The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Biocompatible Magnetic Conjugated Polymer Nanoparticles for Optical and Lifetime Imaging Applications in the First Biological Window.

Conjugated polymers are organic semiconductors that can be used for fluorescence microscopy of living specimens. Here, we report the encapsulation of the bright-red-emitting conjugated polymer, poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}] (CN-FO-DPD), and superparamagnetic iron oxide nanoparticles (SPIONs) within poly(styrene-co-maleic anhydride) (PSMA) micelles. The resulting particles exhibited an emission peak at 657 nm, a fluorescence quantum yield of 21%, an average diameter of 65 nm, and a ζ potential of -30 mV. They are taken up by cells, and we describe their use in fluorescence microscopy of living Hela cells and zebrafish embryos and their associated cytotoxicity in HEK, HeLa, and HCE cells.

Allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants.

In model organisms, RNA-sequencing (RNA-seq) is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often over-represented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that the differential expression of genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

The adaptor protein Grb2b is an essential modulator for lympho-venous sprout formation in the zebrafish trunk.

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI.

The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

A bone to pick with zebrafish.

The development of high-throughput sequencing and genome-wide association studies allows us to deduce the genetic factors underlying diseases much more rapidly than possible through classical genetics, but a true understanding of the molecular mechanisms of these diseases still relies on integrated approaches including in vitro and in vivo model systems. One such model that is particularly suitable for studying bone diseases is the zebrafish (Danio rerio), a small fresh-water teleost that is highly amenable to genetic manipulation and in vivo imaging. Zebrafish physiology and genome organization are in many aspects similar to those of humans, and the skeleton and mineralizing tissues are no exception. In this review, we highlight some of the contributions that have been made through the study of mutant zebrafish that feature bone and/or mineralization disorders homologous to human diseases, including osteogenesis imperfecta, fibrodysplasia ossificans progressiva and generalized arterial calcification of infancy. The genomic and phenotypic similarities between the zebrafish and human cases are illustrated. We show that, despite some systemic physiological differences between mammals and teleosts, and a relative lack of a history as a model for bone research, the zebrafish represents a useful complement to mouse and tissue culture systems in the investigation of genetic bone disorders.

Prolactin stimulates precursor cells in the adult mouse hippocampus.

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.

The latent stem cell population is retained in the hippocampus of transgenic Huntington's disease mice but not wild-type mice.

The demonstration of the brain's ability to initiate repair in response to disease or injury has sparked considerable interest in therapeutic strategies to stimulate adult neurogenesis. In this study we examined the effect of a progressive neurodegenerative condition on neural precursor activity in the subventricular zone (SVZ) and hippocampus of the R6/1 transgenic mouse model of Huntington's disease (HD). Our results revealed an age-related decline in SVZ precursor numbers in both wild-type (WT) and HD mice. Interestingly, hippocampal precursor numbers declined with age in WT mice, although we observed maintenance in hippocampal precursor number in the HD animals in response to advancement of the disease. This maintenance was consistent with activation of a recently identified latent hippocampal precursor population. We found that the small latent stem cell population was also maintained in the HD hippocampus at 33 weeks, whereas it was not present in the WT. Our findings demonstrate that, despite a loss of neurogenesis in the HD hippocampus in vivo, there is a unique maintenance of the precursor and stem cells, which may potentially be activated to ameliorate disease symptoms.

Endogenous interferon gamma directly regulates neural precursors in the non-inflammatory brain.

Although a number of growth factors have been shown to be involved in neurogenesis, the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly, however, these effects were mirrored in vivo, with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis, as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore, NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs, as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen, a downstream marker of IFNgamma-mediated activation. Moreover, the inhibitory effect was ameliorated in the presence of SVZ-derived microglia, with their removal resulting in almost complete inhibition of NP proliferation. Interestingly, in contrast to the results obtained in the adult, exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain, an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain.

Norepinephrine directly activates adult hippocampal precursors via beta3-adrenergic receptors.

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that beta(3)-adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly, systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally, using a novel ex vivo "slice-sphere" assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via beta-adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via beta(3)-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants.