Search

Cognitive theories of depression, and mindfulness theories of well-being, converge on the notion that self-judgment plays a critical role in mental health. However, these theories have rarely been tested via tasks and computational modeling analyses that can disentangle the information processes operative in self-judgments. We applied a drift-diffusion computational model to the self-referential encoding task (SRET) collected before and after an 8-week mindfulness intervention (n = 96). A drift-rate regression parameter representing positive-relative to negative-self-referential judgment strength positively related to mindful awareness and inversely related to depression, both at baseline and over time; however, this parameter did not significantly relate to the interaction between mindful awareness and nonjudgmentalness. At the level of individual depression symptoms, at baseline, a spectrum of symptoms (inversely) correlated with the drift-rate regression parameter, suggesting that many distinct depression symptoms relate to valenced self-judgment between subjects. By contrast, over the intervention, changes in only a smaller subset of anhedonia-related depression symptoms showed substantial relationships with this parameter. Both behavioral and model-derived measures showed modest split-half and test-retest correlations. Results support cognitive theories that implicate self-judgment in depression and mindfulness theories, which imply that mindful awareness should lead to more positive self-views.

How does rumination affect reinforcement learning—the ubiquitous process by which people adjust behavior after error to behave more effectively in the future? In a within-subjects design ( N = 49), we tested whether experimentally manipulated rumination disrupts reinforcement learning in a multidimensional learning task previously shown to rely on selective attention. Rumination impaired performance, yet unexpectedly, this impairment could not be attributed to decreased attentional breadth (quantified using a decay parameter in a computational model). Instead, trait rumination (between subjects) was associated with higher decay rates (implying narrower attention) but not with impaired performance. Our task-performance results accord with the possibility that state rumination promotes stress-generating behavior in part by disrupting reinforcement learning. The trait-rumination finding accords with the predictions of a prominent model of trait rumination (the attentional-scope model). More work is needed to understand the specific mechanisms by which state rumination disrupts reinforcement learning.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller 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-18a(mi5012)), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18a(mi5012) 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-18a(mi5012) 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-18a(mi5012) retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18a(mi5012) 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.

Why has computational psychiatry yet to influence routine clinical practice? One reason may be that it has neglected context and temporal dynamics in the models of certain mental health problems. We develop three heuristics for estimating whether time and context are important to a mental health problem: Is it characterized by a core neurobiological mechanism? Does it follow a straightforward natural trajectory? And is intentional mental content peripheral to the problem? For many problems the answers are no, suggesting that modeling time and context is critical. We review computational psychiatry advances toward this end, including modeling state variation, using domain-specific stimuli, and interpreting differences in context. We discuss complementary network and complex systems approaches. Novel methods and unification with adjacent fields may inspire a new generation of computational psychiatry. Expected final online publication date for the Annual Review of Psychology, Volume 73 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that fully regenerate photoreceptors. MicroRNAs (miRNAs) regulate neurogenesis in the CNS, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miRNA 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 by 1-day post-injury (dpi) and increases through 5 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 bromodeoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess Müller glia-derived progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 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 during photoreceptor regeneration in zebrafish, miR-18a regulates proliferation and photoreceptor regeneration by regulating the inflammatory response.

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., Acute inflammation is necessary for photoreceptor regeneration in zebrafish.MMP‐9 negatively regulates Müller glia‐derived progenitors during photoreceptor regeneration.Mmp‐9 promotes the survival of regenerated cone photoreceptors.

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 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.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), Müller glia function as intrinsic stem cells, producing progenitor cells that regenerate photoreceptors and restore vision. MicroRNAs (miRNAs) critically regulate neurogenesis in the brain and retina, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. The miRNA miR-18a regulates photoreceptor differentiation in the embryonic retina. The purpose of the current study was to determine the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization (ISH) and immunohistochemistry (IHC) showed that miR-18a expression increases throughout the retina by 1-day post-injury (dpi) and continues to increase through 5 dpi. Bromodeoxyuridine (BrdU) labeling showed that at 7 and 10 dpi, when regenerated photoreceptors are normally differentiating, there are more proliferating Müller glia-derived progenitors in homozygous miR-18a mutant (miR-18ami5012) retinas compared with wild type (WT), indicating that miR-18a negatively regulates injury-induced proliferation. At 7 and 10 dpi, miR-18ami5012 retinas have fewer mature photoreceptors than WT, but there is no difference at 14 dpi, revealing that photoreceptor regeneration is delayed. BrdU labeling showed that the excess progenitors in miR-18ami5012 retinas migrate to other retinal layers besides the photoreceptor layer. Inflammation is critical for photoreceptor regeneration and RT-qPCR showed that, in the absence of miR-18a, inflammation is prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that during injury-induced photoreceptor regeneration, miR-18a regulates proliferation and photoreceptor regeneration by regulating key aspects of the inflammatory response during photoreceptor regeneration in zebrafish.

