Darpp-32 is regulated by dopamine and is required for the formation of GABAergic neurons in the developing telencephalon.

DARPP-32 (dopamine and cAMP-regulated phosphoprotein Mr. 32 kDa) is a phosphoprotein that is modulated by multiple receptors integrating intracellular pathways and playing roles in various physiological functions. It is regulated by dopaminergic receptors through the cAMP/protein kinase A (PKA) pathway, which modulates the phosphorylation of threonine 34 (Thr34). When phosphorylated at Thr34, DARPP-32 becomes a potent protein phosphatase-1 (PP1) inhibitor. Since dopamine is involved in the development of GABAergic neurons and DARPP-32 is expressed in the developing brain, it is possible that DARPP-32 has a role in GABAergic neuronal development. We cloned the zebrafish darpp-32 gene (ppp1r1b) gene and observed that it is evolutionarily conserved in its inhibitory domain (Thr34 and surrounding residues) and the docking motif (residues 7-11 (KKIQF)). We also characterized darpp-32 protein expression throughout the 5 days post-fertilization (dpf) zebrafish larval brain by immunofluorescence and demonstrated that darpp-32 is mainly expressed in regions that receive dopaminergic projections (pallium, subpallium, preoptic region, and hypothalamus). We demonstrated that dopamine acutely suppressed darpp-32 activity by reducing the levels of p-darpp-32 in the 5dpf zebrafish larval brain. In addition, the knockdown of darpp-32 resulted in a decrease in the number of GABAergic neurons in the subpallium of the 5dpf larval brain, with a concomitant increase in the number of DAergic neurons. Finally, we demonstrated that darpp-32 downregulation during development reduced the motor behavior of 5dpf zebrafish larvae. Thus, our observations suggest that darpp-32 is an evolutionarily conserved regulator of dopamine receptor signaling and is required for the formation of GABAergic neurons in the developing telencephalon.

In Situ Quantification and Isolation of Müller Glial Cells by Fluorescence-Activated Cell Sorting from the Regenerating Larval Zebrafish Retina.

Müller glia (MG) are a relatively quiescent radial glial cell population capable of dedifferentiating to regenerate cells in the zebrafish retina that are lost due to damage. Here, we provide a protocol to both quantify MG cell dedifferentiation behavior during a regenerative response and isolate MG cells by fluorescence activated cell sorting (FACS). First, the retina is exposed to high-intensity light to induce retinal damage and either processed for immunohistochemistry or live MG cells are isolated by FACS that can be used for subsequent genomic or transcriptomic analyses. This method allows us to correlate MG cell behavior observed in situ with their transcriptomic profile at different stages during the regenerative response.

Usher syndrome type 1-associated gene, pcdh15b, is required for photoreceptor structural integrity in zebrafish.

Blindness associated with Usher syndrome type 1 (USH1) is typically characterized as rod photoreceptor degeneration, followed by secondary loss of cones. The mechanisms leading to blindness are unknown because most genetic mouse models only recapitulate auditory defects. We generated zebrafish mutants for one of the USH1 genes, protocadherin-15b (pcdh15b), a putative cell adhesion molecule. Zebrafish Pcdh15 is expressed exclusively in photoreceptors within calyceal processes (CPs), at the base of the outer segment (OS) and within the synapse. In our mutants, rod and cone photoreceptor integrity is compromised, with early and progressively worsening abnormal OS disc growth and detachment, in part due to weakening CP contacts. These effects were attenuated or exacerbated by growth in dark and bright-light conditions, respectively. We also describe novel evidence for structural defects in synapses of pcdh15b mutant photoreceptors. Cell death does not accompany these defects at early stages, suggesting that photoreceptor structural defects, rather than overt cell loss, may underlie vision deficits. Thus, we present the first genetic animal model of a PCDH15-associated retinopathy that can be used to understand the aetiology of blindness in USH1. This article has an associated First Person interview with the first author of the paper.

Know thy neighbor: Modeling spatiotemporal cell-fate patterns in a neural stem cell niche.

How lineage and the microenvironment influence stem cell homeostasis at a population level remains unresolved. In this issue of Cell Stem Cell, Dray et al. (2021) use in vivo imaging and statistical modeling to discover a key role for local progenitor cell descendants in constraining neural stem cell divisions.

