Heterogeneous pdgfrb+ cells regulate coronary vessel development and revascularization during heart regeneration.

Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrß, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.

Midkine-a Is Required for Cell Cycle Progression of Müller Glia during Neuronal Regeneration in the Vertebrate Retina.

In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of regenerating all classes of retinal neurons and restoring visual function. Understanding the cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish may provide cues to unlock the regenerative potential in the mammalian nervous system. Midkine is a cytokine/growth factor with multiple roles in neural development, tissue repair, and disease. In midkine-a loss-of-function mutants of both sexes, Müller glia initiate the appropriate reprogramming response to photoreceptor death by increasing expression of stem cell-associated genes, and entering the G1 phase of the cell cycle. However, transition from G1 to S phase is blocked in the absence of Midkine-a, resulting in significantly reduced proliferation and selective failure to regenerate cone photoreceptors. Failing to progress through the cell cycle, Müller glia undergo reactive gliosis, a pathological hallmark in the injured CNS of mammals. Finally, we determined that the Midkine-a receptor, anaplastic lymphoma kinase, is upstream of the HLH regulatory protein, Id2a, and of the retinoblastoma gene, p130, which regulates progression through the cell cycle. These results demonstrate that Midkine-a functions as a core component of the mechanisms that regulate proliferation of stem cells in the injured CNS.SIGNIFICANCE STATEMENT The death of retinal neurons and photoreceptors is a leading cause of vision loss. Regenerating retinal neurons is a therapeutic goal. Zebrafish can regenerate retinal neurons from intrinsic stem cells, Müller glia, and are a powerful model to understand how stem cells might be used therapeutically. Midkine-a, an injury-induced growth factor/cytokine that is expressed by Müller glia following neuronal death, is required for Müller glia to progress through the cell cycle. The absence of Midkine-a suspends proliferation and neuronal regeneration. With cell cycle progression stalled, Müller glia undergo reactive gliosis, a pathological hallmark of the mammalian retina. This work provides a unique insight into mechanisms that control the cell cycle during neuronal regeneration.

Defect patterns on the curved surface of fish retinae suggest a mechanism of cone mosaic formation.

The outer epithelial layer of zebrafish retinae contains a crystalline array of cone photoreceptors, called the cone mosaic. As this mosaic grows by mitotic addition of new photoreceptors at the rim of the hemispheric retina, topological defects, called "Y-Junctions", form to maintain approximately constant cell spacing. The generation of topological defects due to growth on a curved surface is a distinct feature of the cone mosaic not seen in other well-studied biological patterns like the R8 photoreceptor array in the Drosophila compound eye. Since defects can provide insight into cell-cell interactions responsible for pattern formation, here we characterize the arrangement of cones in individual Y-Junction cores as well as the spatial distribution of Y-junctions across entire retinae. We find that for individual Y-junctions, the distribution of cones near the core corresponds closely to structures observed in physical crystals. In addition, Y-Junctions are organized into lines, called grain boundaries, from the retinal center to the periphery. In physical crystals, regardless of the initial distribution of defects, defects can coalesce into grain boundaries via the mobility of individual particles. By imaging in live fish, we demonstrate that grain boundaries in the cone mosaic instead appear during initial mosaic formation, without requiring defect motion. Motivated by this observation, we show that a computational model of repulsive cell-cell interactions generates a mosaic with grain boundaries. In contrast to paradigmatic models of fate specification in mostly motionless cell packings, this finding emphasizes the role of cell motion, guided by cell-cell interactions during differentiation, in forming biological crystals. Such a route to the formation of regular patterns may be especially valuable in situations, like growth on a curved surface, where the resulting long-ranged, elastic, effective interactions between defects can help to group them into grain boundaries.

Inflammation and matrix metalloproteinase 9 (Mmp-9) regulate photoreceptor regeneration in 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 photoreceptor ablation, anti-inflammatory treatment suppresses the number of injury-induced progenitors and regenerated photoreceptors. Upon photoreceptor injury, mmp-9 is induced in Müller glia and Müller 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 survival of the regenerated cones. At all time-points studied, the levels of tnf-α are significantly elevated in mutant retinas. Anti-inflammatory treatment in mutants rescues the defects in cone survival. These data provide a link between injury-induced inflammation in the vertebrate CNS, Mmp-9 function during neuronal regeneration and the requirement of Mmp-9 for the survival of regenerated cones.

