Vertebrate CNS Research Articles

ScMultiome analysis identifies a single caudal hindbrain compartment in the developing zebrafish nervous system

BackgroundA key step in nervous system development involves the coordinated control of neural progenitor specification and positioning. A long-standing model for the vertebrate CNS postulates that transient anatomical compartments – known as neuromeres – function to position neural progenitors along the embryonic anteroposterior neuraxis. Such neuromeres are apparent in the embryonic hindbrain – that contains six rhombomeres with morphologically apparent boundaries – but other neuromeres lack clear morphological boundaries and have instead been defined by different criteria, such as differences in gene expression patterns and the outcomes of transplantation experiments. Accordingly, the caudal hindbrain (CHB) posterior to rhombomere (r) 6 has been variably proposed to contain from two to five ‘pseudo-rhombomeres’, but the lack of comprehensive molecular data has precluded a detailed definition of such structures.MethodsWe used single-cell Multiome analysis, which allows simultaneous characterization of gene expression and chromatin state of individual cell nuclei, to identify and characterize CHB progenitors in the developing zebrafish CNS.ResultsWe identified CHB progenitors as a transcriptionally distinct population, that also possesses a unique profile of accessible transcription factor binding motifs, relative to both r6 and the spinal cord. This CHB population can be subdivided along its dorsoventral axis based on molecular characteristics, but we do not find any molecular evidence that it contains multiple pseudo-rhombomeres. We further observe that the CHB is closely related to r6 at the earliest embryonic stages, but becomes more divergent over time, and that it is defined by a unique gene regulatory network.ConclusionsWe conclude that the early CHB represents a single neuromere compartment that cannot be molecularly subdivided into pseudo-rhombomeres and that it may share an embryonic origin with r6.

Transcriptional regulators with broad expression in the zebrafish spinal cord.

The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

GABAergic Mechanisms of the Brain Tolerance to Hypoxia in Lower Vertebrates

Hypoxic/ischemic brain injuries a major medical challenge. One of the approaches to the development of therapeutic interventions is to establish the pathways of survival for neurons in tolerant to O2 deficiency vertebrates, which could suggest the ways to mitigate hypoxic catastrophe for separate cells under oxygen starvation. Metabolic depression is considered to be a universal strategy for the survival of hypoxia tolerant animals; however, the details of the mechanism of brain metabolism limitation with a decrease in PO2 have not hitherto been established. Under oxygen starvation, an increase in the extracellular concentration of inhibitory neurotransmitters can be one of the significant links in the apparatus for suppression of electrical activity, which makes it possible to reduce energy demand. GABA (γ-aminobutyric acid) serves as a universal inhibitory neurotransmitter in the CNS of higher and lower vertebrates, the functioning of which is associated with the metabolism suppression and leveling the consequences of an energy failure. GABA is found in various taxonomic groups of vertebrates. This review considers strategies for GABA involvement in the mechanisms of ensuring a brain tolerance to oxygen starvation in representatives of various taxonomic groups of lower vertebrates (cyclostomes, cartilaginous and bony fish, amphibians, reptiles), which are distinguished by a most pronounced ability to survive under acute and chronic hypoxia/anoxia.

Interorgan crosstalk and metabolism regulation in Drosophila

I will discuss our ongoing studies to identify and characterize communication pathways between major organs in Drosophila. Organ‐to‐organ communications are critical to living systems and play major roles in homeostasis. For example, the vertebrate CNS receives information regarding the status of peripheral metabolic processes via hormonal signaling and direct macromolecular sensing. In addition, skeletal muscles produce various myokines that influence metabolic homeostasis, lifespan, and the progression of age‐related diseases and aging in non‐muscle tissues. Using genetic screening, transcriptome analyses and proteomic approaches, we are identifying and characterizing secreted factors by which organs communicate their physiological state to others in both homeostasis and altered metabolism, as well as tumor models. These studies are providing fundamental insights into how biological processes observed in one tissue/organ (e.g., decreased cellular metabolism, mitochondrial dysfunction) influence the state of other tissues/organs.

