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Motor circuits in the vertebrate hindbrain need to become functional early in development. What are the fundamental mechanisms that establish early synaptic inputs to motor neurons? Previous evidence is consistent with the hypothesis that motor neuron dendrite positioning serves a causal role in early spinal motor circuit development, with initial connectivity determined by the overlap between premotor axons and motor neuron dendrites (perhaps without the need for molecular recognition). Does motor neuron dendrite topography serve a similar role in the hindbrain? In the current study, we provide the first quantitative analysis of the dendrites of facial branchiomotor neurons (FBMNs) in larval zebrafish. We previously demonstrated that FBMNs exhibit functional topography along the dorsoventral axis, with the most ventral cell bodies most likely to exhibit early rhythmic activity-suggesting that FBMNs with ventral cell bodies are most likely to receive inputs from premotor neurons carrying rhythmic respiratory signals. We hypothesized that this functional topography can be explained by differences in dendrite positioning, giving ventral FBMNs preferential access to premotor axons carrying rhythmic signals. If this hypothesis is true, we predicted that FBMN cell body position would be correlated with dendrite position along the dorsoventral axis. To test this prediction, we used single-cell labeling to trace the dendritic arbors of FBMNs in larval zebrafish at 5-days post-fertilization (dpf). FBMN dendrites varied in complexity, and this variation could not be attributed to differences in the relative age of neurons. Most dendrites grew caudally, laterally, and ventrally from the cell body-though FBMN dendrites could extend their dendrites dorsally. Across our sample, FBMN cell body position correlated with dendrite position along the dorsoventral axis, consistent with our hypothesis that differences in dendrite positioning serve as the substrate for differences in activity patterns across neurons. Future studies will build on this foundational data, testing additional predictions of the central hypothesis-to further investigate the mechanisms of early motor circuit development., (© 2024 The Author(s). The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations., Competing Interests: Declaration of interests R.S.D. is a paid consultant for AstraZeneca. H.Y.Z. is a member of the Board of Regeneron, co-founder of Cajal Neuroscience, and on the science advisory board of the Column group. This is work in the Zoghbi Lab and has not been licensed anywhere. The work in this paper has no connection to any of the consulting or companies named here., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The most successful obesity therapeutics, glucagon-like peptide-1 receptor (GLP1R) agonists, cause aversive responses such as nausea and vomiting 1,2 , effects that may contribute to their efficacy. Here, we investigated the brain circuits that link satiety to aversion, and unexpectedly discovered that the neural circuits mediating these effects are functionally separable. Systematic investigation across drug-accessible GLP1R populations revealed that only hindbrain neurons are required for the efficacy of GLP1-based obesity drugs. In vivo two-photon imaging of hindbrain GLP1R neurons demonstrated that most neurons are tuned to either nutritive or aversive stimuli, but not both. Furthermore, simultaneous imaging of hindbrain subregions indicated that area postrema (AP) GLP1R neurons are broadly responsive, whereas nucleus of the solitary tract (NTS) GLP1R neurons are biased towards nutritive stimuli. Strikingly, separate manipulation of these populations demonstrated that activation of NTS GLP1R neurons triggers satiety in the absence of aversion, whereas activation of AP GLP1R neurons triggers strong aversion with food intake reduction. Anatomical and behavioural analyses revealed that NTS GLP1R and AP GLP1R neurons send projections to different downstream brain regions to drive satiety and aversion, respectively. Importantly, GLP1R agonists reduce food intake even when the aversion pathway is inhibited. Overall, these findings highlight NTS GLP1R neurons as a population that could be selectively targeted to promote weight loss while avoiding the adverse side effects that limit treatment adherence., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cerebellar granule neuron progenitors (GNPs) originate from the upper rhombic lip (URL), a germinative niche in which developmental defects produce human diseases. T-cell factor (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity, together with transcripts encoding proneural gene repressors hairy and enhancer of split (Hes/Hey), are detected in the URL; however, their functions and regulatory modes are undeciphered. Here, we established amphibian as a pertinent model for studying vertebrate URL development. The amphibian long-lived URL is TCF active, whereas the external granular layer (EGL) is non-proliferative and expresses hes4 and hes5 genes. Using functional and transcriptomic approaches, we show that TCF activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL, acting partly through direct TCF inhibition. Identification of Barhl1 target genes suggests that, besides TCF, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role for Barhl in maintenance of the URL and/or EGL via co-regulation of TCF, Hes and Hey genes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq 1,2 -a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures 1 . Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq 2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use., (© 2023. The Author(s).)

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions., (© 2021. The Author(s).)

Lowered glucose availability, sensed by the hindbrain, has been suggested to enhance gluconeogenesis and food intake as well as suppress reproductive function. In fact, our previous histological and in vitro studies suggest that hindbrain ependymal cells function as a glucose sensor. The present study aimed to clarify the hindbrain glucose sensor-hypothalamic neural pathway activated in response to hindbrain glucoprivation to mediate counterregulatory physiological responses. Administration of 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, into the fourth ventricle (4V) of male rats for 0.5 hour induced messenger RNA (mRNA) expression of c-fos, a marker for cellular activation, in ependymal cells in the 4V, but not in the lateral ventricle, the third ventricle or the central canal without a significant change in blood glucose and testosterone levels. Administration of 2DG into the 4V for 1 hour significantly increased blood glucose levels, food intake, and decreased blood testosterone levels. Simultaneously, the expression of c-Fos protein was detected in the 4V ependymal cells; dopamine β-hydroxylase-immunoreactive cells in the C1, C2, and A6 regions; neuropeptide Y (NPY) mRNA-positive cells in the C2; corticotropin-releasing hormone (CRH) mRNA-positive cells in the hypothalamic paraventricular nucleus (PVN); and NPY mRNA-positive cells in the arcuate nucleus (ARC). Taken together, these results suggest that lowered glucose availability, sensed by 4V ependymal cells, activates hindbrain catecholaminergic and/or NPY neurons followed by CRH neurons in the PVN and NPY neurons in the ARC, thereby leading to counterregulatory responses, such as an enhancement of gluconeogenesis, increased food intake, and suppression of sex steroid secretion., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development., Competing Interests: The authors have declared that no competing interests exist.

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development., Competing Interests: The authors have declared that no competing interests exist.

Saccades are rapid eye movements that redirect gaze. Their magnitudes and directions are tightly controlled by the oculomotor system, which is capable of generating conjugate, monocular, convergent and divergent saccades. Recent studies suggest a mainly monocular control of saccades in mammals, although the development of binocular control and the interaction of different functional populations is less well understood. For zebrafish, a well-established model in sensorimotor research, the nature of binocular control in this key oculomotor behavior is unknown. Here, we use the optokinetic response and calcium imaging to characterize how the developing zebrafish oculomotor system encodes the diverse repertoire of saccades. We find that neurons with phasic saccade-associated activity (putative burst neurons) are most frequent in dorsal regions of the hindbrain and show elements of both monocular and binocular encoding, revealing a mix of the response types originally hypothesized by Helmholtz and Hering. Additionally, we observed a certain degree of behavior-specific recruitment in individual neurons. Surprisingly, calcium activity is only weakly tuned to saccade size. Instead, saccade size is apparently controlled by a push-pull mechanism of opposing burst neuron populations. Our study reveals the basic layout of a developing vertebrate saccade system and provides a perspective into the evolution of the oculomotor system.

In vertebrate animals, motor and sensory efferent neurons carry information from the central nervous system (CNS) to peripheral targets. These two types of efferent systems sometimes bear a close resemblance, sharing common segmental organization, axon pathways, and chemical messengers. Here, we focus on the development of the octavolateral efferent neurons (OENs) and their interactions with the closely-related facial branchiomotor neurons (FBMNs) in zebrafish. Using live-imaging approaches, we investigate the birth, migration, and projection patterns of OENs. We find that OENs are born in two distinct groups: a group of rostral efferent neurons (RENs) that arises in the fourth segment, or rhombomere (r4), of the hindbrain and a group of caudal efferent neurons (CENs) that arises in r5. Both RENs and CENs then migrate posteriorly through the hindbrain between 18 and 48 hrs postfertilization, alongside the r4-derived FBMNs. Like the FBMNs, migration of the r4-derived RENs depends on function of the segmental identity gene hoxb1a; unlike the FBMNs, however, both OEN populations move independently of prickle1b. Further, we investigate whether the previously described "pioneer" neuron that leads FBMN migration through the hindbrain is an r4-derived FBMN/REN or an r5-derived CEN. Our experiments verify that the pioneer is an r4-derived neuron and reaffirm its role in leading FBMN migration across the r4/5 border. In contrast, the r5-derived CENs migrate independently of the pioneer. Together, these results indicate that the mechanisms OENs use to navigate the hindbrain differ significantly from those employed by FBMNs., (© 2020 Wiley Periodicals LLC.)

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss of CXCR4 signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss of RET signaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.

The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423 , which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance ( Lmx1a and Otx2 ); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis ( Gmnc ). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Embryonic boundaries were first described in Drosophila, and then in vertebrate embryos, as cellular interfaces between compartments. They display signaling properties and in vertebrates might allocate cells fated to different anatomical structures, or cells that will play different functions over time. One of the vertebrate embryonic structures with boundaries is the hindbrain, the posterior brain vesicle, which is transitory segmented upon morphogenesis. The hindbrain is formed by iterative units called rhombomeres that constitute units of gene expression and cell-lineage compartments. Rhombomeric cells are segregated by interhombomeric boundaries, which are prefigured by sharp gene expression borders. Hindbrain boundaries were first described as static groups of cells. However, later discoveries demonstrated the dynamic behavior of this specific cell population. They play distinct functional properties during brain morphogenesis that partially overlap on time, starting as a mechanical barrier to prevent cell intermingling, becoming a signaling hub, to finally constitute a group of proliferating progenitors providing differentiated neurons to the system. In this review, I try to give a more functional overview of this segmentation process and in particular of hindbrain boundaries. I will discuss the new challenges in the field on how to integrate cell fate specification and morphogenesis during brain embryonic development., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rationale: Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration., Objective: To examine the contribution of EMPs to intraembryonic endothelial cells., Methods and Results: We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc) + circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development., Conclusions: Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.

Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLC VGLUT2 neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLC VGLUT2 neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLC VGLUT2 neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLC VGLUT2 neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLC VGLUT2 neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity., Competing Interests: Declaration of Interests S.M.S. has served on the Scientific Advisory Board of Inscopix, Inc. and owns stock in the company., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of the dorsal hindbrain, which expands from r2 to r7, and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei. Although there is information about the contribution of specific proneural-progenitor populations to specific deep nuclei, and the distinct rhombomeric contribution, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated. In this work, we investigated the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1a-derivatives by using complementary strategies in the zebrafish embryo. We unveiled that atoh1a is necessary and sufficient for specification of LRL cells by activating atoh1b, which worked as a differentiation gene to transition progenitor cells towards neuron differentiation in a Notch-dependent manner. This cell state transition involved the release of atoh1a-derivatives from the LRL: atoh1a progenitors contributed first to atoh1b cells, which are committed non-proliferative precursors, and to the lhx2b-neuronal lineage as demonstrated by cell fate studies and functional analyses. Using in vivo cell lineage approaches we revealed that the proliferative cell capacity, as well as the mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. We showed that atoh1a may behave as the cell fate selector gene, whereas atoh1b functions as a neuronal differentiation gene, contributing to the lhx2b neuronal population. atoh1a-progenitor cell dynamics (cell proliferation, cell differentiation, and neuronal migration) relies on their position, demonstrating the challenges that progenitor cells face in computing positional information from a dynamic two-dimensional grid in order to generate the stereotyped neuronal structures in the embryonic hindbrain., Competing Interests: The authors have declared that no competing interests exist.

The mammalian claustrum, owing to its widespread connectivity with other forebrain structures, has been hypothesized to mediate functions that range from decision-making to consciousness 1 . Here we report that a homologue of the claustrum, identified by single-cell transcriptomics and viral tracing of connectivity, also exists in a reptile-the Australian bearded dragon Pogona vitticeps. In Pogona, the claustrum underlies the generation of sharp waves during slow-wave sleep. The sharp waves, together with superimposed high-frequency ripples 2 , propagate to the entire neighbouring pallial dorsal ventricular ridge (DVR). Unilateral or bilateral lesions of the claustrum suppress the production of sharp-wave ripples during slow-wave sleep in a unilateral or bilateral manner, respectively, but do not affect the regular and rapidly alternating sleep rhythm that is characteristic of sleep in this species 3 . The claustrum is thus not involved in the generation of the sleep rhythm itself. Tract tracing revealed that the reptilian claustrum projects widely to a variety of forebrain areas, including the cortex, and that it receives converging inputs from, among others, areas of the mid- and hindbrain that are known to be involved in wake-sleep control in mammals 4-6 . Periodically modulating the concentration of serotonin in the claustrum, for example, caused a matching modulation of sharp-wave production there and in the neighbouring DVR. Using transcriptomic approaches, we also identified a claustrum in the turtle Trachemys scripta, a distant reptilian relative of lizards. The claustrum is therefore an ancient structure that was probably already present in the brain of the common vertebrate ancestor of reptiles and mammals. It may have an important role in the control of brain states owing to the ascending input it receives from the mid- and hindbrain, its widespread projections to the forebrain and its role in sharp-wave generation during slow-wave sleep.

Astrocytes generate robust cytoplasmic calcium signals in response to reductions in extracellular glucose. This calcium signal, in turn, drives purinergic gliotransmission, which controls the activity of catecholaminergic (CA) neurons in the hindbrain. These CA neurons are critical to triggering glucose counter-regulatory responses (CRRs) that, ultimately, restore glucose homeostasis via endocrine and behavioral means. Although the astrocyte low-glucose sensor involvement in CRR has been accepted, it is not clear how astrocytes produce an increase in intracellular calcium in response to a decrease in glucose. Our ex vivo calcium imaging studies of hindbrain astrocytes show that the glucose type 2 transporter (GLUT2) is an essential feature of the astrocyte glucosensor mechanism. Coimmunoprecipitation assays reveal that the recombinant GLUT2 binds directly with the recombinant Gq protein subunit that activates phospholipase C (PLC). Additional calcium imaging studies suggest that GLUT2 may be connected to a PLC-endoplasmic reticular-calcium release mechanism, which is amplified by calcium-induced calcium release (CICR). Collectively, these data help outline a potential mechanism used by astrocytes to convert information regarding low-glucose levels into intracellular changes that ultimately regulate the CRR.

Background: Elevated circulating levels of the divergent transforming growth factor-beta (TGFb) family cytokine, growth differentiation factor 15 (GDF15), acting through its CNS receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), can cause anorexia and weight loss leading to anorexia/cachexia syndrome of cancer and other diseases. Preclinical studies suggest that administration of drugs based on recombinant GDF15 might be used to treat severe obesity. However, the role of the GDF15-GFRAL pathway in the physiological regulation of body weight and metabolism is unclear. The critical site of action of GFRAL in the CNS has also not been proven beyond doubt. To investigate these two aspects, we have inhibited the actions of GDF15 in mice started on high-fat diet (HFD)., Methods: The actions of GDF15 were inhibited using two methods: (1) Groups of 8 mice under HFD had their endogenous GDF15 neutralised by monoclonal antibody treatment, (2) Groups of 15 mice received AAV-shRNA to knockdown GFRAL at its hypothesised major sites of action, the hindbrain area postrema (AP) and the nucleus of the solitary tract (NTS). Metabolic measurements were determined during both experiments., Conclusions: Treating mice with monoclonal antibody to GDF15 shortly after commencing HFD results in more rapid gain of body weight, adiposity and hepatic lipid deposition than the control groups. This is accompanied by reduced glucose and insulin tolerance and greater expression of pro-inflammatory cytokines in adipose tissue. Localised AP and NTS shRNA-GFRAL knockdown in mice commencing HFD similarly caused an increase in body weight and adiposity. This effect was in proportion to the effectiveness of GFRAL knockdown, indicated by quantitative analysis of hindbrain GFRAL staining. We conclude that the GDF15-GFRAL axis plays an important role in resistance to obesity in HFD-fed mice and that the major site of action of GDF15 in the CNS is GFRAL-expressing neurons in the AP and NTS.

During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440., (©AlphaMed Press 2019.)

Although electromagnetic brain stimulation is a promising treatment in neurology and psychiatry, clinical outcomes are variable, and underlying mechanisms are ill-defined, which impedes the development of new effective stimulation protocols. Here, we show, in vivo and ex vivo, that repetitive transcranial magnetic stimulation at low-intensity (LI-rTMS) induces axon outgrowth and synaptogenesis to repair a neural circuit. This repair depends on stimulation pattern, with complex biomimetic patterns being particularly effective, and the presence of cryptochrome, a putative magnetoreceptor. Only repair-promoting LI-rTMS patterns up-regulated genes involved in neuronal repair; almost 40% of were cryptochrome targets. Our data open a new framework to understand the mechanisms underlying structural neuroplasticity induced by electromagnetic stimulation. Rather than neuronal activation by induced electric currents, we propose that weak magnetic fields act through cryptochrome to activate cellular signaling cascades. This information opens new routes to optimize electromagnetic stimulation and develop effective treatments for different neurological diseases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Cells perceive their microenvironment through chemical and physical cues. However, how the mechanical signals are interpreted during embryonic tissue deformation to result in specific cell behaviors is largely unknown. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, and to cell shape and actomyosin cytoskeletal changes. In this study, we demonstrate the role of Yap/Taz-TEAD activity as a sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during zebrafish hindbrain compartmentalization. Monitoring of in vivo Yap/Taz activity during hindbrain segmentation indicated that boundary cells responded to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decreased their proliferative activity when Yap/Taz-TEAD activity ceased, which preceded changes in their cell fate from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining progenitor features in the hindbrain boundary cell population., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Engraftment of oligodendrocyte progenitor cells (OPCs), which form myelinating oligodendrocytes, has the potential to treat demyelinating diseases such as multiple sclerosis. However, conventional strategies for generating oligodendrocytes have mainly focused on direct differentiation into forebrain- or spinal cord-restricted oligodendrocytes without establishing or amplifying stem/progenitor cells. Taking advantage of a recently established culture system, we generated expandable EN1- and GBX2-positive glial-restricted progenitor-like cells (GPLCs) near the anterior hindbrain. These cells expressed PDGFRα, CD9, S100β, and SOX10 and mostly differentiated into GFAP-positive astrocytes and MBP-positive oligodendrocytes. RNA-seq analysis revealed that the transcriptome of GPLCs was similar to that of O4-positive OPCs, but distinct from that of rosette-type neural stem cells. Notably, engrafted GPLCs not only differentiated into GFAP-positive astrocytes but also myelinated the brains of adult shiverer mice 8 weeks after transplantation. Our strategy for establishing anterior hindbrain-specific GPLCs with gliogenic potency will facilitate their use in the treatment of demyelinating diseases and studies of the molecular mechanisms underlying glial development in the hindbrain.

Hindbrain astrocytes are emerging as critical components in the regulation of homeostatic functions by either modulating synaptic activity or serving as primary detectors of physiological parameters. Recent studies have suggested that the glucose counter-regulation response (CRR), a critical defense against hypoglycemic emergencies, is dependent on glucoprivation-sensitive astrocytes in the hindbrain. This subpopulation of astrocytes produces a robust calcium signal in response to glucopenic stimuli. Both ex vivo and in vivo evidence suggest that low-glucose sensitive astrocytes utilize purinergic gliotransmission to activate catecholamine neurons in the hindbrain that are critical to the generation of the integrated CRR. Lastly, reports in the clinical literature suggest that an uncontrolled activation of CRR may as part of the pathology of severe traumatic injury. Work in our laboratory also suggests that this pathological hyperglycemia resulting from traumatic injury may be caused by the action of thrombin (generated by tissue trauma or bleeding) on hindbrain astrocytes. Similar to their glucopenia-sensitive neighbors, these hindbrain astrocytes may trigger hyperglycemic responses by their interactions with catecholaminergic neurons., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine β-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Little is known about the molecular and cellular mechanisms involved in the formation of the cranial peripheral sensory system in vertebrates. To identify genes involved in the formation of these circuits, we performed a forward genetic screen utilizing a transgenic zebrafish line ( p2rx3.2:gfp sl1 ) that expresses green fluorescent protein (gfp) in sensory neurons of the Vth, VIIth, IXth and Xth cranial ganglia. Here, we describe a novel zebrafish mutant in which a missense mutation in the adam19b gene selectively affects the epibranchial sensory circuits.

Neurons in the brainstem dorsal vagal complex integrate neural and humoral signals to coordinate autonomic output to viscera that regulate a variety of physiological functions, but how this circuitry regulates metabolism is murky. We tested the hypothesis that premotor, GABAergic neurons in the nucleus tractus solitarius (NTS) form a hindbrain micro-circuit with preganglionic parasympathetic motorneurons of the dorsal motor nucleus of the vagus (DMV) that is capable of modulating systemic blood glucose concentration. In vitro, neuronal activation or inhibition using either excitatory or inhibitory designer receptor exclusively activated by designer drugs (DREADDs) constructs expressed in GABAergic NTS neurons increased or decreased, respectively, action potential firing of GABAergic NTS neurons and downstream synaptic inhibition of the DMV. In vivo, DREADD-mediated activation of GABAergic NTS neurons increased systemic blood glucose concentration, whereas DREADD-mediated silencing of these neurons was without effect. The DREADD-induced hyperglycemia was abolished by blocking peripheral muscarinic receptors, consistent with the hypothesis that altered parasympathetic drive mediated the response. This effect was paralleled by elevated serum glucagon and hepatic phosphoenolpyruvate carboxykinase 1 (PEPCK1) expression, without affecting insulin levels or muscle metabolism. Activity in a hindbrain inhibitory microcircuit is sufficient to modulate systemic glucose concentration, independent of insulin secretion or utilization.

