Zebrafish Hypothalamus Research Articles

Social isolation modulates appetite and avoidance behavior via a common oxytocinergic circuit in larval zebrafish

Animal brains have evolved to encode social stimuli and transform these representations into advantageous behavioral responses. The commonalities and differences of these representations across species are not well-understood. Here, we show that social isolation activates an oxytocinergic (OXT), nociceptive circuit in the larval zebrafish hypothalamus and that chemical cues released from conspecific animals are potent modulators of this circuit’s activity. We delineate an olfactory to subpallial pathway that transmits chemical social cues to OXT circuitry, where they are transformed into diverse outputs simultaneously regulating avoidance and feeding behaviors. Our data allow us to propose a model through which social stimuli are integrated within a fundamental neural circuit to mediate diverse adaptive behaviours.

Brief Developmental Exposure to Fluoxetine Causes Life-Long Alteration of the Brain Transcriptome in Zebrafish.

Fluoxetine (FLX) and other selective serotonin reuptake inhibitors are widely used to treat depressive disorders during pregnancy. Early-life exposure to FLX is known to disrupt the normal function of the stress axis in humans, rodents, and teleosts. We used a zebrafish line with a cortisol-inducible fluorescent transgene to study the effects of developmental daily exposure to FLX (54 µg/L) on the transcriptomic profile of brain tissues in exposed larvae and later as 6-month-old adults. High throughput RNA sequencing was conducted on brain tissues in unstressed and stressed conditions. Long-lasting effects of FLX were observed in telencephalon (Tel) and hypothalamus (Hyp) of adult zebrafish with 1927 and 5055 genes significantly (≥1.2 fold-change, false-discovery p-value < 0.05) dysregulated in unstressed condition, respectively. Similar findings were observed in Hyp with 1245 and 723 genes being significantly dysregulated in stressed adults, respectively. Differentially expressed genes converted to Homo sapiens orthologues were used for Ingenuity Pathway Analysis. The results showed alteration of pathways involved in neuroendocrine signaling, cholesterol metabolism and synaptogenesis. Enriched networks included lipid metabolism, molecular transport, and nervous system development. Analysis of putative upstream transcription regulators showed potential dysregulation of clocka and nr3c1 which control circadian rhythm, stress response, cholesterol metabolism and histone modifications. Several genes involved in epigenetic regulation were also affected by FLX, including dnmt3a, adarb1, adarb2, hdac4, hdac5, hdac8, and atf2. We report life-long disruptive effects of FLX on pathways associated with neuroendocrine signaling, stress response and the circadian rhythm, and all of which are implicated in the development of depressive disorders in humans. Our results raise concern for the persistent endocrine-disrupting potential of brief antidepressant exposure during embryonic development.

Gonadotropin Releasing Hormone (Gnrh) Triggers Neurogenesis in the Hypothalamus of Adult Zebrafish.

Recently, it has been shown in adult mammals that the hypothalamus can generate new cells in response to metabolic changes, and tanycytes, putative descendants of radial glia, can give rise to neurons. Previously we have shown in vitro that neurospheres generated from the hypothalamus of adult zebrafish show increased neurogenesis in response to exogenously applied hormones. To determine whether adult zebrafish have a hormone-responsive tanycyte-like population in the hypothalamus, we characterized proliferative domains within this region. Here we show that the parvocellular nucleus of the preoptic region (POA) labels with neurogenic/tanycyte markers vimentin, GFAP/Zrf1, and Sox2, but these cells are generally non-proliferative. In contrast, Sox2+ proliferative cells in the ventral POA did not express vimentin and GFAP/Zrf1. A subset of the Sox2+ cells co-localized with Fezf2:GFP, a transcription factor important for neuroendocrine cell specification. Exogenous treatments of GnRH and testosterone were assayed in vivo. While the testosterone-treated animals showed no significant changes in proliferation, the GnRH-treated animals showed significant increases in the number of BrdU-labeled cells and Sox2+ cells. Thus, cells in the proliferative domains of the zebrafish POA do not express radial glia (tanycyte) markers vimentin and GFAP/Zrf1, and yet, are responsive to exogenously applied GnRH treatment.

Involvement of Cxcl12a/Cxcr4b Chemokine System in Mediating the Stimulatory Effect of Embryonic Ethanol Exposure on Neuronal Density in Zebrafish Hypothalamus.