A self-renewing division of zebrafish Müller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons.

Müller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Müller glia partially dedifferentiate, re-express neuroepithelial markers and re-enter the cell cycle. We show that the immunoglobulin superfamily adhesion molecule Alcama is a novel marker of multipotent retinal stem cells, including injury-induced Müller glia, and that each Müller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Müller glia involves interkinetic nuclear migration, but mitosis of retinal progenitors occurs in situ. Rapidly dividing retinal progenitors form neurogenic clusters tightly associated with Alcama/N-cadherin-labeled Müller glial radial processes. Genetic suppression of N-cadherin function interferes with basal migration of retinal progenitors and subsequent regeneration of HuC/D(+) inner retinal neurons.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes.

Inflammation Regulates the Multi-Step Process of Retinal Regeneration in Zebrafish.

The ability to regenerate tissues varies between species and between tissues within a species. Mammals have a limited ability to regenerate tissues, whereas zebrafish possess the ability to regenerate almost all tissues and organs, including fin, heart, kidney, brain, and retina. In the zebrafish brain, injury and cell death activate complex signaling networks that stimulate radial glia to reprogram into neural stem-like cells that repair the injury. In the retina, a popular model for investigating neuronal regeneration, Müller glia, radial glia unique to the retina, reprogram into stem-like cells and undergo a single asymmetric division to generate multi-potent retinal progenitors. Müller glia-derived progenitors then divide rapidly, numerically matching the magnitude of the cell death, and differentiate into the ablated neurons. Emerging evidence reveals that inflammation plays an essential role in this multi-step process of retinal regeneration. This review summarizes the current knowledge of the inflammatory events during retinal regeneration and highlights the mechanisms whereby inflammatory molecules regulate the quiescence and division of Müller glia, the proliferation of Müller glia-derived progenitors and the survival of regenerated neurons.

Growth-associated protein43 (GAP43) is a biochemical marker for the whole period of fish optic nerve regeneration.

In adult visual system, goldfish can regrow their axons and fully restore their visual function even after optic nerve transection. The optic nerve regeneration process in goldfish is very long and it takes about a half year to fully recover visual function via synaptic refinement. Therefore, we investigated time course of growth-associated protein 43 (GAP43) expression in the goldfish retina for over 6 months after axotomy. In the control retina, very weak immunoreactivity could be seen in the retinal ganglion cells (RGCs). The immunoreactivity of GAP43 started to increase in the RGCs at 5 days, peaked at 7-20 days and then gradually decreased at 30-40 days after axotomy. The weak but significant immunoreactivity of GAP43 in the RGCs continued during 50-90 days and slowly returned to the control level by 180 days after lesion. The levels of GAP43 mRNA showed a biphasic pattern; a short-peak increase (9-folds) at 1-3 weeks and a long plateau increase (5-folds) at 50-120 days after axotomy. Thereafter, the levels declined to the control value by 180 days after axotomy. The changes of chasing behavior of pair of goldfish with bilaterally axotomized optic nerve also showed a slow biphasic recovery pattern in time course. Although further experiment is needed to elucidate the role of GAP43 in the regrowing axon terminals, the GAP43 is a good biochemical marker for monitoring the whole period of optic nerve regeneration in fish.

A hypoplastic retinal lamination in the purpurin knock down embryo in zebrafish.