Benzothiadiazines derivatives as novel allosteric modulators of kainic acid receptors.

The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainite receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.

The Akt-mTOR Pathway Drives Myelin Sheath Growth by Regulating Cap-Dependent Translation.

In the vertebrate CNS, oligodendrocytes produce myelin, a specialized membrane, to insulate and support axons. Individual oligodendrocytes wrap multiple axons with myelin sheaths of variable lengths and thicknesses. Myelin grows at the distal ends of oligodendrocyte processes, and multiple lines of work have provided evidence that mRNAs and RNA binding proteins localize to myelin, together supporting a model where local translation controls myelin sheath growth. What signal transduction mechanisms could control this? One strong candidate is the Akt-mTOR pathway, a major cellular signaling hub that coordinates transcription, translation, metabolism, and cytoskeletal organization. Here, using zebrafish as a model system, we found that Akt-mTOR signaling promotes myelin sheath growth and stability during development. Through cell-specific manipulations to oligodendrocytes, we show that the Akt-mTOR pathway drives cap-dependent translation to promote myelination and that restoration of cap-dependent translation is sufficient to rescue myelin deficits in mTOR loss-of-function animals. Moreover, an mTOR-dependent translational regulator was phosphorylated and colocalized with mRNA encoding a canonically myelin-translated protein in vivo, and bioinformatic investigation revealed numerous putative translational targets in the myelin transcriptome. Together, these data raise the possibility that Akt-mTOR signaling in nascent myelin sheaths promotes sheath growth via translation of myelin-resident mRNAs during development.SIGNIFICANCE STATEMENT In the brain and spinal cord, oligodendrocytes extend processes that tightly wrap axons with myelin, a protein- and lipid-rich membrane that increases electrical impulses and provides trophic support. Myelin membrane grows dramatically following initial axon wrapping in a process that demands protein and lipid synthesis. How protein and lipid synthesis is coordinated with the need for myelin to be generated in certain locations remains unknown. Our study reveals that the Akt-mTOR signaling pathway promotes myelin sheath growth by regulating protein translation. Because we found translational regulators of the Akt-mTOR pathway in myelin, our data raise the possibility that Akt-mTOR activity regulates translation in myelin sheaths to deliver myelin on demand to the places it is needed.

Slow AMPA Receptors in Hippocampal Principal Cells

Glutamate receptor ion channels such as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediate the majority of fast excitatory neurotransmission in the vertebrate CNS. AMPA receptors canonically provide the fast, millisecond component of the synaptic current. However, we found that about two-thirds of principal cells in the mouse hippocampus express AMPA receptors that do not desensitize and stay active for up to half a second. These receptors are expressed at synapses with a sparse but flat spatial distribution. The resulting increase in charge transfer allows single connections to reliably trigger action potentials. Biophysical and pharmacological observations imply that slow AMPA receptors incorporate γ-8 and other auxiliary proteins, and their activation lengthens individual miniature synaptic currents. Synaptic connections harboring slow AMPARs should have unique roles in hippocampal function.

Stac protein regulates release of neuropeptides.

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Formation of Neuronal and Neuroglial Populations during Pre- and Postnatal Development of the CNS in Vertebrates

This article provides a brief review of concepts of the features of the pre- and postnatal development of the central nervous system in vertebrates. Particular attention is paid to questions of the origins of neuron populations at different periods of nervous system development. Neuron and glial populations are shown to arise from different sources: neural stem cells (NSC) give rise to the neurogenic epithelium by vertical migration in the brain wall, while closer to birth, their successors give rise to cells of the so-called radial glia (RG) and intermediate precursor cells (IPC). Replenishment of the neuron population in some parts of the brain occurs as a result of tangential migration of neuroblasts from the neurogenic zones located at great distances from the final site of neuron differentiation. The processes of neuro- and gliogenesis are affected by a large number of different growth, neurotrophic, and transcription factors. Questions of the features of postnatal neurogenesis in the adult nervous system in vertebrates and the potential of using model objects for studying this process in humans are discussed.

Improved tools to study astrocytes.