Embryonic exposure to ethanol (EtOH) produces marked disturbances in neuronal development and alcohol-related behaviors, with low-moderate EtOH doses stimulating neurogenesis without producing apoptosis and high doses having major cytotoxic effects while causing gross morphological abnormalities. With the pro-inflammatory chemokine system, Cxcl12, and its main receptor Cxcr4, known to promote processes of neurogenesis, we examined here this neuroimmune system in the embryonic hypothalamus to test directly if it mediates the stimulatory effects low-moderate EtOH doses have on neuronal development. We used the zebrafish (Danio rerio) model, which develops externally and allows one to investigate the developing brain in vivo with precise control of dose and timing of EtOH delivery in the absence of maternal influence. Zebrafish were exposed to low-moderate EtOH doses (0.1, 0.25, 0.5% v/v), specifically during a period of peak hypothalamic development from 22 to 24hours postfertilization, and in some tests were pretreated from 2 to 22 hpf with the Cxcr4 receptor antagonist, AMD3100. Measurements in the hypothalamus at 26 hpf were taken of cxcl12a and cxcr4b transcription, signaling, and neuronal density using qRT-PCR, RNAscope, and live imaging of transgenic zebrafish. Embryonic EtOH exposure, particularly at the 0.5% dose, significantly increased levels of cxcl12a and cxcr4b mRNA in whole embryos, number of cxcl12a and cxcr4b transcripts in developing hypothalamus, and internalization of Cxcr4b receptors in hypothalamic cells. Embryonic EtOH also caused an increase in the number of hypothalamic neurons and coexpression of cxcl12a and cxcr4b transcripts within these neurons. Each of these stimulatory effects of EtOH in the embryo was blocked by pretreatment with the Cxcr4 antagonist AMD3100. These results provide clear evidence that EtOH's stimulatory effects at low-moderate doses on the number of hypothalamic neurons early in development are mediated, in part, by increased transcription and intracellular activation of this chemokine system, likely due to autocrine signaling of Cxcl12a at its Cxcr4b receptor within the neurons.

Hedgehog Signaling Regulates Neurogenesis in the Larval and Adult Zebrafish Hypothalamus.

Neurogenesis is now known to play a role in adult hypothalamic function, yet the cell-cell mechanisms regulating this neurogenesis remain poorly understood. Here, we show that Hedgehog (Hh)/Gli signaling positively regulates hypothalamic neurogenesis in both larval and adult zebrafish and is necessary and sufficient for normal hypothalamic proliferation rates. Hh-responsive radial glia represent a relatively highly proliferative precursor population that gives rise to dopaminergic, serotonergic, and GABAergic neurons. In situ and transgenic reporter analyses revealed substantial heterogeneity in cell-cell signaling within the hypothalamic niche, with slow cycling Nestin-expressing cells residing among distinct and overlapping populations of Sonic Hh (Shh)-expressing, Hh-responsive, Notch-responsive, and Wnt-responsive radial glia. This work shows for the first time that Hh/Gli signaling is a key component of the complex cell-cell signaling environment that regulates hypothalamic neurogenesis throughout life.

Hypothalamic Dopamine Neurons Control Sensorimotor Behavior by Modulating Brainstem Premotor Nuclei in Zebrafish

Dopamine (DA)-producing neurons are critically involved in the production of motor behaviors in multiple circuits that are conserved from basal vertebrates to mammals. Although there is increasing evidence that DA neurons in the hypothalamus play a locomotor role, their precise contributions to behavior and the circuit mechanisms by which they are achieved remain unclear. Here, we demonstrate that tyrosine-hydroxylase-2-expressing (th2+) DA neurons in the zebrafish hypothalamus fire phasic bursts of activity to acutely promote swimming and modulate audiomotor behaviors on fast timescales. Their anatomy and physiology reveal two distinct functional DA modules within the hypothalamus. The first comprises an interconnected set of cerebrospinal-fluid-contacting DA nuclei surrounding the 3rd ventricle, which lack distal projections outside of the hypothalamus and influence locomotion through unknown means. The second includes neurons in the preoptic nucleus, which send long-range projections to targets throughout the brain, including the mid- and hindbrain, where they activate premotor circuits involved in swimming and sensorimotor integration. These data suggest a broad regulation of motor behavior by DA neurons within multiple hypothalamic nuclei and elucidate a novel functional mechanism for the preoptic DA neurons in the initiation of movement.