Endothelial cells emerge from the atrioventricular canal (AVC) to form nascent coronary blood vessels in the juvenile zebrafish heart. We found that pdgfrβ is first expressed in the epicardium around the AVC and later becomes localized mainly in the mural cells. pdgfrβ mutant fish display severe defects in mural cell recruitment and coronary vessel development. pdgfrβ+ mural cells are heterogeneous and those associated with coronary arteries also express cxcl12b. Mural cells positive for both pdgfrβ and cxcl12b transgenic reporters had elevated expression of smooth muscle cell genes. Interestingly, these mural cells were associated with coronary arteries even in the absence of Pdgfrβ, although smooth muscle gene expression was downregulated in these cells. We found that pdgfrβ expression dynamically changes in the epicardium derived cells, which we found to be a heterogeneous population. mdka was identified as a gene upregulated in subpopulations of pdgfrβ+ cells during heart regeneration. However, pdgfrβ but not mdka mutants showed defects in heart regeneration. Our results demonstrated that pdgfrβ+ cells and Pdgfrβ signaling are essential for coronary development and heart regeneration.SUMMARY STATEMENTHeterogeneous pdgfrβ positive cells are present in developing and regenerating zebrafish hearts and are required for development of mural cells and their association with the nascent coronary vessels during zebrafish heart development and 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 Pdgfrβ, 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.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller 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.

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.

During embryonic retinal development, six types of retinal neurons are generated from 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, in the developing zebrafish retina, miR-18a regulates NeuroD 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 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 3' UTR 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.

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.

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.

We evaluated the expression and function of the microglia-specific growth factor, Progranulin-a (Pgrn-a) during developmental neurogenesis in the embryonic retina of zebrafish. At 24 hpf pgrn-a is expressed throughout the forebrain, but by 48 hpf pgrn-a is exclusively expressed by microglia and/or microglial precursors within the brain and retina. Knockdown of Pgrn-a does not alter the onset of neurogenic programs or increase cell death, however, in its absence, neurogenesis is significantly delayed-retinal progenitors fail to exit the cell cycle at the appropriate developmental time and postmitotic cells do not acquire markers of terminal differentiation, and microglial precursors do not colonize the retina. Given the link between Progranulin and cell cycle regulation in peripheral tissues and transformed cells, we analyzed cell cycle kinetics among retinal progenitors following Pgrn-a knockdown. Depleting Pgrn-a results in a significant lengthening of the cell cycle. These data suggest that Pgrn-a plays a dual role during nervous system development by governing the rate at which progenitors progress through the cell cycle and attracting microglial progenitors into the embryonic brain and retina. Collectively, these data show that Pgrn-a governs neurogenesis by regulating cell cycle kinetics and the transition from proliferation to cell cycle exit and differentiation. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 77: 1114-1129, 2017.

SummaryIn zebrafish, Müller 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 Müller 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 Müller 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, Müller glia that fail to divide undergo reactive gliosis, a pathological hallmark of Müller glia in mammals. Finally, we show that activation of the Midkine-a receptor, ALK, is required for Müller glia proliferation. These data provide mechanistic insights into Müller glia stem cells in the vertebrate retina and suggest avenues for eliciting neuronal regeneration in mammals.

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.

During embryonic retinal development, six types of retinal neurons are generated from 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, in the developing zebrafish retina, miR‐18a regulates NeuroD 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 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 3′ UTR 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.