Retinal Stem Cell 'Retirement Plans': Growth, Regulation and Species Adaptations in the Retinal Ciliary Marginal Zone.

The vertebrate retina develops from a specified group of precursor cells that adopt distinct identities and generate lineages of either the neural retina, retinal pigmented epithelium, or ciliary body. In some species, including teleost fish and amphibians, proliferative cells with stem-cell-like properties capable of continuously supplying new retinal cells post-embryonically have been characterized and extensively studied. This region, termed the ciliary or circumferential marginal zone (CMZ), possibly represents a conserved retinal stem cell niche. In this review, we highlight the research characterizing similar CMZ-like regions, or stem-like cells located at the peripheral margin, across multiple different species. We discuss the proliferative parameters, multipotency and growth mechanisms of these cells to understand how they behave in vivo and how different molecular factors and signalling networks converge at the CMZ niche to regulate their activity. The evidence suggests that the mature retina may have a conserved propensity for homeostatic growth and plasticity and that dysfunction in the regulation of CMZ activity may partially account for dystrophic eye growth diseases such as myopia and hyperopia. A better understanding of the properties of CMZ cells will enable important insight into how an endogenous generative tissue compartment can adapt to altered retinal physiology and potentially even restore vision loss caused by retinal degenerative conditions.

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

Using Teleost Fish to Discern Developmental Signatures of Evolutionary Adaptation From Phenotypic Plasticity in Brain Structure.

Traditionally, the impact of evolution on the central nervous system has been studied by comparing the sizes of brain regions between species. However, more recent work has demonstrated that environmental factors, such as sensory experience, modulate brain region sizes intraspecifically, clouding the distinction between evolutionary and environmental sources of neuroanatomical variation in a sampled brain. Here, we review how teleost fish have played a central role in shaping this traditional understanding of brain structure evolution between species as well as the capacity for the environment to shape brain structure similarly within a species. By demonstrating that variation measured by brain region size varies similarly both inter- and intraspecifically, work on teleosts highlights the depth of the problem of studying brain evolution using neuroanatomy alone: even neurogenesis, the primary mechanism through which brain regions are thought to change size between species, also mediates experience-dependent changes within species. Here, we argue that teleost models also offer a solution to this overreliance on neuroanatomy in the study of brain evolution. With the advent of work on teleosts demonstrating interspecific evolutionary signatures in embryonic gene expression and the growing understanding of developmental neurogenesis as a multi-stepped process that may be differentially regulated between species, we argue that the tools are now in place to reframe how we compare brains between species. Future research can now transcend neuroanatomy to leverage the experimental utility of teleost fishes in order to gain deeper neurobiological insight to help us discern developmental signatures of evolutionary adaptation from phenotypic plasticity.

The role of neuro-epithelial-like and radial-glial stem and progenitor cells in development, plasticity, and repair.

Neural stem and progenitor cells (NSPCs) are the primary source of new neurons in the brain and serve critical roles in tissue homeostasis and plasticity throughout life. Within the vertebrate brain, NSPCs are located within distinct neurogenic niches differing in their location, cellular composition, and proliferative behaviour. Heterogeneity in the NSPC population is hypothesized to reflect varying capacities for neurogenesis, plasticity and repair between different neurogenic zones. Since the discovery of adult neurogenesis, studies have predominantly focused on the behaviour and biological significance of adult NSPCs (aNSPCs) in rodents. However, compared to rodents, who show lifelong neurogenesis in only two restricted neurogenic niches, zebrafish exhibit constitutive neurogenesis across multiple stem cell niches that provide new neurons to every major brain division. Accordingly, zebrafish are a powerful model to probe the unique cellular and molecular profiles of NSPCs and investigate how these profiles govern tissue homeostasis and regenerative plasticity within distinct stem cell populations over time. Amongst the NSPC populations residing in the zebrafish central nervous system (CNS), proliferating radial-glia, quiescent radial-glia and neuro-epithelial-like cells comprise the majority. Here, we provide insight into the extent to which these distinct NSPC populations function and mature during development, respond to experience, and contribute to successful CNS regeneration in teleost fish. Together, our review brings to light the dynamic biological roles of these individual NSPC populations and showcases their diverse regenerative modes to achieve vertebrate brain repair later in life.

Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish.

The postembryonic brain exhibits experience-dependent development, in which sensory experience guides normal brain growth. This neuroplasticity is thought to occur primarily through structural and functional changes in pre-existing neurons. Whether neurogenesis also mediates the effects of experience on brain growth is unclear. Here, we characterized the importance of motor experience on postembryonic neurogenesis in larval zebrafish. We found that movement maintains an expanded pool of forebrain neural precursors by promoting progenitor self-renewal over the production of neurons. Physical cues associated with swimming (bodily movement) increase neurogenesis and these cues appear to be conveyed by dorsal root ganglia (DRG) in the zebrafish body: DRG-deficient larvae exhibit attenuated neurogenic responses to movement and targeted photoactivation of DRG in immobilized larvae expands the pallial pool of proliferative cells. Our results demonstrate the importance of movement in neurogenic brain growth and reveal a fundamental sensorimotor association that may couple early motor and brain development.

Visual Experience Facilitates BDNF-Dependent Adaptive Recruitment of New Neurons in the Postembryonic Optic Tectum.

Postembryonic brain development is sensitive to environmental input and sensory experience, but the mechanisms underlying healthy adaptive brain growth are poorly understood. Here, we tested the importance of visual experience on larval zebrafish (Danio rerio) postembryonic development of the optic tectum (OT), a midbrain structure involved in visually guided behavior. We first characterized postembryonic neurogenic growth in OT, in which new neurons are generated along the caudal tectal surface and contribute appositionally to anatomical growth. Restricting visual experience during development by rearing larvae in dim light impaired OT anatomical and neurogenic growth, specifically by reducing the survival of new neurons in the medial periventricular gray zone. Neuronal survival in the OT was reduced only when visual experience was restricted for the first 5 d following new neuron generation, suggesting that tectal neurons exhibit an early sensitive period in which visual experience protects these cells from subsequent neuronal loss. The effect of dim rearing on neuronal survival was mimicked by treatment with an NMDA receptor antagonist early, but not later, in a new neuron's life. Both dim rearing and antagonist treatment reduced BDNF production in the OT, and supplementing larvae with exogenous BDNF during dim rearing prevented neuronal loss, suggesting that visual experience protects new tectal neurons through neural activity-dependent BDNF expression. Collectively, we present evidence for a sensitive period of neurogenic adaptive growth in the larval zebrafish OT that relies on visual experience-dependent mechanisms.SIGNIFICANCE STATEMENT Early brain development is shaped by environmental factors via sensory input; however, this form of experience-dependent neuroplasticity is traditionally studied as structural and functional changes within preexisting neurons. Here, we found that restricting visual experience affects development of the larval zebrafish optic tectum, a midbrain structure involved in visually guided behavior, by limiting the survival of newly generated neurons. We found that new tectal neurons exhibit a sensitive period soon after cell birth in which adequate visual experience, likely mediated by neuronal activity driving BDNF production within the tectum, would protect them from subsequent neuronal loss over the following week. Collectively, we present evidence for neurogenic adaptive tectal growth under different environmental lighting conditions.

The expanding role of the Ehmt2/G9a complex in neurodevelopment.

Epigenetic regulators play a crucial role in neurodevelopment. One such epigenetic complex, Ehmt1/2 (G9a/GLP), is essential for repressing gene transcription by methylating H3K9 in a highly tissue- and temporal-specific manner. Recently, data has emerged suggesting that this complex plays additional roles in regulating the activity of numerous other non-histone proteins. While much is known about the downstream effects of Ehmt1/2 function, evidence is only beginning to come to light suggesting the control of Ehmt1/2 function may be, at least in part, due to context-dependent binding partners. Here we review emerging roles for the Ehmt1/2 complex suggesting that it may play a much larger role than previously recognized, and discuss binding partners that we and others have recently characterized which act to coordinate its activity during early neurodevelopment.

Cone-like rhodopsin expressed in the all-cone retina of the colubrid pine snake as a potential adaptation to diurnality.