A transgene targeted to the zebrafish nkx2.4b locus drives specific green fluorescent protein expression and disrupts thyroid development.

With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b, a gene expressed during hypothalamic and thyroid development. We combined transcription activator-like effector nucleases (TALENs)-mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus. Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes. By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss-of-function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development.

Multiple convergent hypothalamus-brainstem circuits drive defensive behavior.

The hypothalamus is composed of many neuropeptidergic cell populations and directs multiple survival behaviors, including defensive responses to threats. However, the relationship between the peptidergic identity of neurons and their roles in behavior remains unclear. Here, we address this issue by studying the function of multiple neuronal populations in the zebrafish hypothalamus during defensive responses to a variety of homeostatic threats. Cellular registration of large-scale neural activity imaging to multiplexed in situ gene expression revealed that neuronal populations encoding behavioral features encompass multiple overlapping sets of neuropeptidergic cell classes. Manipulations of different cell populations showed that multiple sets of peptidergic neurons play similar behavioral roles in this fast-timescale behavior through glutamate co-release and convergent output to spinal-projecting premotor neurons in the brainstem. Our findings demonstrate that homeostatic threats recruit neurons across multiple hypothalamic cell populations, which cooperatively drive robust defensive behaviors.

Phoenixin-20 Stimulates mRNAs Encoding Hypothalamo-Pituitary-Gonadal Hormones, is Pro-Vitellogenic, and Promotes Oocyte Maturation in Zebrafish

Phoenixin-20 (PNX-20) is a bioactive peptide with hormone-like actions in vertebrates. In mammals, PNX stimulates hypothalamo-pituitary-gonadal hormones and regulate reproductive processes. Our immunohisto/cytochemical studies show PNX-like and the putative PNX receptor, SREB3-like immunoreactivity in the gonads of zebrafish, and in zebrafish liver (ZFL) cells. Intraperitoneal injection of zebrafish PNX-20 upregulates mRNAs encoding both salmon gonadotropin-releasing hormone (GnRH), and chicken GnRH-II and kisspeptin and its receptor in zebrafish hypothalamus. Similarly, luteinizing hormone receptor mRNA expression in the testis, follicle-stimulating hormone receptor in the ovary, and the kisspeptin system were upregulated in the gonads of PNX-20 injected fish. We also observed the upregulation of genes involved in the sex steroidogenic pathway (cyp11a1, cyp17a1, 17βhsd, cyp19a1a) in the gonads of PNX-20 administered fish. PNX-20 upregulates the expression of vitellogenin isoforms and estrogen receptor (esr2a and 2b) mRNAs in ZFL cells in vitro. Meanwhile, siRNA-mediated knockdown of PNX-20 resulted in the downregulation of all vitellogenin transcripts, further suggesting its possible role in vitellogenesis. PNX-20 treatment resulted in a significant increase in germinal vesicle breakdown in zebrafish follicles in vitro. Collectively, these results provide strong evidence for PNX-20 effects on the HPG axis and liver to promote reproduction in zebrafish.

Conserved Genoarchitecture of the Basal Hypothalamus in Zebrafish Embryos.

Analyses of genoarchitecture recently stimulated substantial revisions of anatomical models for the developing hypothalamus in mammalian and other vertebrate systems. The prosomeric model proposes the hypothalamus to be derived from the secondary prosencephalon, and to consist of alar and basal regions. The basal hypothalamus can further be subdivided into tuberal and mamillary regions, each with distinct subregions. Albeit being a widely used model system for neurodevelopmental studies, no detailed genoarchitectural maps exist for the zebrafish (Danio rerio) hypothalamus. Here, we compare expression domains of zebrafish genes, including arxa, shha, otpa, isl1, lhx5, nkx2.1, nkx2.2a, pax6, and dlx5a, the orthologs of which delimit specific subregions within the murine basal hypothalamus. We develop the highly conserved brain-specific homeobox (bsx) gene as a novel marker for genoarchitectural analysis of hypothalamic regions. Our comparison of gene expression patterns reveals that the genoarchitecture of the basal hypothalamus in zebrafish embryos 48 hours post fertilization is highly similar to mouse embryos at E13.5. We found the tuberal hypothalamus in zebrafish embryos to be relatively large and to comprise previously ill-defined regions around the posterior hypothalamic recess. The mamillary hypothalamus is smaller and concentrates to rather medial areas in proximity to the anterior end of the neural tube floor plate. Within the basal hypothalamus we identified longitudinal and transverse tuberal and mamillary subregions topologically equivalent to those previously described in other vertebrates. However, the hypothalamic diencephalic boundary region and the posterior tuberculum still provide a challenge. We applied the updated prosomeric model to the developing zebrafish hypothalamus to facilitate cross-species comparisons. Accordingly, we applied the mammalian nomenclature of hypothalamic organization to zebrafish and propose it to replace some controversial previous nomenclature.