This article summarizes the outcomes of the second national conference on the Future of Bioscience Graduate and Postdoctoral Training. Five topics were addressed during the conference: diversity in leadership positions; mentoring; modernizing the curriculum; experiential learning; and the need for better data on trainees. The goal of the conference was to develop a consensus around these five topics and to recommend policies that can be implemented by academic and research institutions and federal funding agencies in the United States.

The functional role of midkine during development, following injury and in disease has been studied in a variety of tissues. In this review, we summarize what is known about midkine in the vertebrate retina, focusing largely on recent studies utilizing the zebrafish (Danio rerio) as an animal model. Zebrafish are a valuable animal model for studying the retina, due to its very rapid development and amazing ability for functional neuronal regeneration following neuronal cell death. The zebrafish genome harbours two midkine paralogues, midkine-a (mdka) and midkine-b (mdkb), which, during development, are expressed in nested patterns among different cell types. mdka is expressed in the retinal progenitors and mdkb is expressed in newly post-mitotic cells. Interestingly, studies of loss-and gain-of-function in zebrafish larvae indicate that midkine-a regulates cell cycle kinetics. Moreover, both mdka and mdkb are expressed in different cell types in the normal adult zebrafish retina, but after light-induced death of photoreceptors, both are up-regulated and expressed in proliferating Muller glia and photoreceptor progenitors, suggesting an important and (perhaps) coincident role for these cytokines during stem cell-based neuronal regeneration. Based on its known role in other tissues and the expression and function of the midkine paralogues in the zebrafish retina, we propose that midkine has an important functional role both during development and regeneration in the retina. Further studies are needed to understand this role and the mechanisms that underlie it. Linked Articles This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4

This report emerges from a workshop convened by the National Eye Institute (NEI) as part of the "Audacious Goals Initiative" (AGI). The workshop addressed the replacement of retinal ganglion cells (RGCs) from exogenous and endogenous sources, and sought to identify the gaps in our knowledge and barriers to progress in devising cellular replacement therapies for diseases where RGCs die. Here, we briefly review relevant literature regarding common diseases associated with RGC death, the genesis of RGCs in vivo, strategies for generating transplantable RGCs in vitro, and potential endogenous cellular sources to regenerate these cells. These topics provided the clinical and scientific context for the discussion among the workshop participants and are relevant to efforts that may lead to therapeutic approaches for replacing RGCs. This report also summarizes the content of the workshop discussion, which focused on: (1) cell sources for RGC replacement and regeneration, (2) optimizing integration, survival, and synaptogenesis of new RGCs, and (3) approaches for assessing the outcomes of RGC replacement therapies. We conclude this report with a summary of recommendations, based on the workshop discussions, which may guide vision scientists seeking to develop therapies for replacing RGCs in humans.

We evaluated the expression and function of the microglia‐specific growth factor, Progranulin‐a (Pgrn‐a) during developmental neurogenesis in the embryonic retina of zebrafish. At 24 hpf pgrn‐a is expressed throughout the forebrain, but by 48 hpf pgrn‐a is exclusively expressed by microglia and/or microglial precursors within the brain and retina. Knockdown of Pgrn‐a does not alter the onset of neurogenic programs or increase cell death, however, in its absence, neurogenesis is significantly delayed—retinal progenitors fail to exit the cell cycle at the appropriate developmental time and postmitotic cells do not acquire markers of terminal differentiation, and microglial precursors do not colonize the retina. Given the link between Progranulin and cell cycle regulation in peripheral tissues and transformed cells, we analyzed cell cycle kinetics among retinal progenitors following Pgrn‐a knockdown. Depleting Pgrn‐a results in a significant lengthening of the cell cycle. These data suggest that Pgrn‐a plays a dual role during nervous system development by governing the rate at which progenitors progress through the cell cycle and attracting microglial progenitors into the embryonic brain and retina. Collectively, these data show that Pgrn‐a governs neurogenesis by regulating cell cycle kinetics and the transition from proliferation to cell cycle exit and differentiation. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 77: 1114–1129, 2017