An important model for axon pathfinding is provided by guidance of embryonic commissural axons from dorsal spinal cord to ventral midline floor plate (FP). FP cells produce a chemoattractive activity, comprised largely of netrin1 (FP-netrin1) and Sonic hedgehog (Shh), that can attract the axons at a distance in vitro. netrin1 is also produced by ventricular zone (VZ) progenitors along the axons' route (VZ-netrin1). Recent studies using region-specific netrin1 deletion suggested that FP-netrin1 is dispensable and VZ-netrin1 sufficient for netrin guidance activity in vivo. We show that removing FP-netrin1 actually causes guidance defects in spinal cord consistent with long-range action (i.e., over hundreds of micrometers), and double mutant analysis supports that FP-netrin1 and Shh collaborate to attract at long range. We further provide evidence that netrin1 may guide via chemotaxis or haptotaxis. These results support the model that netrin1 signals at both short and long range to guide commissural axons in spinal cord., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During many behaviors in vertebrates, the CNS generates asymmetric activities between the left and right sides to produce asymmetric body movements. For asymmetrical activations of the CNS, reciprocal inhibition between the left and right sides is believed to play a key role. However, the complexity of the CNS makes it difficult to identify the reciprocal inhibition circuits at the level of individual cells and the contribution of each neuron to the asymmetric activity. Using larval zebrafish, we examined this issue by investigating reciprocal inhibition circuits between a pair of Mauthner (M) cells, giant reticulospinal neurons that trigger fast escapes. Previous studies have shown that a class of excitatory neurons, called cranial relay neurons, is involved in the reciprocal inhibition pathway between the M cells. Using transgenic fish, in which two of the cranial relay neurons (Ta1 and Ta2) expressed GFP, we showed that Ta1 and Ta2 constitute major parts of the pathway. In larvae in which Ta1/Ta2 were laser-ablated, the amplitude of the reciprocal IPSPs dropped to less than one-third. Calcium imaging and electrophysiological recording showed that the occurrence probability of bilateral M-cell activation upon sound/vibration stimuli was greatly increased in the Ta1/Ta2-ablated larvae. Behavioral experiments revealed that the Ta1/Ta2 ablation resulted in shallower body bends during sound/vibration-evoked escapes, which is consistent with the observation that increased occurrence of bilateral M-cell activation impaired escape performance. Our study revealed major components of the reciprocal inhibition circuits in the M cell system and the behavioral importance of the circuits. SIGNIFICANCE STATEMENT Reciprocal inhibition between the left and right side of the CNS is considered imperative for producing asymmetric movements in animals. It has been difficult, however, to identify the circuits at the individual cell level and their role in behavior. Here, we address this problem by examining the reciprocal inhibition circuits of the hindbrain Mauthner (M) cell system in larval zebrafish. We determined that two paired interneurons play a critical role in the reciprocal inhibition between the paired M cells and that the reciprocal inhibition prevents bilateral firing of the M cells and is thus necessary for the full body bend during M cell-initiated escape. Further, we discussed the cooperation of multiple reciprocal inhibitions working in the hindbrain and spinal cord to ensure high-performance escapes., (Copyright © 2019 the authors 0270-6474/19/391182-13$15.00/0.)

Vestibular function was established early in vertebrates and has remained, for the most part, unchanged. In contrast, each group of tetrapods underwent independent evolutionary processes to solve the problem of hearing on land, resulting in a remarkable mixture of conserved, divergent and convergent features that define extant auditory systems. The vestibuloacoustic nuclei of the hindbrain develop from a highly conserved ground plan and provide an ideal framework on which to address the participation of developmental processes to the evolution of neuronal circuits. We employed an electroporation strategy to unravel the contribution of two dorsoventral and four axial lineages to the development of the chick hindbrain vestibular and auditory nuclei. We compare the chick developmental map with recently established genetic fate-maps of the developing mouse hindbrain. Overall, we find considerable conservation of developmental origin for the vestibular nuclei. In contrast, a comparative analysis of the developmental origin of hindbrain auditory structures echoes the complex evolutionary history of the auditory system. In particular, we find that the developmental origin of the chick auditory interaural time difference circuit supports its emergence from an ancient vestibular network, unrelated to the analogous mammalian counterpart., Competing Interests: ML, RW No competing interests declared, (© 2018, Lipovsek and Wingate.)