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling

Background: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. Materials and Methods: In order to study the role of Nts, nts mutant (nts–/–), and overexpressing zebrafish were generated. Results: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts–/– larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts–/– larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts–/– larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts–/– larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts–/– larvae. Conclusion: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

MRNA expression and metabolic regulation of npy and agrp1/2 in the zebrafish brain

Neuropeptide Y (NPY) is an evolutionarily conserved neuropeptide implicated in feeding regulation in vertebrates. In mammals, NPY neurons coexpress Agouti-related protein (AgRP) in the arcuate nucleus of the hypothalamus, and NPY/AgRP neurons activate orexigenic signaling to increase food intake. Zebrafish express npy and two agrp genes, agrp1 and agrp2, in the brain. Similar to mammals, NPY and AgRP1 act as orexigenic factors in zebrafish, but the exact distribution of NPY and AgRP neurons in the zebrafish brain and the regulation of these genes by metabolic states remain unclear. In this study, we analyzed the tissue distribution of npy, agrp1, and agrp2 mRNA in the brain of larval and adult zebrafish. We detected the expression of agrp1, but not npy, in the hypothalamus of larval zebrafish. In the adult zebrafish brain, npy mRNA expression was detected in the dorsal area of the periventricular and lateral hypothalamus, but fasting induced upregulation of npy only in the lateral hypothalamus, indicating that NPY neurons in this area are implicated in feeding regulation. However, consistent with the findings in larval zebrafish, NPY neurons in the hypothalamus did not coexpress AgRP1. In contrast, fasting resulted in a dramatic increase in AgRP1 neurons in the ventral periventricular hypothalamus, which do not coexpress NPY. In addition, we found for the first time that npy- and agrp1-expressing neurons function as GABAergic inhibitory neurons in zebrafish, as they do in mammals. Taken together, our results show that the zebrafish NPY/AgRP system is involved in appetite regulation. In addition, our data suggest that although npy and agrp1 were initially expressed in distinct neurons, evolution has resulted in their coexpression in mammalian hypothalamic neurons.

Effects of starvation on the expression of feeding related neuropeptides in the larval zebrafish hypothalamus.

Vertebrate feeding behavior is regulated by neuropeptide Y (NPY), GALANIN and GMAP prepropeptide (GAL), agouti related neuropeptide (AGRP) and proopiomelanocortin (POMC) in the hypothalamus. However, there are few studies on the relationship between these neuropeptides and feeding in zebrafish larvae. In the present study, real-time quantitative PCR and in situ hybridization were applied to examine the expression levels of npy, galanin, agrp and pomca in the hypothalamus of zebrafish larvae after starvation and re-feeding. The results showed the expression of agrp and galanin increased significantly after starvation compared to the control group, whilst the expression of pomca decreased significantly compared to control. If the animals were re-fed for two days after starvation, the expression of pomca, agrp and galanin showed no significant difference from the control. Expression of npy did not alter in either condition. These results indicate that starvation increases expression levels of agrp and galanin, and reduces the pomca expression. In addition, these starvation-induced changes can be reversed by re-feeding.

QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish.

The hypothalamus is thought to play a key role in regulating sleep in vertebrate animals, but few sleep-promoting signaling pathways that function in the hypothalamus have been identified. Here we use the zebrafish, a diurnal vertebrate, to functionally and anatomically characterize the neuropeptide QRFP. We show that QRFP is exclusively expressed in a small number of neurons in the larval zebrafish hypothalamus that project widely in the brain. We also show that QRFP overexpression reduces locomotor activity, whereas animals that lack QRFP signaling are more active and sleep less. These results suggest that QRFP signaling participates in the hypothalamic regulation of sleep.

