Dynamic spatiotemporal coordination of neural stem cell fate decisions occurs through local feedback in the adult vertebrate brain.

Neural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis and coordinate specific spatiotemporal correlations.

Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis.

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium.

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1-/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation.

Advances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridisation (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild-type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.

Large-scale live imaging of adult neural stem cells in their endogenous niche.

Live imaging of adult neural stem cells (aNSCs) in vivo is a technical challenge in the vertebrate brain. Here, we achieve long-term imaging of the adult zebrafish telencephalic neurogenic niche and track a population of >1000 aNSCs over weeks, by taking advantage of fish transparency at near-infrared wavelengths and of intrinsic multiphoton landmarks. This methodology enables us to describe the frequency, distribution and modes of aNSCs divisions across the entire germinal zone of the adult pallium, and to highlight regional differences in these parameters.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene.

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

Long-range evolutionary constraints reveal cis-regulatory interactions on the human X chromosome.

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression.

Notch3 signaling gates cell cycle entry and limits neural stem cell amplification in the adult pallium.

Maintaining the homeostasis of germinal zones in adult organs is a fundamental but mechanistically poorly understood process. In particular, what controls stem cell activation remains unclear. We have previously shown that Notch signaling limits neural stem cell (NSC) proliferation in the adult zebrafish pallium. Combining pharmacological and genetic manipulations, we demonstrate here that long-term Notch invalidation primarily induces NSC amplification through their activation from quiescence and increased occurrence of symmetric divisions. Expression analyses, morpholino-mediated invalidation and the generation of a notch3-null mutant directly implicate Notch3 in these effects. By contrast, abrogation of notch1b function results in the generation of neurons at the expense of the activated NSC state. Together, our results support a differential involvement of Notch receptors along the successive steps of NSC recruitment. They implicate Notch3 at the top of this hierarchy to gate NSC activation and amplification, protecting the homeostasis of adult NSC reservoirs under physiological conditions.

Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate.

Neurogenesis is widespread in the zebrafish adult brain through the maintenance of active germinal niches. To characterize which progenitor properties correlate with this extensive neurogenic potential, we set up a method that allows progenitor cell transduction and tracing in the adult zebrafish brain using GFP-encoding retro- and lentiviruses. The telencephalic germinal zone of the zebrafish comprises quiescent radial glial progenitors and actively dividing neuroblasts. Making use of the power of clonal viral vector-based analysis, we demonstrate that these progenitors follow different division modes and fates: neuroblasts primarily undergo a limited amplification phase followed by symmetric neurogenic divisions; by contrast, radial glia are capable at the single cell level of both self-renewing and generating different cell types, and hence exhibit bona fide neural stem cell (NSC) properties in vivo. We also show that radial glial cells predominantly undergo symmetric gliogenic divisions, which amplify this NSC pool and may account for its long-lasting maintenance. We further demonstrate that blocking Notch signaling results in a significant increase in proliferating cells and in the numbers of clones, but does not affect clone composition, demonstrating that Notch primarily controls proliferation rather than cell fate. Finally, through long-term tracing, we illustrate the functional integration of newborn neurons in forebrain adult circuitries. These results characterize fundamental aspects of adult progenitor cells and neurogenesis, and open the way to using virus-based technologies for stable genetic manipulations and clonal analyses in the zebrafish adult brain.

Chronic alcohol consumption affects gastrointestinal motility and reduces the proportion of neuronal NOS-immunoreactive myenteric neurons in the murine jejunum.

Alcohol consumption interferes with gastrointestinal transit causing symptoms in alcoholic patients. Nitric oxide (NO), synthesized by neuronal nitric oxide synthase (nNOS) plays an important role in the control of gastrointestinal motility. Our aim was to investigate whether chronic alcohol intake in a murine model induces gastrointestinal motility disturbances and affects the nitrergic myenteric neurons in the stomach and jejunum. Gastric emptying, small intestinal transit and geometric centre were measured in vivo after intragastric gavage of Evans blue. Nitrergic relaxations to electrical field stimulation (EFS) and exogenous NO were recorded in jejunal muscle strips in vitro. The proportion of nNOS-immunopositive myenteric neurons was assessed using PGP9.5 and nNOS immunostaining. After chronic alcohol consumption, gastric emptying and small intestinal transit were delayed compared with control mice, and the nitrergic nerve-mediated relaxations to EFS in the jejunum were decreased, whereas relaxations to exogenous NO did not differ. The proportion of nNOS-immunoreactive neurons did not change in the stomach, whereas in the jejunum the percentage decreased from 33% to 27% (P < 0.001) after chronic alcohol intake. The total number of myenteric neurons remained unchanged. These results suggest that chronic alcohol consumption disturbs gastric and small intestinal motility in vivo and in vitro and is associated with a decrease in the proportion of nNOS-immunoreactive myenteric neurons in the murine jejunum.

Chronic ethanol exposure impairs neuronal nitric oxide synthase in the rat intestine.

BACKGROUND: Nitric oxide (NO), synthesized by neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric oxide synthases, plays an essential role in the physiological functions of the gastrointestinal (GI) tract. Chronic ethanol intake has been shown to interfere with several of these physiological functions, leading to the pathological alterations observed in alcoholic individuals. Our aim therefore was to investigate the effects of chronic ethanol consumption on NOS isoforms in different GI segments. METHODS: Rats received either 20% aqueous ethanol solution or water for 8 weeks. Tissue samples of the duodenum, jejunum, ileum, and colon of the rats were used for measurement of the NOS activity, protein content, and nNOS immunohistochemistry. Anti-HuC/D immunohistochemistry was used to determine the total number of neurons. RESULTS: Measurement of the physiological constitutive NOS (cNOS) activity revealed a 20 times higher activity in the colon than in the small intestine and after chronic ethanol treatment demonstrated a significant decrease in the jejunum, ileum, and colon, while in the duodenum it remained unchanged compared with the control group. The physiological iNOS activity was higher in the ileum and colon than in the duodenum and jejunum, and these levels were not significantly affected by ethanol. Neuronal nitric oxide synthase immunohistochemistry revealed a significant decrease in the numbers of immunostained cells in all investigated intestinal segments, while the total number of myenteric neurons remained constant. The nNOS protein content measured by Western blotting indicated a significant decrease in the colon after ethanol consumption, while in other intestinal segments change was not detectable. CONCLUSIONS: This study has demonstrated for the first time that chronic ethanol consumption has a differential effect on NOS activity, NOS protein content, and the number of nitrergic neurons in different intestinal segments, suggesting that chronic ethanol administration affects the NO pathways in the enteric nervous system.