The retina displays numerous processes that follow a circadian rhythm. These processes are coordinated through the direct action of light on photoreceptive molecules and, in the absence of light, through autocrine/paracrine actions of extracellular neuromodulators. We previously described the expression of the genes encoding the secreted heparin-binding growth factors,midkine-a(mdka) andmidkine-b(mdkb), in the retina of the zebrafish. Here, we provide evidence that the expression ofmdkaandmdkbfollows a daily rhythm, which is independent of the presence or absence of light, and we propose that the expression ofmdkais regulated by the circadian clock. Both qualitative and quantitative measures show that formdka, the levels of mRNA and protein decrease during the night and increase during the subjective day. Qualitative measures show that the expression ofmdkbincreases during the second half of the subjective night and decreases during the second half of the subjective day. Within horizontal cells, the two midkine paralogs show asynchronous circadian regulation. Though intensely studied in the contexts of physiology and disease, this is the first study to provide evidence for the circadian regulation of midkines in the vertebrate nervous system.

In the retina of adult teleosts, stem cells are sustained in two specialized niches: the ciliary marginal zone (CMZ) and the microenvironment surrounding adult Muller glia. Recently, Muller glia were identified as the regenerative stem cells in the teleost retina. Secreted signaling molecules that regu- late neuronal regeneration in the retina are largely unknown. In a microarray screen to discover such factors, we identi- fied midkine-b (mdkb). Midkine is a highly conserved heparin-binding growth factor with numerous biological functions. The zebrafish genome encodes two distinct mid- kine genes: mdka and mdkb. Here we describe the cellular expression of mdka and mdkb during retinal development and the initial, proliferative phase of photoreceptor regen- eration. The results show that in the embryonic and larval retina mdka and mdkb are expressed in stem cells, retinal progenitors, and neurons in distinct patterns that suggest different functions for the two molecules. Following the se- lective death of photoreceptors in the adult, mdka and mdkb are coexpressed in horizontal cells and proliferating Muller glia and their neurogenic progeny. These data reveal that Mdka and Mdkb are signaling factors present in the retinal stem cell niches in both embryonic and mature retinas, and that their cellular expression is actively modulated during retinal development and regeneration. J. Comp. Neurol. 514

Retinal development in teleosts can broadly be divided into three epochs. The first is the specification of cellular domains in the larval forebrain that give rise to the retinal primordia and undergo early morphogenetic movements. The second is the neurogenic events within the retina proper-proliferation, cell fate determination, and pattern formation-that establish neuronal identities and form retinal laminae and cellular mosaics. The third, which is unique to teleosts and occurs in the functioning eye, is stretching of the retina and persistent neurogenesis that allows the growth of the retina to keep pace with the growth of the eye and other tissues. The first two events are rapid, complete by about 3 days postfertilization in the zebrafish embryo. The third is life-long and accounts for the bulk of retinal growth and the vast majority of adult retinal neurons. In addition, but clearly related to the retina's developmental history, lesions that kill retinal neurons elicit robust neuronal regeneration that originates from cells intrinsic to the retina. This paper reviews recent studies of retinal development in teleosts, focusing on those that shed light on the genetic and molecular regulation of retinal specification and morphogenesis in the embryo, retinal neurogenesis in larvae and adults, and injury-induced neuronal regeneration.

The retina of the adult teleost fish is an important model for studying persistent and injury-induced neurogenesis in the vertebrate central nervous system. All neurons, with the exception of rod photoreceptors, are continually appended to the extant retina from an annulus of progenitors at the margin. Rod photoreceptors, in contrast, are added to differentiated retina only from a lineage of progenitors dedicated to making rods. Further, when the retina is lesioned, the lineage that produces only rods ceases this activity and regenerates retinal neurons of all types. The progenitors that supply neurons at the retinal margin and rod photoreceptors and regenerated neurons in the mature tissue originate from multipotent stem cells. Recent data suggest that the growth-associated neurogenic activity in the retina is regulated as part of the growth hormone/insulin-like growth factor-I axis. This paper reviews recent evidence for the presence of stem cells in the teleost retina and the molecular regulation of neurogenesis and presents a consensus cellular model that describes persistent and injury-induced neurogenesis in the retinas of teleost fish.

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.