Wilhelm His (1831-1904) provided lasting insights into the development of the central and peripheral nervous system using innovative technologies such as the microtome, which he invented. 150 years after his resurrection of the classical germ layer theory of Wolff, von Baer and Remak, his description of the developmental origin of cranial and spinal ganglia from a distinct cell population, now known as the neural crest, has stood the test of time and more recently sparked tremendous advances regarding the molecular development of these important cells. In addition to his 1868 treatise on 'Zwischenstrang' (now neural crest), his work on the development of the human hindbrain published in 1890 provided novel ideas that more than 100 years later form the basis for penetrating molecular investigations of the regionalization of the hindbrain neural tube and of the migration and differentiation of its constituent neuron populations. In the first part of this review we briefly summarize the major discoveries of Wilhelm His and his impact on the field of embryology. In the second part we relate His' observations to current knowledge about the molecular underpinnings of hindbrain development and evolution. We conclude with the proposition, present already in rudimentary form in the writings of His, that a primordial spinal cord-like organization has been molecularly supplemented to generate hindbrain 'neomorphs' such as the cerebellum and the auditory and vestibular nuclei and their associated afferents and sensory organs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The folding of epithelial tissues is crucial for development of three-dimensional structure and function. Understanding this process can assist in determining the etiology of developmental disease and engineering of tissues for the future of regenerative medicine. Folding of epithelial tissues towards the apical surface has long been studied, but the molecular mechanisms that mediate epithelial folding towards the basal surface are just emerging. Here, we utilize zebrafish neuroepithelium to identify mechanisms that mediate basal tissue folding to form the highly conserved embryonic midbrain-hindbrain boundary. Live imaging revealed Wnt5b as a mediator of anisotropic epithelial cell shape, both apically and basally. In addition, we uncovered a Wnt5b-mediated mechanism for specific regulation of basal anisotropic cell shape that is microtubule dependent and likely to involve JNK signaling. We propose a model in which a single morphogen can differentially regulate apical versus basal cell shape during tissue morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The noradrenergic (NA) system of vertebrates is implicated in learning, memory, arousal, and neuroinflammatory responses, but is difficult to access experimentally. Small and optically transparent, larval zebrafish offer the prospect of exploration of NA structure and function in an intact animal. We made multiple transgenic zebrafish lines using the CRISPR/Cas9 system to insert fluorescent reporters upstream of slc6a2, the norepinephrine transporter gene. These lines faithfully express reporters in NA cell populations, including the locus coeruleus (LC), which contains only about 14 total neurons. We used the lines in combination with two-photon microscopy to explore the structure and projections of the NA system in the context of the columnar organization of cell types in the zebrafish hindbrain. We found robust alignment of NA projections with glutamatergic neurotransmitter stripes in some hindbrain segments, suggesting orderly relations to neuronal cell types early in life. We also quantified neurite density in the rostral spinal cord in individual larvae with as much as 100% difference in the number of LC neurons, and found no correlation between neuronal number in the LC and projection density in the rostral spinal cord. Finally, using light sheet microscopy, we performed bilateral calcium imaging of the entire LC. We found that large-amplitude calcium responses were evident in all LC neurons and showed bilateral synchrony, whereas small-amplitude events were more likely to show interhemispheric asynchrony, supporting the potential for targeted LC neuromodulation. Our observations and new transgenic lines set the stage for a deeper understanding of the NA system., (© 2018 Wiley Periodicals, Inc.)

The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.

Neural stem cells (NSCs) are self-renewing multipotent cells that line the neural-tube and generate all the nervous system. Understanding NSC biology is fundamental for neurodevelopmental research and therapy. Many studies emphasized the need to culture NSCs, which are typically purified from mammalian embryonic/adult brains. These sources are somewhat limited in terms of quantity, availability and animal ethical guidelines. Therefore, new sources are needed. The chick is a powerful system for experimental embryology which contributed enormously to neurodevelopmental concepts. Its accessibility, genetic/molecular manipulations, and homology to other vertebrates, makes it valuable for developmental biology research. Recently, we identified a population of NSCs in the chick hindbrain. It resides in rhombomere-boundaries, expresses Sox2 and generates progenitors and neurons. Here, we investigated whether these cells can recapitulate hindbrain development in culture. By developing approaches to propagate and image cells, manipulate their growth-conditions and separate them into subpopulations, we demonstrate the ordered formation of multipotent and self-renewing neurospheres that maintain regional identity and display differential stem/differentiation/proliferation properties. Live imaging revealed new cellular dynamics in the culture. Collectively, these NSC cultures reproduce major aspects of hindbrain development in-vitro, proposing the chick as a model for culturing hindbrain-NSCs that can be directly applied to other neural-tube domains and species.