Motor Behavior Mediated by Continuously Generated Dopaminergic Neurons in the Zebrafish Hypothalamus Recovers after Cell Ablation

Postembryonic neurogenesis has been observed in several regions of the vertebrate brain, including the dentate gyrus and rostral migratory stream in mammals, and is required for normal behavior [1-3]. Recently, the hypothalamus has also been shown to undergo continuous neurogenesis as a way to mediate energy balance [4-10]. As the hypothalamus regulates multiple functional outputs, it is likely that additional behaviors may be affected by postembryonic neurogenesis in this brain structure. Here, we have identified a progenitor population inthe zebrafish hypothalamus that continuously generates neurons that express tyrosine hydroxylase 2 (th2). We develop and use novel transgenic tools tocharacterize the lineage of th2(+) cells and demonstrate that they are dopaminergic. Through genetic ablation and optogenetic activation, we then show that th2(+) neurons modulate the initiation of swimming behavior in zebrafish larvae. Finally, we find that the generation of new th2(+) neurons following ablation correlates with restoration of normal behavior. This work thus identifies for the first time a population of dopaminergic neurons that regulates motor behavior capable of functional recovery.

Nkx2.1 and nkx2.4 genes function partially redundant during development of the zebrafish hypothalamus, preoptic region, and pallidum.

During ventral forebrain development, orthologs of the homeodomain transcription factor Nkx2.1 control patterning of hypothalamus, preoptic region, and ventral telencephalon. However, the relative contributions of Nkx2.1 and Nkx2.4 to prosencephalon development are poorly understood. Therefore, we analyzed functions of the previously uncharacterized nkx2.4-like zgc:171531 as well as of the presumed nkx2.1 orthologs nkx2.1a and nkx2.1b in zebrafish forebrain development. Our results show that zgc:171531 and nkx2.1a display overlapping expression patterns and a high sequence similarity. Together with a high degree of synteny conservation, these findings indicate that both these genes indeed are paralogs of nkx2.4. As a result, we name zgc:171531 now nkx2.4a, and changed the name of nkx2.1a to nkx2.4b, and of nkx2.1b to nkx2.1. In nkx2.1, nkx2.4a, and nkx2.4b triple morpholino knockdown (nkx2TKD) embryos we observed a loss of the rostral part of prosomere 3 and its derivative posterior tubercular and hypothalamic structures. Furthermore, there was a loss of rostral and intermediate hypothalamus, while a residual preoptic region still develops. The reduction of the ventral diencephalon was accompanied by a ventral expansion of the dorsally expressed pax6, revealing a dorsalization of the basal hypothalamus. Within the telencephalon we observed a loss of pallidal markers, while striatum and pallium are forming. At the neuronal level, nkx2TKD morphants lacked several neurosecretory neuron types, including avp, crh, and pomc expressing cells in the hypothalamus, but still form oxt neurons in the preoptic region. Our data reveals that, while nkx2.1, nkx2.4a, and nkx2.4b genes act partially redundant in hypothalamic development, nkx2.1 is specifically involved in the development of rostral ventral forebrain including the pallidum and preoptic regions, whereas nkx2.4a and nkx2.4b control the intermediate and caudal hypothalamus.

Enhancer turnover and conserved regulatory function in vertebrate evolution

Mutations in regulatory regions including enhancers are an important source of variation and innovation during evolution. Enhancers can evolve by changes in the sequence, arrangement and repertoire of transcription factor binding sites, but whole enhancers can also be lost or gained in certain lineages in a process of turnover. The proopiomelanocortin gene (Pomc), which encodes a prohormone, is expressed in the pituitary and hypothalamus of all jawed vertebrates. We have previously described that hypothalamic Pomc expression in mammals is controlled by two enhancers-nPE1 and nPE2-that are derived from transposable elements and that presumably replaced the ancestral neuronal Pomc regulatory regions. Here, we show that nPE1 and nPE2, even though they are mammalian novelties with no homologous counterpart in other vertebrates, nevertheless can drive gene expression specifically to POMC neurons in the hypothalamus of larval and adult transgenic zebrafish. This indicates that when neuronal Pomc enhancers originated de novo during early mammalian evolution, the newly created cis- and trans-codes were similar to the ancestral ones. We also identify the neuronal regulatory region of zebrafish pomca and confirm that it is not homologous to the mammalian enhancers. Our work sheds light on the process of gene regulatory evolution by showing how a locus can undergo enhancer turnover and nevertheless maintain the ancestral transcriptional output.