The retina of the adult goldfish grows throughout the life of the animal, in part, by the continual addition of new neurons. Further, destruction of extant neurons in this tissue stimulates neuronal regeneration. In an attempt to identify growth factors that regulate both normal and injury-stimulated neurogenesis, we used organ culture techniques and tested nine peptide growth factors for their ability to modulate cell proliferation in both normal retinas and retinas with lesions. Of the growth factors tested, only the insulin-related peptides (insulin and insulin-like growth factors I and II) consistently stimulated proliferation, and this was restricted to the retinal progenitors within the circumferential germinal zone. None of the growth factors tested stimulated proliferation of rod precursors (cells in the mature retina whose progeny are exclusively rod photoreceptors) or the injury-stimulated retinal progenitors. Although the negative data are subject to multiple interpretations, these data suggest that in the retina of the adult goldfish, insulin-related peptides regulate proliferation of retinal progenitors within the circumferential germinal zone, but molecules that modulate the proliferation of the rod precursors or injury-induced retinal progenitors in the retina of the adult goldfish have yet to be identified. J. Comp. Neurol. 394:386‐394, 1998. r 1998 Wiley-Liss, Inc.

Results of the previous study suggest that insulin-related peptides regulate proliferation of retinal progenitors in the adult goldfish. Because of their known roles in retinal neurogenesis, we have chosen to focus future studies on insulin-like growth factor I (IGF-I) and the IGF-I receptor. In the study described here, we characterized the spatial distribution and specificity of IGF-I binding sites in the retina of the adult goldfish by performing receptor-binding autoradiography with [ 125 I]-IGF-I alone and with unlabeled IGF-I-related molecules (IGF-I, IGF-II, insulin, and des-[1-3]-IGF-I) as competitive inhibitors of [ 125 I]-IGF-I binding. The results of these experiments show that IGF-I binds in two locations in the retina of the adult goldfish, within the inner plexiform layer of the differentiated retina and the circumferential germinal zone. The competition experiments suggest that [ 125 I]-IGF-I binds at sites specific for IGF-I, and that both IGF-I receptors and IGF-I binding proteins are present in the retina. J. Comp. Neurol. 394:395‐401, 1998. r 1998 Wiley-Liss, Inc. Indexing terms: teleost fish; autoradiography; growth factors; growth factor receptors; IGF-I

The mouse neurological mutant lethargic (lh) is characterized by ataxia, focal myoclonus, and absence epilepsy due to a loss-of-function mutation in the beta4 subunit of the voltage-gated calcium channel. To evaluate the role of this channel subunit in human neurological disease, we determined the chromosomal location and intron/exon structure of the human CACNB4 gene. The 1560-bp open reading frame of the CACNB4 cDNA predicts a 58-kDa protein with an amino acid sequence that is 99% identical to the rat protein. The 13 coding exons of CACNB4 span >55 kb of genomic DNA. Human cerebellar RNA contains one major CACNB4 transcript that is 9 kb in length. Expression of CACNB4 was detected in cerebellum, kidney, testis, retina, lymphoblasts, and circulating lymphocytes. Retinal transcripts were localized by in situ hybridization to ganglion cells and the inner nuclear layer. Analysis of the GeneBridge 4 radiation hybrid mapping panel localized CACNB4 to position 791 cR on human chromosome 2, in a conserved linkage group on human 2q22-q31 and mouse chromosome 2. We localized CACNB4 to the 1.3-Mb YAC clone 952F10 in Whitehead contig WC861, along with the polymorphic markers D2S2236 and D2S2299. The chromosomal linkage of three of the four beta subunit genes to homeobox gene clusters associates the evolutionary origin of the beta gene family with the events that generated the four HOX clusters early in vertebrate evolution.

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.

Major depressive disorder (MDD) is characterized by cognitive biases in attention, memory and language use. Language use biases often parallel depression symptoms, and contain over-representations of both negative emotive and death words as well as low levels of positive emotive words. This study further explores cognitive biases in depression by comparing the effect of current depression status to cumulative depression history on an elaborated verbal recall of emotional photographs.Following a negative mood induction, fifty-two individuals (42 women) with partially-remitted depression viewed - then recalled and verbally described - slides from the International Affective Picture System (IAPS). Descriptions were transcribed and frequency of depression-related word use (positive emotion, negative emotion, sex, ingestion and death) was analyzed using the Linguistic Inquiry and Word Count program (LIWC).Contrary to expectations and previous findings, current depression status did not affect word use in any categories of interest. However, individuals with more than 5 years of previous depression used fewer words related to positive emotion (t(50) = 2.10, p = .04, (d = 0.57)), and sex (t(48) = 2.50, p = .013 (d = 0.81)), and there was also a trend for these individuals to use fewer ingestion words (t(50) = 1.95, p = .057 (d = 0.58)), suggesting a deficit in appetitive processing.Our findings suggest that depression duration affects appetitive information processing and that appetitive word use may be a behavioral marker for duration related brain changes which may be used to inform treatment.