Studies of the vertebrate hindbrain have revealed parallel mechanisms that establish sharp segments with a distinct and homogeneous regional identity. Recent work has revealed roles of cell identity regulation and its relationships with cell segregation. At early stages, there is overlapping expression at segment borders of the Egr2 and Hoxb1 transcription factors that specify distinct identities, which is resolved by reciprocal repression. Computer simulations show that this dynamic regulation of cell identity synergises with cell segregation to generate sharp borders. Some intermingling between segments occurs at early stages, and ectopic egr2-expressing cells switch identity to match their new neighbours. This switching is mediated by coupling between egr2 expression and the level of retinoic acid signalling, which acts in a community effect to maintain homogeneous segmental identity. These findings reveal an interplay between cell segregation and the dynamic regulation of cell identity in the formation of sharp patterns in the hindbrain and raise the question of whether similar mechanisms occur in other tissues., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Hindbrain catecholaminergic (CA) neurons are required for critical autonomic, endocrine, and behavioral counterregulatory responses (CRRs) to hypoglycemia. Recent studies suggest that CRR initiation depends on hindbrain astrocyte glucose sensors (McDougal DH, Hermann GE, Rogers RC. Front Neurosci 7: 249, 2013; Rogers RC, Ritter S, Hermann GE. Am J Physiol Regul Integr Comp Physiol 310: R1102-R1108, 2016). To test the proposition that hindbrain CA responses to glucoprivation are astrocyte dependent, we utilized transgenic mice in which the calcium reporter construct (GCaMP5) was expressed selectively in tyrosine hydroxylase neurons (TH-GCaMP5). We conducted live cell calcium-imaging studies on tissue slices containing the nucleus of the solitary tract (NST) or the ventrolateral medulla, critical CRR initiation sites. Results show that TH-GCaMP5 neurons are robustly activated by a glucoprivic challenge and that this response is dependent on functional astrocytes. Pretreatment of hindbrain slices with fluorocitrate (an astrocytic metabolic suppressor) abolished TH-GCaMP5 neuronal responses to glucoprivation, but not to glutamate. Pharmacologic results suggest that the astrocytic connection with hindbrain CA neurons is purinergic via P2 receptors. Parallel imaging studies on hindbrain slices of NST from wild-type C57BL/6J mice, in which astrocytes and neurons were prelabeled with a calcium reporter dye and an astrocytic vital dye, show that both cell types are activated by glucoprivation but astrocytes responded significantly sooner than neurons. Pretreatment of these hindbrain slices with P2 antagonists abolished neuronal responses to glucoprivation without interruption of astrocyte responses; pretreatment with fluorocitrate eliminated both astrocytic and neuronal responses. These results support earlier work suggesting that the primary detection of glucoprivic signals by the hindbrain is mediated by astrocytes.

Molecular and functional diversity within midbrain dopaminergic (mDA) and hindbrain serotonergic (5-HT) neurons has emerged as a relevant feature that could underlie selective vulnerability of neurons in clinical disorders. We have investigated the role of transforming growth factor beta (TGF-β) during development of mDA and 5-HT subgroups. We have generated TβRII flox/flox ::En1 cre/+ mice where type II TGF-β receptor is conditionally deleted from engrailed 1-expressing cells and have investigated the hindbrain serotonergic system of these mice together with Tgf-β2 -/- mice. The results show a significant decrease in the number of 5-HT neurons in TGF-β2-deficient mice at embryonic day (E) 12 and a selective significant decrease in the hindbrain paramedian raphe 5-HT neurons at E18, compared to wild type. Moreover, conditional deletion of TGF-β signaling from midbrain and rhombomere 1 leads to inactive TGF-β signaling in cre-expressing cells, impaired development of mouse mDA neuron subgroups and of dorsal raphe 5-HT neuron subgroups in a temporal manner. These results highlight a selective growth factor dependency of individual rostral hindbrain serotonergic subpopulations, emphasize the impact of TGF-β signaling during development of mDA and 5-HT subgroups, and suggest TGF-βs as potent candidates to establish diversity within the hindbrain serotonergic system. Thus, the data contribute to a better understanding of development and degeneration of mDA neurons and 5-HT-associated clinical disorders., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

The patterning of tissues to form subdivisions with distinct and homogeneous regional identity is potentially disrupted by cell intermingling. Transplantation studies suggest that homogeneous segmental identity in the hindbrain is maintained by identity switching of cells that intermingle into another segment. We show that switching occurs during normal development and is mediated by feedback between segment identity and the retinoic acid degrading enzymes, cyp26b1 and cyp26c1. egr2, which specifies the segmental identity of rhombomeres r3 and r5, underlies the lower expression level of cyp26b1 and cyp26c1 in r3 and r5 compared with r2, r4, and r6. Consequently, r3 or r5 cells that intermingle into adjacent segments encounter cells with higher cyp26b1/c1 expression, which we find is required for downregulation of egr2b expression. Furthermore, egr2b expression is regulated in r2, r4, and r6 by non-autonomous mechanisms that depend upon the number of neighbors that express egr2b. These findings reveal that a community regulation of retinoid signaling maintains homogeneous segmental identity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous studies have demonstrated that both Wnt5a and Sonic hedgehog (Shh) are involved in regulating the pathfinding of descending serotonergic (5-HT, 5-hydroxytryptamine) axons in an opposite manner in the brainstem. Shh and Wnt signaling pathways interact to guide post-crossing commissural axons, where Shh acts as a repellent directly and shaping the Wnt gradient indirectly by regulating the gradient expression of the frizzled-related protein 1 (Sfrp1). Whether such a mechanism functions in descending 5-HT axon guidance remains unknown. Here, we found that the core components of the Shh and Wnt planar cell polarity signaling pathways are expressed in caudal 5-HT neurons, and the expression gradients of Shh, Sfrp1, and Wnt5a exist simultaneously in hindbrain. Dunn chamber assays revealed that Sfrp1 suppressed the attractive Wnt gradient. Moreover, we found that Shh overexpression led to pathfinding defects in 5-HT axon descending, and the axonal pathfinding defects could be partially rescued by administration of an Sfrp1 antagonist in vivo. Biochemical evidence showed Shh overexpression upregulated the expression of the Sfrp1 gene and interrupted Wnt5a binding to Frizzled-3. Taken together, our results indicate that Shh, Sfrp1, and Wnt5a collaborate to direct the pathfinding of descending 5-HT axons in the brainstem., (Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.)