Recently, we cloned a photoreceptor-specific purpurin cDNA from axotomized goldfish retina. In the present study, we investigate the structure of zebrafish purpurin genomic DNA and its function during retinal development. First, we cloned a 3.7-kbp genomic DNA fragment including 1.4-kbp 5'-flanking region and 2.3-kbp full-length coding region. In the 1.4-kbp 5'-upstream region, there were some cone-rod homeobox (crx) protein binding motifs. The vector of the 1.4-kbp 5'-flanking region combined with the reporter GFP gene showed specific expression of this gene only in the photoreceptors. Although the first appearance time of purpurin mRNA expression was a little bit later (40 hpf) than that of crx (17-24 hpf), the appearance site was identical to the ventral part of the retina. Next, we made purpurin or crx knock down embryos with morpholino antisense oligonucleotides. The both morphants (purpurin and crx) showed similar abnormal phenotypes in the eye development; small size of eyeball and lacking of retinal lamination. Furthermore, co-injection of crx morpholino and purpurin mRNA significantly rescued these abnormalities. These data strongly indicate that purpurin is a key molecule for the cell differentiation during early retinal development in zebrafish under transcriptional crx regulation.

Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging.

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Reprogramming Müller Glia to Regenerate Retinal Neurons.

In humans, various genetic defects or age-related diseases, such as diabetic retinopathies, glaucoma, and macular degeneration, cause the death of retinal neurons and profound vision loss. One approach to treating these diseases is to utilize stem and progenitor cells to replace neurons in situ, with the expectation that new neurons will create new synaptic circuits or integrate into existing ones. Reprogramming non-neuronal cells in vivo into stem or progenitor cells is one strategy for replacing lost neurons. Zebrafish have become a valuable model for investigating cellular reprogramming and retinal regeneration. This review summarizes our current knowledge regarding spontaneous reprogramming of Müller glia in zebrafish and compares this knowledge to research efforts directed toward reprogramming Müller glia in mammals. Intensive research using these animal models has revealed shared molecular mechanisms that make Müller glia attractive targets for cellular reprogramming and highlighted the potential for curing degenerative retinal diseases from intrinsic cellular sources.

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.

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo.

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish (Carassius auratus). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5-40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5-40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo. None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.

Changes of phospho-growth-associated protein 43 (phospho-GAP43) in the zebrafish retina after optic nerve injury: a long-term observation.

The major model animal of optic nerve regeneration in fish is goldfish. A closely related zebrafish is the most popular model system for genetic and developmental studies of vertebrate central nervous system. A few challenging works of optic nerve regeneration have been done with zebrafish. However, knowledge concerning the long term of optic nerve regeneration apparently lacks in zebrafish. In the present study, therefore, we followed changes of zebrafish behavior and phosphorylated form of growth-associated protein 43 (phospho-GAP43) expression in the zebrafish retina over 100 days after optic nerve transection. Optomotor response was fast recovered by 20-25 days after axotomy whereas chasing behavior (a schooling behavior) was slowly recovered by 80-100 days after axotomy. The temporal pattern of phospho-GAP43 expression showed a biphasic increase, a short-peak (12 folds) at 1-2 weeks and a long-plateau (4 folds) at 1-2 months after axotomy. The recovery of optomotor response well correlated with projection of growing axons to the tectum, whereas the recovery of chasing behavior well correlated with synaptic refinement of retinotectal topography. The present data strongly suggest that phospho-GAP43 plays an active role in both the early and late stages of optic nerve regeneration in fish.

Novel Animal Model of Crumbs-Dependent Progressive Retinal Degeneration That Targets Specific Cone Subtypes.

Purpose: Human Crb1 is implicated in some forms of retinal degeneration, suggesting a role in photoreceptor maintenance. Multiple Crumbs (Crb) polarity genes are expressed in vertebrate retina, although their functional roles are not well understood. To gain further insight into Crb and photoreceptor maintenance, we compared retinal cell densities between wild-type and Tg(RH2-2:Crb2b-sfEX/RH2-2:GFP)pt108b transgenic zebrafish, in which the extracellular domain of Crb2b-short form (Crb2b-sfEX) is expressed in the retina as a secreted protein, which disrupts the planar organization of RGB cones (red, green, and blue) by interfering with Crb2a/2b-based cone-cone adhesion. Methods: We used standard morphometric techniques to assess age-related changes in retinal cell densities in adult zebrafish (3 to 27 months old), and to assess effects of the Crb2b-sfEX transgene on retinal structure and photoreceptor densities. Linear cell densities were measured in all retinal layers in radial sections with JB4-Feulgen histology. Planar (surface) densities of cones were determined in retinal flat-mounts. Cell counts from wild-type and pt108b transgenic fish were compared with both a "photoreceptor maintenance index" and statistical analysis of cell counts. Results: Age-related changes in retinal cell linear densities and cone photoreceptor planar densities in wild-type adult zebrafish provided a baseline for analysis. Expression of Crb2b-sfEX caused progressive and selective degeneration of RGB cones, but had no effect on ultraviolet-sensitive (UV) cones, and increased numbers of rod photoreceptors. Conclusions: These differential responses of RGB cones, UV cones, and rods to sustained exposure to Crb2b-sfEX suggest that Crb-based photoreceptor maintenance mechanisms are highly selective.