Background Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina’s stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. Results The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. Conclusions These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.

Objectives: Converging research suggests that mindfulness training exerts its therapeutic effects on depression by reducing rumination. Theoretically, rumination is a multifaceted construct that aggregates multiple neurocognitive aspects of depression, including poor executive control, negative and overgeneral memory bias, and persistence or stickiness of negative mind states. Current measures of rumination, most often self-reports, do not capture these different aspects of ruminative tendencies, and therefore are limited in providing detailed information about the mechanisms of mindfulness. Methods: We developed new insight into the potential mechanisms of rumination, based on three model-based metrics of free recall dynamics. These three measures reflect the patterns of memory retrieval of valenced information: The probability of first recall (Pstart) which represents initial affective bias, the probability of staying with the same valence category rather than switching, which indicates strength of positive or negative association networks (Pstay); and probability of stopping (Pstop) or ending recall within a given valence, which indicates "drift" persistence or stickiness of a mind state. We investigated the effects of MBCT (N=29) vs wait-list control (N=23) on these recall dynamics in a Randomized Controlled Trial (RCT) in individuals with recurrent depression. Participants completed a standard laboratory stressor, the Trier Social Stress Test (TSST), to induce negative mood and activate ruminative tendencies. Following that, participants completed a free recall task consisting of three word lists. This assessment was conducted both before and after treatment or wait-list. Results: While MBCT participant's Pstart remained relatively stable, controls showed multiple indications of depression-related deterioration towards more negative and less positive bias. Following the intervention, MBCT participants decreased in their tendency to sustain trains of negative words and increased their tendency to sustain trains of positive words. Conversely, controls showed the opposite tendency: controls stayed in trains of negative words for longer, and stayed in trains of positive words for less time relative to T1 scores. MBCT participants tended to stop recall less often with negative words, which indicates less persistence or stickiness of negatively-valenced mental context. Conclusion: MBCT participants showed a decrease in patterns that may perpetuate rumination on all three types of recall dynamics (Pstart, Pstay and Pstop), compared to controls. MBCT may weaken the strength of self-perpetuating negative associations networks that are responsible for the persistent and "sticky" negative mind states observed in depression, and increase in positive associations that are lacking in depression. This study also offers a novel, objective method of measuring several indices of ruminative tendencies indicative of the underlying mechanisms of rumination. © 2012 Van vugt, Hitchcock, Shahar and Britton.

Microglia are cells from non-neuronal lineages that reside in the central nervous system. In zebrafish, early macrophages migrate from the yolk sac to the brain and retina at 26–30 hours post fertilization (hpf) and transform into microglia at 55–60 hpf. The migration of macrophages into the central nervous system requires signaling by macrophage colony stimulating factor-1 receptor (csf-1r), which is encoded by the gene fms. In this study, we show that the targeted knockdown of csf-1r with morpholino oligonucleotides delays migration of macrophages from the yolk sac to the retina, and this delay in macrophage migration results in microphthalmia, delay in cell cycle withdrawal among retinal progenitors and the absence of neuronal differentiation. When embryos were allowed to survive beyond the time when morpholino-dependent translation inhibition is lost, microglia re-occupy the retina and neuronal differentiation partially recovers. Our data demonstrate that microglia are required for normal retinal growth and neurogenesis. This study provides new insight into the neurogenic role of microglia during retinal development in zebrafish.

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transscleral surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2'-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithial-like cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.