Colubridae is the largest and most diverse family of snakes, with visual systems that reflect this diversity, encompassing a variety of retinal photoreceptor organizations. The transmutation theory proposed by Walls postulates that photoreceptors could evolutionarily transition between cell types in squamates, but few studies have tested this theory. Recently, evidence for transmutation and rod-like machinery in an all-cone retina has been identified in a diurnal garter snake (Thamnophis), and it appears that the rhodopsin gene at least may be widespread among colubrid snakes. However, functional evidence supporting transmutation beyond the existence of the rhodopsin gene remains rare. We examined the all-cone retina of another colubrid, Pituophis melanoleucus, thought to be more secretive/burrowing than Thamnophis We found that P. melanoleucus expresses two cone opsins (SWS1, LWS) and rhodopsin (RH1) within the eye. Immunohistochemistry localized rhodopsin to the outer segment of photoreceptors in the all-cone retina of the snake and all opsin genes produced functional visual pigments when expressed in vitro Consistent with other studies, we found that P. melanoleucus rhodopsin is extremely blue-shifted. Surprisingly, P. melanoleucus rhodopsin reacted with hydroxylamine, a typical cone opsin characteristic. These results support the idea that the rhodopsin-containing photoreceptors of P. melanoleucus are the products of evolutionary transmutation from rod ancestors, and suggest that this phenomenon may be widespread in colubrid snakes. We hypothesize that transmutation may be an adaptation for diurnal, brighter-light vision, which could result in increased spectral sensitivity and chromatic discrimination with the potential for colour vision.

A second visual rhodopsin gene, rh1-2, is expressed in zebrafish photoreceptors and found in other ray-finned fishes.

Rhodopsin (rh1) is the visual pigment expressed in rod photoreceptors of vertebrates that is responsible for initiating the critical first step of dim-light vision. Rhodopsin is usually a single copy gene; however, we previously discovered a novel rhodopsin-like gene expressed in the zebrafish retina, rh1-2, which we identified as a functional photosensitive pigment that binds 11-cis retinal and activates in response to light. Here, we localized expression of rh1-2 in the zebrafish retina to a subset of peripheral photoreceptor cells, which indicates a partially overlapping expression pattern with rh1 We also expressed, purified and characterized Rh1-2, including investigation of the stability of the biologically active intermediate. Using fluorescence spectroscopy, we found the half-life of the rate of retinal release of Rh1-2 following photoactivation to be more similar to that of the visual pigment rhodopsin than to the non-visual pigment exo-rhodopsin (exorh), which releases retinal around 5 times faster. Phylogenetic and molecular evolutionary analyses show that rh1-2 has ancient origins within teleost fishes, is under similar selective pressure to rh1, and likely experienced a burst of positive selection following its duplication and divergence from rh1 These findings indicate that rh1-2 is another functional visual rhodopsin gene, which contradicts the prevailing notion that visual rhodopsin is primarily found as a single copy gene within ray-finned fishes. The reasons for retention of this duplicate gene, as well as possible functional consequences for the visual system, are discussed.

Coordinating progenitor cell cycle exit and differentiation in the developing vertebrate retina.

The proper development of the vertebrate retina relies heavily on producing the correct number and type of differentiated retinal cell types. To achieve this, proliferating retinal progenitor cells (RPCs) must exit the cell cycle at an appropriate time and correctly express a subset of differentiation markers that help specify retinal cell fate. Homeobox genes, which encode a family of transcription factors, have been accredited to both these processes, implicated in the transcriptional regulation of important cell cycle components, such as cyclins and cyclin-dependent kinases, and proneural genes. This dual regulation of homeobox genes allows these factors to help co-ordinate the transition from the proliferating RPC to postmitotic, differentiated cell. However, understanding the exact molecular targets of these factors remains a challenging task. This commentary highlights the current knowledge we have about how these factors regulate cell cycle progression and differentiation, with particular emphasis on a recent discovery from our lab demonstrating an antagonistic relationship between Vsx2 and Dmbx1 to control RPC proliferation. Future studies should aim to further understand the direct transcriptional targets of these genes, additional co-factors/interacting proteins and the possible recruitment of epigenetic machinery by these homeobox genes.

G9a and ZNF644 Physically Associate to Suppress Progenitor Gene Expression during Neurogenesis.

