Biology subjects

Hitchcock, P. F.

Publications and source records attributed to Hitchcock, P. F..

6 recordsLinked to original sources

bioRxiv2026

Spatially distinct microglia coordinate the response to photoreceptor injury in zebrafish

Microglia exhibit substantial molecular and functional heterogeneity, however, how the local tissue environment influences their responses to neuronal injury remains poorly understood. The zebrafish retina provides a unique opportunity to examine this relationship, because in addition to microglia within the retinal parenchyma, it contains a distinct population that resides within the subretinal space. In this study, we characterize the anatomical distribution and molecular heterogeneity of retinal microglia in zebrafish and examine their response to selective photoreceptor injury. Retinal microglia occupied distinct anatomical niches and exhibited diverse transcriptional states, including complement associated, proliferative, and apoc1/apoeb-enriched populations, suggesting that microglial heterogeneity reflects both tissue context and cellular. Following damage both subretinal and parenchymal microglia rapidly alter their morphology and migrate toward the injured site, resulting in their accumulation in the subretinal space. Microglia subsequently proliferate predominantly within this compartment. By 14 days post lesion, when photoreceptor regeneration is nearly completed, microglial morphology, distribution, and the number largely return to their unlesioned state. In contrast to the dynamic response of retinal microglia, response of microglia within the optic tectum was negligible. To investigate the mechanisms underlying the maintenance of these distinct microglial populations and their injury-induced responses, we examined retinal microglia in csf1ra mutants. In mutants lacking colony stimulating factor 1 receptor a (Csf1ra), parenchymal microglia are markedly reduced, whereas the number of subretinal microglia is preserved. Photoreceptor death following photolytic injury was comparable between csf1ra mutants and wildtype animals, and both parenchymal and subretinal microglia responded to the injury. However, proliferation of microglia in csf1ra mutants was severely impaired, and the normal redistribution of parenchymal microglia was not restored. These findings identify distinct anatomical and molecular features of retinal microglia and demonstrate that Csf1ra signaling differentially regulates microglial maintenance and injury-induced dynamics during photoreceptor regeneration.

neuroscience↗

bioRxiv2021

The microRNA miR-18a links proliferation and inflammation during photoreceptor regeneration in the injured zebrafish retina

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Muller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.

neuroscience↗

bioRxiv2020

Midkine-a functions as a universal regulator of proliferation during epimorphic regeneration in adult zebrafish

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.

developmental biology↗

bioRxiv2019

Midkine-a is required for cell cycle progression of Müller glia during neuronal regeneration

In zebrafish, Muller glia function as intrinsic retinal stem cells that can regenerate ablated neurons. Understanding the mechanisms governing neuronal stem cells may provide clues to regenerate neurons in mammals. We report that in Muller glia the cytokine/growth factor, Midkine-a, functions as a core autocrine regulator of the cell cycle. Utilizing midkine-a mutants, we determined that Midkine-a regulates elements of an Id2a-retinoblastoma network in reprogrammed Muller glia that controls the expression of cell cycle genes and is required for transition from G1 to S phases of the cell cycle. In mutants, Muller glia that fail to divide undergo reactive gliosis, a pathological hallmark of Muller glia in mammals. Finally, we show that activation of the Midkine-a receptor, ALK, is required for Muller glia proliferation. These data provide mechanistic insights into Muller glia stem cells in the vertebrate retina and suggest avenues for eliciting neuronal regeneration in mammals.

developmental biology↗

bioRxiv2019

Inflammation and matrix metalloproteinase 9 (Mmp-9) regulate photoreceptor regeneration in the adult zebrafish

Brain injury activates complex inflammatory signals in dying neurons, surviving neurons, and glia. Here, we establish that inflammation regulates the regeneration of photoreceptors in the zebrafish retina and determine the cellular expression and function of the inflammatory protease, matrix metalloproteinase 9 (Mmp-9), during this regenerative neurogenesis. Following sterile photoreceptor ablation anti-inflammatory treatment suppresses both the number of injury-induced progenitors and regenerated photoreceptors. Upon photoreceptor injury, mmp-9 is induced in Muller glia, the intrinsic retinal stem cell, and Muller glia-derived photoreceptor progenitors. Deleting mmp-9 results in over production of injury-induced progenitors and regenerated photoreceptors, but over time the absence of Mmp-9 compromises the maturation and survival of the regenerated cones. Anti-inflammatory treatment in mutants rescues the defects in cone maturation and survival. These data provide a link between injury-induced inflammation in the vertebrate CNS, Mmp-9 function during photoreceptor regeneration and the requirement of Mmp-9 for the survival of regenerated cones. Significance StatementThe innate immune system is activated by neuronal death, and recent studies demonstrate that in zebrafish neuroinflammation is required for neuronal regeneration. The roles of inflammatory cytokines are being investigated, however, the function of the inflammatory protease, matrix metalloprotease Mmp-9, in neuronal regeneration is unknown. We show herein that in adult zebrafish retinal inflammation governs the proliferative phase of the stem cell-based regeneration of rod and cone photoreceptors and determine the specific roles for Mmp-9 in photoreceptor regeneration. This study provides the first mechanistic insights into the potential role of Mmp-9 and serves to link neuroinflammation, stem cell-based regeneration of photoreceptors and human photoreceptor disease.

neuroscience↗

bioRxiv2018

The microRNA, miR-18a, regulates NeuroD and photoreceptor differentiation in the retina of the zebrafish

During embryonic retinal development, six types of retinal neurons are generated from a pool of multipotent progenitors in a strict spatiotemporal pattern. This pattern requires cell cycle exit (i.e. neurogenesis) and differentiation to be precisely regulated in a lineage-specific manner. In zebrafish, the bHLH transcription factor NeuroD governs photoreceptor genesis through Notch signaling but also governs photoreceptor differentiation though distinct mechanisms that are currently unknown. Also unknown are the mechanisms that regulate NeuroD and the spatiotemporal pattern of photoreceptor development. Members of the miR-17-92 microRNA cluster regulate CNS neurogenesis, and a member of this cluster, miR-18a, is predicted to target neuroD mRNA. The purpose of this study was to determine if miR-18a regulates NeuroD in the retina and if it plays a role in photoreceptor development. Quantitative RT-PCR showed that, of the three miR-18 family members (miR-18a, b and c), miR-18a expression most closely parallels neuroD expression. Morpholino oligonucleotides and CRISPR/Cas9 gene editing were used for miR-18a loss-of-function (LOF) and both approaches resulted in larvae with more mature photoreceptors at 70 hpf without affecting cell proliferation. Western blot showed that miR-18a LOF increases NeuroD protein levels and in vitro dual luciferase assay showed that miR-18a directly interacts with the 32UTR of neuroD. Finally, tgif1 mutants have increased miR-18a expression, less NeuroD protein and fewer mature photoreceptors, and the photoreceptor deficiency is rescued by miR-18a knockdown. Together these results show that, independent of neurogenesis, miR-18a regulates the timing of photoreceptor differentiation and indicate that this occurs through post-transcriptional regulation of NeuroD.

developmental biology↗