During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3β (GSK3β) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

During brainstem development, newborn neurons originating from the rhombic lip embark on exceptionally long migrations to generate nuclei important for audition, movement, and respiration. Along the way, this highly motile population passes several cranial nerves yet remains confined to the CNS. We found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps at nerve entry sites. In mice null for Ntn1 or its receptor DCC, hindbrain neurons enter cranial nerves and migrate into the periphery. CNS neurons also escape when Ntn1 is selectively lost from the sub-pial region (SPR), and conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. These findings identify a permissive role for Ntn1 in maintaining the CNS-PNS boundary. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Heterogeneity of firing rate statistics is known to have severe consequences on neural coding. Recent experimental recordings in weakly electric fish indicate that the distribution-width of superficial pyramidal cell firing rates (trial- and time-averaged) in the electrosensory lateral line lobe (ELL) depends on the stimulus, and also that network inputs can mediate changes in the firing rate distribution across the population. We previously developed theoretical methods to understand how two attributes (synaptic and intrinsic heterogeneity) interact and alter the firing rate distribution in a population of integrate-and-fire neurons with random recurrent coupling. Inspired by our experimental data, we extend these theoretical results to a delayed feedforward spiking network that qualitatively capture the changes of firing rate heterogeneity observed in in-vivo recordings. We demonstrate how heterogeneous neural attributes alter firing rate heterogeneity, accounting for the effect with various sensory stimuli. The model predicts how the strength of the effective network connectivity is related to intrinsic heterogeneity in such delayed feedforward networks: the strength of the feedforward input is positively correlated with excitability (threshold value for spiking) when firing rate heterogeneity is low and is negatively correlated with excitability with high firing rate heterogeneity. We also show how our theory can be used to predict effective neural architecture. We demonstrate that neural attributes do not interact in a simple manner but rather in a complex stimulus-dependent fashion to control neural heterogeneity and discuss how it can ultimately shape population codes.

While the peripheral auditory system of fish has been well studied, less is known about how the fish's brain and central auditory system process complex social acoustic signals. The plainfin midshipman fish, Porichthys notatus, has become a good species for investigating the neural basis of acoustic communication because the production and reception of acoustic signals is paramount for this species' reproductive success. Nesting males produce long-duration advertisement calls that females detect and localize among the noise in the intertidal zone to successfully find mates and spawn. How female midshipman are able to discriminate male advertisement calls from environmental noise and other acoustic stimuli is unknown. Using the immediate early gene product cFos as a marker for neural activity, we quantified neural activation of the ascending auditory pathway in female midshipman exposed to conspecific advertisement calls, heterospecific white seabass calls, or ambient environment noise. We hypothesized that auditory hindbrain nuclei would be activated by general acoustic stimuli (ambient noise and other biotic acoustic stimuli) whereas auditory neurons in the midbrain and forebrain would be selectively activated by conspecific advertisement calls. We show that neural activation in two regions of the auditory hindbrain, i.e., the rostral intermediate division of the descending octaval nucleus and the ventral division of the secondary octaval nucleus, did not differ via cFos immunoreactive (cFos-ir) activity when exposed to different acoustic stimuli. In contrast, female midshipman exposed to conspecific advertisement calls showed greater cFos-ir in the nucleus centralis of the midbrain torus semicircularis compared to fish exposed only to ambient noise. No difference in cFos-ir was observed in the torus semicircularis of animals exposed to conspecific versus heterospecific calls. However, cFos-ir was greater in two forebrain structures that receive auditory input, i.e., the central posterior nucleus of the thalamus and the anterior tuberal hypothalamus, when exposed to conspecific calls versus either ambient noise or heterospecific calls. Our results suggest that higher-order neurons in the female midshipman midbrain torus semicircularis, thalamic central posterior nucleus, and hypothalamic anterior tuberal nucleus may be necessary for the discrimination of complex social acoustic signals. Furthermore, neurons in the central posterior and anterior tuberal nuclei are differentially activated by exposure to conspecific versus other acoustic stimuli., (© 2018 S. Karger AG, Basel.)

Hoxa1 has important functional roles in neural crest specification, hindbrain patterning and heart and ear development, yet the enhancers and genes that are targeted by Hoxa1 are largely unknown. In this study, we performed a comprehensive analysis of Hoxa1 target genes using genome-wide Hoxa1 binding data in mouse ES cells differentiated with retinoic acid (RA) into neural fates in combination with differential gene expression analysis in Hoxa1 gain- and loss-of-function mouse and zebrafish embryos. Our analyses reveal that Hoxa1-bound regions show epigenetic marks of enhancers, occupancy of Hox cofactors and differential expression of nearby genes, suggesting that these regions are enriched for enhancers. In support of this, 80 of them mapped to regions with known reporter activity in transgenic mouse embryos based on the Vista enhancer database. Two additional enhancers in Dok5 and Wls1 were shown to mediate neural expression in developing mouse and zebrafish. Overall, our analysis of the putative target genes indicate that Hoxa1 has input to components of major signaling pathways, including Wnt, TGF-β, Hedgehog and Hippo, and frequently does so by targeting multiple components of a pathway such as secreted inhibitors, ligands, receptors and down-stream components. We also identified genes implicated in heart and ear development, neural crest migration and neuronal patterning and differentiation, which may underlie major Hoxa1 mutant phenotypes. Finally, we found evidence for a high degree of evolutionary conservation of many binding regions and downstream targets of Hoxa1 between mouse and zebrafish. Our genome-wide analyses in ES cells suggests that we have enriched for in vivo relevant target genes and pathways associated with functional roles of Hoxa1 in mouse development., (Copyright © 2017 Elsevier Inc. All rights reserved.)