Relationships between radial glial progenitors and 5‐HT neurons in the paraventricular organ of adult zebrafish – potential effects of serotonin on adult neurogenesis

In non-mammalian vertebrates, serotonin (5-HT)-producing neurons exist in the paraventricular organ (PVO), a diencephalic structure containing cerebrospinal fluid (CSF)-contacting neurons exhibiting 5-HT or dopamine (DA) immunoreactivity. Because the brain of the adult teleost is known for its neurogenic activity supported, for a large part, by radial glial progenitors, this study addresses the origin of newborn 5-HT neurons in the hypothalamus of adult zebrafish. In this species, the PVO exhibits numerous radial glial cells (RGCs) whose somata are located at a certain distance from the ventricle. To study relationships between RGCs and 5-HT CSF-contacting neurons, we performed 5-HT immunohistochemistry in transgenic tg(cyp19a1b-GFP) zebrafish in which RGCs are labelled with GFP under the control of the cyp19a1b promoter. We show that the somata of the 5-HT neurons are located closer to the ventricle than those of RGCs. RGCs extend towards the ventricle cytoplasmic processes that form a continuous barrier along the ventricular surface. In turn, 5-HT neurons contact the CSF via processes that cross this barrier through small pores. Further experiments using proliferating cell nuclear antigen or 5-bromo-2'-deoxyuridine indicate that RGCs proliferate and give birth to 5-HT neurons migrating centripetally instead of centrifugally as in other brain regions. Furthermore, treatment of adult zebrafish with tryptophan hydroxylase inhibitor causes a significant decrease in the number of proliferating cells in the PVO, but not in the mediobasal hypothalamus. These data point to the PVO as an intriguing region in which 5-HT appears to promote genesis of 5-HT neurons that accumulate along the brain ventricles and contact the CSF.

Wnt Signaling Regulates Postembryonic Hypothalamic Progenitor Differentiation

Previous studies have raised the possibility that Wntsignaling may regulate both neural progenitor maintenance and neuronal differentiation within a single population. Here we investigate the role of Wnt/β-catenin activity in the zebrafish hypothalamus and find that the pathway is first required for the proliferation of unspecified hypothalamic progenitors in the embryo. At later stages, including adulthood, sequential activation and inhibition of Wnt activity is required for the differentiation of neural progenitors and negatively regulates radial glia differentiation. The presence of Wnt activity is conserved in hypothalamic progenitors of the adult mouse, where it plays a conserved role in inhibiting the differentiation of radial glia. This study establishes the vertebrate hypothalamus as a model for Wnt-regulated postembryonic neural progenitor differentiation and defines specific roles for Wnt signaling in neurogenesis.

The Metabolic Regulator PGC-1α Directly Controls the Expression of the Hypothalamic Neuropeptide Oxytocin

The transcriptional coactivator PGC-1α is a key regulator of cellular energy expenditure in peripheral tissues. Recent studies report that PGC-1α-null mice develop late-onset obesity and that the neuronal inactivation of PGC-1α causes increased food intake. However, the exact role of PGC-1α in the CNS remains unclear. Here we show that PGC-1α directly regulates the expression of the hypothalamic neuropeptide oxytocin, a known central regulator of appetite. We developed a unique genetic approach in the zebrafish, allowing us to monitor and manipulate PGC-1α activity in oxytocinergic neurons. We found that PGC-1α is coexpressed with oxytocin in the zebrafish hypothalamus. Targeted knockdown of the zebrafish PGC-1α gene activity caused a marked decrease in oxytocin mRNA levels and inhibited the expression of a transgenic GFP reporter driven by the oxytocin promoter. The effect of PGC-1α loss of function on oxytocin gene activity was rescued by tissue-specific re-expression of either PGC-1α or oxytocin precursor in zebrafish oxytocinergic neurons. PGC-1α activated the oxytocin promoter in a heterologous cell culture system, and overexpression of PGC-1α induced ectopic expression of oxytocin in muscles and neurons. Finally, PGC-1α forms an in vivo complex with the oxytocin promoter in fed but not fasted animals. These findings demonstrate that PGC-1α is both necessary and sufficient for the production of oxytocin, implicating hypothalamic PGC-1α in the direct activation of a hypothalamic hormone known to control energy intake.