Astrocytes are a type of glial cell that tile the CNS. They interact with multiple cell types, including neurons, glial cells and blood vessels, and are involved or implicated in brain disorders. Progress has been made in understanding astrocytes, but the field lacks detailed information concerning how they perform their multifarious functions, and how and when they influence the operations of the neural circuits with which they interact. One recognized bottleneck to progress has been the paucity of reliable tools with which to explore astrocytes within the adult vertebrate CNS in vivo. However, improved tools for molecular, genetic, morphological and physiological assessments have been developed recently or have been adapted from their original purposes to study neurons and are now being used to systematically document and interrogate astrocyte biology in vivo. These tools, their uses and limitations, and the insights that they afford are summarized in this Review.

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

Brain injury activates complex inflammatory signals in dying neurons, surviving neurons, and glia. Here, we establish that inflammation regulates the regeneration of photoreceptors in the zebrafish retina and determine the cellular expression and function of the inflammatory protease, matrix metalloproteinase 9 (Mmp‐9), during this regenerative neurogenesis. Following photoreceptor ablation, anti‐inflammatory treatment suppresses the number of injury‐induced progenitors and regenerated photoreceptors. Upon photoreceptor injury, mmp‐9 is induced in Müller glia and Müller glia‐derived photoreceptor progenitors. Deleting mmp‐9 results in over production of injury‐induced progenitors and regenerated photoreceptors, but over time the absence of Mmp‐9 compromises the survival of the regenerated cones. At all time‐points studied, the levels of tnf‐α are significantly elevated in mutant retinas. Anti‐inflammatory treatment in mutants rescues the defects in cone survival. These data provide a link between injury‐induced inflammation in the vertebrate CNS, Mmp‐9 function during neuronal regeneration and the requirement of Mmp‐9 for the survival of regenerated cones.

Action Potential Activity and Membrane Structure in Neurons of the Goldfish Retina Undergo Seasonal Changes

Seasonal changes in the structure of cell membranes may occur as an adaptive measure to withstand exposure to prolonged, environmental stress. Little is known, however, about how such biophysical changes may impact sensory systems and other aspects of the CNS in vertebrates. We compared membrane properties of neurons from the retina of goldfish (Carassius auratus), an organism well-suited to environmental change, during the summer and winter months. We isolated horizontal cells, which modulate output from light-sensitive photoreceptors in the retina and display spontaneous Ca2+-based action potentials in vitro and in situ. Measurement of intracellular Ca2+ concentration using Fura-2 microspectrofluorometry demonstrated that action potentials were of lower frequency and higher amplitude in cells isolated from goldfish during the winter months. Analysis of action potential activity using current-clamp electrophysiology correlated changes in frequency and amplitude with resting membrane potential, such that membrane hyperpolarization gave rise to a winter phenotype. Indeed, voltage-clamp recording of horizontal cells isolated during the winter confirmed that neurons displayed a resting membrane potential that was hyperpolarized by approximately 20 mV, compared to summer neurons, suggestive of a seasonal effect on membrane excitability. Analysis of whole-retina phospholipid composition using mass spectrometry revealed a significant change in membrane structure during the winter, including a 2-fold increase in phosphatidylethanolamine species. These studies indicate significant biophysical modification that occurs in retinal neurons of goldfish throughout the year, and suggests profound seasonal variation in visual processing and CNS physiology in organisms adapted to survive environmental change.

Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-β, the toxic driver of Alzheimer’s disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain

Glial phagocytosis is critical for the development and maintenance of the CNS in vertebrates and flies and relies on the function of phagocytic receptors to remove apoptotic cells and debris. Glial phagocyticability declines with age, which correlates with neuronal dysfunction, suggesting that increased glial phagocytosis may prevent neurodegeneration. Contradicting this hypothesis, we provide experimental evidence showing that an elevated expression of the phagocytic receptors Six-Microns-Under (SIMU) and Draper (Drpr) in adult Drosophila glia leads to a loss of both dopaminergic and GABAergic neurons, accompanied by motor dysfunction and a shortened lifespan. Importantly, this reduction in neuronal number is not linked to neuronal apoptosis, but rather to phosphatidylserine-mediated phagoptosis of live neurons by hyper-phagocytic glia. Altogether, our study reveals that the level of glial phagocytic receptors must be tightly regulated for proper brain function and that neurodegeneration occurs not only by defective, but also excessive glial cell function.

Identification of Peptidyl-Prolyl Cis-Trans Isomerase-Like 4 as a Disease Causing Gene in Intracranial Aneurysms and its Role in Vertebrate CNS Specific Angiogenesis

Abstract INTRODUCTION Intracranial aneurysm (IA) is a significant health burden affecting nearly 6 million people in the United States with an estimated prevalence of 3.2% worldwide. Using whole exome sequencing (WES) of 728 European IA cases, we identified damaging mutations in and RNA binding protein peptidyl-prolyl cis-trans isomerase-like 4 (PPIL4) in both familial and sporadic IA cases. METHODS We performed WES in 728 European IA cases followed by a mutational burden analysis between IA cases and a European (Finnish + Non-Finnish) control group of 1443 unaffected individuals, and European subjects (non-Finnish + Finnish) in the ExAC and gnomAD databases. Fisher exact test was used to determine the significance of the enrichment of rare (ExAC MAF <0.0001) and deleterious variants consisting of loss of function and deleterious missense mutations (MetSVM-D). Mutant ppil4 lines were generated using Crispr-CAS9 in zebrafish and X.tropicalis. RNA-seq and Slam-Seq were performed to identify transcriptome-wide changes in RNA half-life mediated by PPIL4. RESULTS Burden analysis revealed a significant enrichment of rare and deleterious PPIL4 variants in European IA cases when compared to 1443 controls (OR = INF., Fisher P = 3.17 × 10–4), ExAC (OR = 12.26, Fisher P = 5 × 10–4) or gnomAD (OR = 13.14, Fisher P = 3.4 × 10–4). PPIL4 mutants both in zebrafish and X.tropicalis models displayed cerebral hemorrhage and cerebrovascular simplification. Slam-seq analysis revealed that PPIL4 promotes mRNA stability of the genes implicated in VEGF signaling (FDR = 3.75E-07) and Focal adhesion pathways (FDR = 2.04E-06). CONCLUSION In this study, we identified PPIL4 as a candidate gene in IA pathogenesis and demonstrated morphological and hemodynamic impairment in vivo upon abrogating PPIL4 in 2 vertebrate models. Furthermore, we demonstrated that PPIL4 plays a major role in CNS angiogenesis and IA pathogenesis through promoting mRNA stability of key angiogenesis related genes, emphasizing the role of post-transcriptional RNA modification in cerebrovascular development and pathologies.

Nervous System Regionalization Entails Axial Allocation before Neural Differentiation

SummaryNeural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This “primary regionalization” allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.

Tropomodulin Isoform-Specific Regulation of Dendrite Development and Synapse Formation.

Neurons of the CNS elaborate highly branched dendritic arbors that host numerous dendritic spines, which serve as the postsynaptic platform for most excitatory synapses. The actin cytoskeleton plays an important role in dendrite development and spine formation, but the underlying mechanisms remain incompletely understood. Tropomodulins (Tmods) are a family of actin-binding proteins that cap the slow-growing (pointed) end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown. In this study, we present evidence that Tmod1 and Tmod2 exhibit distinct roles in regulating spine development and dendritic arborization, respectively. Using rat hippocampal tissues from both sexes, we find that Tmod1 and Tmod2 are expressed with distinct developmental profiles: Tmod2 is expressed early during hippocampal development, whereas Tmod1 expression coincides with synaptogenesis. We then show that knockdown of Tmod2, but not Tmod1, severely impairs dendritic branching. Both Tmod1 and Tmod2 are localized to a distinct subspine region where they regulate local F-actin stability. However, the knockdown of Tmod1, but not Tmod2, disrupts spine morphogenesis and impairs synapse formation. Collectively, these findings demonstrate that regulation of the actin cytoskeleton by different members of the Tmod family plays an important role in distinct aspects of dendrite and spine development.SIGNIFICANCE STATEMENT The Tropomodulin family of molecules is best known for controlling the length and stability of actin myofilaments in skeletal muscles. While several Tropomodulin members are expressed in the brain, fundamental knowledge about their role in neuronal function is limited. In this study, we show the unique expression profile and subcellular distribution of Tmod1 and Tmod2 in hippocampal neurons. While both Tmod1 and Tmod2 regulate F-actin stability, we find that they exhibit isoform-specific roles in dendrite development and synapse formation: Tmod2 regulates dendritic arborization, whereas Tmod1 is required for spine development and synapse formation. These findings provide novel insight into the actin regulatory mechanisms underlying neuronal development, thereby shedding light on potential pathways disrupted in a number of neurological disorders.

Interactive Repression of MYRF Self-Cleavage and Activity in Oligodendrocyte Differentiation by TMEM98 Protein.

Myelin sheath formed by oligodendrocytes (OLs) is essential for the rapid propagation of action potentials in the vertebrate CNS. Myelin regulatory factor (MYRF) is one of the critical factors that control OL differentiation and myelin maintenance. Previous studies showed that MYRF is a membrane-bound transcription factor associated with the endoplasmic reticulum (ER). After self-cleavage, the N-fragment of MYRF is released from the ER and translocated into the nucleus where it functions as a transcription factor to activate myelin gene expression. At present, it remains unknown whether MYRF self-cleavage and functional activation can be regulated during OL differentiation. Here, we report that TMEM98, an ER-associated transmembrane protein, is capable of binding to the C-terminal of MYRF and inhibiting its self-cleavage and N-fragment nuclear translocation. In the developing CNS, TMEM98 is selectively expressed in early maturing OLs in mouse pups of either sex. Forced expression of TMEM98 in embryonic chicken spinal cord of either sex suppresses endogenous OL differentiation and MYRF-induced ectopic expression of myelin genes. These results suggest that TMEM98, through inhibiting the self-cleavage of MYRF, functions as a negative feedback regulator of MYRF in oligodendrocyte differentiation and myelination.SIGNIFICANCE STATEMENT MYRF protein is initially synthesized as an ER-associated membrane protein that undergoes autoproteolytic cleavage to release the N-fragment, which is then transported into the nucleus and activates the transcription of myelin genes. To date, the molecular mechanisms that regulate the self-cleavage and function of MYRF in regulating oligodendrocyte differentiation have remained unknown. In this study, we present the molecular and functional evidence that TMEM98 membrane protein physically interacts with MYRF in the ER and subsequently blocks its self-cleavage, N-terminal nuclear translocation, and functional activation of myelin gene expression. To our knowledge, this is the first report on the regulation of MYRF self-proteolytic activity and function by an interacting protein, providing new insights into the molecular regulation of OL differentiation and myelinogenesis.

Molecular Landscapes of Embryonic and Post-Embryonic Neurogenesis in the Vertebrate Retina

Zebrafish retinas, undergoing life-long neurogenesis, provide an integrating model to resolve molecular landscapes of embryonic and post-embryonic neurogenesis. By single-cell RNAseq of zebrafish embryonic retinal progenitors and post-embryonic ciliary marginal zone (CMZ) progenitors, we define 7 distinguished states, 4 independent states (S1/S3/S5/S7) separated by 3 transitional states (S2/S4/S6), that develop over time and reveal aspects of lineage evolution. Genes that define earlier states are more critical for making retinas and post-embryonic CMZ, while genes that define later states are more critical for specifying individual cell fates. Remarkably, transitional and independent states expressed different cell-cycle phase genes, suggesting state transition relies on cell cycle. Intriguingly, similar analysis CMZ cells revealed similar set of states, suggesting embryonic and post-embryonic retinogensis conform to the same molecular program, although some interesting differences in rod genesis. Thus, our findings exemplify molecular landscapes of embryonic and post-embryonic neurogenesis in a vertebrate CNS tissue.

Knockdown of epigenetic transcriptional co-regulator Brd2a disrupts apoptosis and proper formation of hindbrain and midbrain-hindbrain boundary (MHB) region in zebrafish

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.