Purpurin expression in the zebrafish retina during early development and after optic nerve lesion in adults.

Purpurin, a retina-specific protein, is known to play a role in cell adhesion during development of the chicken retina. Although purpurin has been significantly detected in adult chicken retina, its function in the matured retina is not well understood. Therefore, to determine the expression pattern of purpurin in the retina, we simultaneously investigated expression patterns of purpurin in the zebrafish retina during development in larvae and optic nerve regeneration after nerve transection in adults. In early development, levels of purpurin suddenly increased in the zebrafish retina 3 to 5 days after fertilization, and purpurin-positive immunoreactivity was diffusely located in all retinal layers. In contrast, levels of purpurin mRNA rapidly increased in the adult retina 1-3 days after optic nerve transection, and rapidly declined by 10 days after injury. Signal for purpurin mRNA was seen only in photoreceptors. Immunohistochemistry showed that levels of purpurin protein were also increased in the retina 1-3 days after nerve injury, but positive staining was located in photoreceptors and ganglion cells, and the staining in ganglion cells was stronger than that in photoreceptors. Thus, the transient expression of purpurin protein was greatly different during development and optic nerve regeneration. In the former, purpurin may be required in all retinal layers, whereas in the latter, purpurin may be required for injured ganglion cells.

Anisotropic Müller glial scaffolding supports a multiplex lattice mosaic of photoreceptors in zebrafish retina.

BACKGROUND: The multiplex, lattice mosaic of cone photoreceptors in the adult fish retina is a compelling example of a highly ordered epithelial cell pattern, with single cell width rows and columns of cones and precisely defined neighbor relationships among different cone types. Cellular mechanisms patterning this multiplex mosaic are not understood. Physical models can provide new insights into fundamental mechanisms of biological patterning. In earlier work, we developed a mathematical model of photoreceptor cell packing in the zebrafish retina, which predicted that anisotropic mechanical tension in the retinal epithelium orients planar polarized adhesive interfaces to align the columns as cone photoreceptors are generated at the retinal margin during post-embryonic growth. METHODS: With cell-specific fluorescent reporters and in vivo imaging of the growing retinal margin in transparent juvenile zebrafish we provide the first view of how cell packing, spatial arrangement, and cell identity are coordinated to build the lattice mosaic. With targeted laser ablation we probed the tissue mechanics of the retinal epithelium. RESULTS: Within the lattice mosaic, planar polarized Crumbs adhesion proteins pack cones into a single cell width column; between columns, N-cadherin-mediated adherens junctions stabilize Müller glial apical processes. The concentration of activated pMyosin II at these punctate adherens junctions suggests that these glial bands are under tension, forming a physical barrier between cone columns and contributing to mechanical stress anisotropies in the epithelial sheet. Unexpectedly, we discovered that the appearance of such parallel bands of Müller glial apical processes precedes the packing of cones into single cell width columns, hinting at a possible role for glia in the initial organization of the lattice mosaic. Targeted laser ablation of Müller glia directly demonstrates that these glial processes support anisotropic mechanical tension in the planar dimension of the retinal epithelium. CONCLUSIONS: These findings uncovered a novel structural feature of Müller glia associated with alignment of photoreceptors into a lattice mosaic in the zebrafish retina. This is the first demonstration, to our knowledge, of planar, anisotropic mechanical forces mediated by glial cells.