), which is a highly conserved glycosylation modification in humans and vertebrates. In the retina, polySia is present on Muller glia predominantly in the form of polySia-NCAM. We used GFP-EndoNDM and mAb735 to investigate polySia distribution in Muller glia of fish, amphibians, reptiles, birds, rodents, retinal organoids, and humans. In adult retinas of most species, polySia was localized to the Muller glia and spanned outer to inner retina, with inner plexiform layer (IPL) sublaminae ramifications. Gliosis was also detectable in degenerating murine retinas. Notable species differences were that only outer retinal regions of Muller glia were labelled in adult zebrafish, whereas the outer Muller glia body up to the first IPL sublamina was labelled in adult turquoise killifish. There was no significant retinal polySia labeling in larval zebrafish, but it was present in the brain. Larval turquoise killifish have polySia throughout the retina, similar to other adult vertebrates. Labelling polySia expands the scientific toolbox for Muller glia markers, and offers a versatile way to visualize and monitor structural changes in non-reactive and reactive Muller glia across most vertebrate species. HighlightsO_LIPolySia is a highly conserved Muller glia marker in vertebrate retina and retinal organoids C_LIO_LILabeling Muller glia with markers of polySia, facilitates the analysis of fine Muller glia processes in the IPL sublaminae, whole Muller glia morphology, and degenerative gliosis C_LIO_LIPolySia can be labeled with an engineered lectin conjugate (GFP-EndoNDM) and a commercially-available monoclonal antibody C_LI

cell biology↗

Development and characteristics of photoreceptor actin-based apical processes in zebrafish (Danio rerio)

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). While CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the processes remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, while heat shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically-oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Muller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development, and surrounding environment of photoreceptor microvilli in the zebrafish retina.

cell biology↗

A Cell Atlas of Microbe-Responsive Processes in the Zebrafish Intestine

Gut microbial products direct growth, differentiation, and development in the animal host. Disruptions to host-microbe interactions have profound health consequences, that include onset of chronic inflammatory illnesses. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled transcriptional activity in individual cells from the intestine, and associated tissue, of zebrafish larvae that we raised in the presence or absence of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome that included regulatory effects on patterning, metabolic and immune activity. For example, we showed that removal of microbes hindered pro-angiogenic signals in the developing vasculature, resulting in impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type. Furthermore, we provide a web-based resource for single-cell gene expression visualization under conventional and germ-free conditions to facilitate exploration of this dataset.

developmental biology↗