The retina of the goldfish grows by a balloon-like expansion and by the addition of new neurons at the margin. It has been proposed that as a consequence of this expansion the dendritic arbors of ganglion cells in central retina grow in a uniform manner without the addition of new branches. In the present study, we have examined this proposal by comparing the geometries of individual dendritic arbors of large-field ganglion cells from the retinas of small/young and large/old fish. These comparisons were based on measurements of several parameters of dendritic morphology, including number of segments and branches, branch angles, changes in diameter at branch points, and proximal versus distal distribution of arbor length. In addition, we used passive, steady-state cable modeling as an independent method of estimating the functional architectures of small and large dendritic arbors. Our morphometric data indicate that, though they are very different in absolute size, dendritic arbors of small and large ganglion cells have remarkably similar architectures. Analysis with steady-state cable equations indicates that the arbors from small and large cells have equivalent electrotonic lengths and show comparable propagation of synaptic currents. These data are consistent with the hypothesis that dendritic arbors of small and large ganglion cells are scaled versions of one another. We conclude that the growth of these cells during the expansion of the retina is the result of the addition of dendritic mass to an arbor whose basic geometry remains unchanged.

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD(EGFP) fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rodhomeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation. J. Comp. Neurol. 501:1–12, 2007. © 2007 Wiley-Liss, Inc. Indexing terms: neurogenesis; development; gene expression; photoreceptors

The vertebrate retina is composed of seven cell types, generated in a conserved sequence from a pool of multipotent, mitotic progenitors.1 Several basic helix-loop-helix (bHLH) transcription factors have prominent roles in the retina to regulate cell fate specification,2–5 cell cycle exit, and neuronal differentiation and maturation.6,7 NeuroD is a proneural bHLH transcription factor identified in vivo as a neuronal determination factor,8 and in vitro as a link between cell cycle exit and neuronal differentiation.9 In the mouse retina, NeuroD is required for survival of rods10 and regulates opsin selection among cones.11 In the chick, NeuroD is required for photoreceptor development and survival.12 In medaka fish, NeuroD and Six6 coregulate each other to control amacrine cell and photoreceptor genesis.6 In embryonic zebrafish, neurod is expressed in mitotic photoreceptor progenitors,13,14 and this expression is regulated by the zinc finger protein, Insm1a.15 Within the photoreceptor lineages NeuroD governs cell cycle exit and photoreceptor maturation.7 The Notch pathway mediates cell-to-cell communication through receptor-ligand interactions. Notch receptors are expressed on the cell surface and interact with membrane-bound ligands (e.g., Delta, Jagged), regulating transcription in apposing cells.16,17 In vertebrate retinal development, Notch signaling regulates the balance between neurogenesis and gliogenesis,18–20 maintains progenitors in an undifferentiated, proliferative state,19,21 specifies cell fates, and governs the onset of neurogenesis.22 These events can be regulated in the retina through transcriptional control of Notch signaling molecules. For example, in the chick and mouse, the bHLH transcription factor Ascl1a governs cell cycle exit and differentiation through regulation of the Notch ligand dll,23,24 and in zebrafish the HLH-binding cofactor Id2a regulates the transition from proliferation to differentiation through regulation of Notch ligands and receptors.21 Following retinal damage in zebrafish, Muller glia function as intrinsic stem cells that dedifferentiate, reenter the cell cycle, and produce neurogenic progenitors that regenerate lost neurons.25–33 Recent studies in zebrafish have identified mechanisms that govern the Muller glia response to retinal injury,34–37 but information is lacking around the pathways that govern photoreceptor regeneration from Muller glia–derived progenitor cells. The mechanisms that govern retinal regeneration are expected to largely recapitulate embryonic development,28,29,38 though few molecules have been functionally studied during both events. NeuroD function has not been studied in the adult zebrafish retina, but following photoreceptor ablation neurod is expressed in Muller glia–derived mitotic progenitors,39 suggesting that NeuroD has a role in photoreceptor regeneration. The goal of the current study is to identify the mechanisms that govern photoreceptor genesis from the pool of multipotent progenitors in the embryo and from stem cell-derived progenitors in the adult, by elucidating the pathways through which NeuroD functions during photoreceptor development and regeneration, respectively. In embryos, reciprocal transplant chimeric analysis shows that for cell cycle exit and photoreceptor maturation, NeuroD function is non–cell-autonomous. Knockdown of NeuroD and CRISPR/Cas9 targeted mutation of neurod prevent cell cycle exit and photoreceptor maturation, and increase expression of Notch pathway molecules. Inhibition of Notch signaling rescues deficiencies in cell cycle exit but not photoreceptor maturation. In adults, NeuroD knockdown prevents cell cycle exit among injury-induced progenitors and photoreceptor regeneration, and this, too, is rescued by Notch inhibition. These data demonstrated a conserved function for NeuroD during photoreceptor genesis and regeneration, and identified Notch signaling as a molecular mechanism that links these events.