Proliferating progenitor cells undergo changes in competence to give rise to post-mitotic progeny of specialized function. These cell-fate transitions typically involve dynamic regulation of gene expression by histone methyltransferase (HMT) complexes. However, the composition, roles, and regulation of these assemblies in regulating cell-fate decisions in vivo are poorly understood. Using unbiased affinity purification and mass spectrometry, we identified the uncharacterized C2H2-like zinc finger protein ZNF644 as a G9a/GLP-interacting protein and co-regulator of histone methylation. In zebrafish, functional characterization of ZNF644 orthologs, znf644a and znf644b, revealed complementary roles in regulating G9a/H3K9me2-mediated gene silencing during neurogenesis. The non-overlapping requirements for znf644a and znf644b during retinal differentiation demarcate critical aspects of retinal differentiation programs regulated by differential G9a-ZNF644 associations, such as transitioning proliferating progenitor cells toward differentiation. Collectively, our data point to ZNF644 as a critical co-regulator of G9a/H3K9me2-mediated gene silencing during neuronal differentiation.

Restrictions on the Importation of Zebrafish into Canada Associated with Spring Viremia of Carp Virus.

The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake.

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the "transmutation" theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single "cones." Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.

Nonsense mutation in the WDR73 gene is associated with Galloway-Mowat syndrome.

BACKGROUND: Neuroanatomical defects are often present in children with severe developmental delay and intellectual disabilities. Few genetic loci have been associated with disorders of neurodevelopment. Our objective of the present study was to analyse a consanguineous Arab family showing some of the hallmark signs of a rare cerebellar hypoplasia-related neurodevelopmental syndrome as a strategy for discovering a causative genetic mutation. METHODS: We used whole exome sequencing to identify the causative mutation in two female siblings of a consanguineous Arab family showing some of the hallmark signs of a cerebellar-hypoplasia-related neurodevelopmental disorder. Direct Sanger sequencing was used to validate the candidate mutations that cosegregated with the phenotype. Gene expression and loss of function studies were carried out in the zebrafish model system to examine the role of the candidate gene in neurodevelopment. RESULTS: Patients presented with severe global developmental delay, intellectual disability, hypoplasia of the cerebellum and biochemical findings suggestive of nephrotic disease. Whole exome sequencing of the two patients revealed a shared nonsense homozygous variant in WDR73 (p.Q235X (c.703C>T)) resulting in loss of the last 144 amino acids of the protein. The variant segregated according to a recessive mode of inheritance in this family and was absent from public and our inhouse databases. We examined the developmental role of WDR73 using a loss-of-function paradigm in zebrafish. There was a significant brain growth and morphogenesis defect in wdr73 knockdown embryos resulting in a poorly differentiated midbrain and cerebellum. CONCLUSIONS: The results provide new insight into the functional role of WDR73 in brain development and show that perturbation of its function in an inherited disorder in humans is associated with cerebellar hypoplasia as well as nephrotic disease, consistent with Galloway-Mowat Syndrome.

Mutual antagonism of the paired-type homeobox genes, vsx2 and dmbx1, regulates retinal progenitor cell cycle exit upstream of ccnd1 expression.

Understanding the mechanisms that regulate the transition between the proliferative and a post-mitotic state of retinal progenitor cells (RPCs) is key to advancing our knowledge of retinal growth and maturation. In the present study we determined that during zebrafish embryonic retinal neurogenesis, two paired-type homeobox genes - vsx2 and dmbx1 - function in a mutually antagonistic manner. We demonstrate that vsx2 gene expression requires active Fgf signaling and that this in turn suppresses dmbx1 expression and maintains cells in an undifferentiated, proliferative RPC state. This vsx2-dependent RPC state can be prolonged cell-autonomously by knockdown of dmbx1, or it can be suppressed prematurely by the over-expression of dmbx1, which we show can inhibit vsx2 expression and lead to precocious neuronal differentiation. dmbx1 loss of function also results in altered expression of canonical cell cycle genes, and in particular up-regulation of ccnd1, which correlates with our previous finding of a prolonged RPC cell cycle. By knocking down ccnd1 and dmbx1 simultaneously, we show that RPCs can overcome this phenotype to exit the cell cycle on time and differentiate normally into retinal neurons. Collectively, our data provide novel insight into the mechanism that enables RPCs to exit the cell cycle through a previously unrecognized antagonistic interaction of two paired-type homeobox genes that are central regulators of an Fgf-vsx2-dmbx1-ccnd1 signaling axis.