To identify the gene responsible for a complex ocular phenotype of late-onset macular degeneration, long anterior zonules (LAZ), and elevated intraocular pressure (IOP) and to study its expression.Ocular examination, visual field, fluorescein angiography, and electrophysiology testing were performed. One affected individual was treated with vitamin A. DNA from 55 family members (UM:H389) was used for linkage, mapping, and mutation analysis. Linkage analysis of macular degeneration and LAZ phenotypes was performed independently. Mutations in candidate genes were screened by sequencing. mRNA expression of CTRP5 and MFRP, which are bicistronic genes, was studied by semiquantitative RT-PCR (qRT-PCR) in various human tissues. CTRP5 expression was also evaluated by in situ hybridization.Affected members had LAZ detectable by the third decade and/or macular degeneration by the fourth to fifth decade. A six-month treatment with vitamin A shortened dark adaptation considerably in one affected member. Both conditions mapped independently with zero recombination to 11q23, with maximum lod scores of 3.31 for macular degeneration and 5.41 for LAZ. The same CTRP5 missense mutation was identified in all affected individuals. Retinal pigment epithelium (RPE) and ciliary epithelium (CE) showed highest CTRP5 transcript expression, which was also true for MFRP. CTRP5 tissue expression was confirmed by in situ hybridization.A single locus at 11q23 is implicated in a complex ocular phenotype involving RPE and CE, tissues of neuroectodermal origin. All individuals with either LAZ and/or macular degeneration carry the same CTRP5 S163R mutation, which is transmitted in autosomal dominant manner.

Persistent rod genesis in the retinas of teleost fish was first described over 2 decades ago, but little is known regarding the underlying genetic and molecular mechanisms that govern this phenomenon. Because of its function in the developing mammalian retina and persistently mitotic adult tissues, we sought to characterize the cellular expression of the basic helix-loop-helix (bHLH) transcription factor neuroD in the persistently neurogenic retina of adult teleosts. We show here that, in the adult retina of the goldfish, neuroD is expressed by putative amacrine cells, nascent cones, and the mitotically active cells of the rod lineage. neuroD is the first gene shown to be expressed by rod precursors, the immediate antecedents of rod photoreceptors. In contrast to the vertebrate classes described previously, neuroD is not expressed in multipotent progenitors in the teleost retina. Combining neuroD in situ hybridizations with cell-cycle-specific markers suggests that, in rod precursors, neuroD expression is cell cycle specific. J. Comp. Neurol. 477:108 –117, 2004. © 2004 Wiley-Liss, Inc.

Based on results from previous studies (J. Comp. Neurol. 394 (1998) 386, 395), it was hypothesized that the persistent neurogenesis in the retina of teleost fish is modulated by insulin-like growth factor-I (IGF-I), which, in turn, is regulated by growth hormone (GH). Two approaches were undertaken to test this hypothesis. First, a variety of techniques were used to determine if IGF-I and the IGF-I receptor (IGF-IR) are expressed in the retina. Second, GH was injected into animals to stimulate IGF-I synthesis in target tissues, and IGF-I expression and cell proliferation in the retina was quantitatively assayed. Reverse transcriptase-polymerase chain reaction, screening a retinal cDNA library and Northern analysis showed that genes encoding IGF-I and IGF-IR are expressed in the retina of goldfish. In situ hybridization showed that IGF-IR is expressed by retinal progenitors and all differentiated retinal neurons. Intraperitoneal injections of GH elevate IGF-I mRNA levels in the liver, brain and retina and produce a dose-dependent change in the proliferation of stem cells and progenitors in the retina. These data indicate that the principal components of the IGF-I signaling cascade are present in the retinas of teleosts, and we suggest these elements mediate the persistent, growth-associated neurogenesis in this tissue.