Developmental and functional relationships between hypothalamic tanycytes and embryonic radial glia

The hypothalamus is a key regulator of several homeostatic processes, such as circadian rhythms, energy balance, thirst, and thermoregulation. Recently, the hypothalamic third ventricle has emerged as a site of postnatal neurogenesis and gliogenesis. This hypothalamic neural stem potential resides in a heterogeneous population of cells known as tanycytes, which, not unlike radial glia, line the floor and ventrolateral walls of the third ventricle and extend a long process into the hypothalamic parenchyma. Here, we will review historical and recent data regarding tanycyte biology across the lifespan, focusing on the developmental emergence of these diverse cells from embryonic radial glia and their eventual role contributing to a fascinating, but relatively poorly characterized, adult neural stem cell niche

Neuroendocrine transcriptional programs adapt dynamically to the supply and demand for neuropeptides as revealed in NSF mutant zebrafish

<p>Abstract</p> <p>Background</p> <p>Regulated secretion of specialized neuropeptides in the vertebrate neuroendocrine system is critical for ensuring physiological homeostasis. Expression of these cell-specific peptide markers in the differentiating hypothalamus commences prior to birth, often predating the physiological demand for secreted neuropeptides. The conserved function and spatial expression of hypothalamic peptides in vertebrates prompted us to search for critical neuroendocrine genes in newly hatched zebrafish larvae.</p> <p>Results</p> <p>We screened mutant 5 days post-fertilization zebrafish larvae that fail to undergo visually mediated background adaptation for disruption in hypothalamic <it>pomc </it>expression. To our surprise, the ATPase <it>N-ethylmaleimide sensitive factor </it>(<it>nsf</it>) was identified as an essential gene for maintenance of neuroendocrine transcriptional programs during the embryo-to-larva transition. Despite normal hypothalamic development in <it>nsf</it>^{<it>st</it>53}mutants, neuropeptidergic cells exhibited a dramatic loss of cell-specific markers by 5 days post-fertilization that is accompanied by elevated intracellular neuropeptide protein. Consistent with the role of NSF in vesicle-membrane fusion events and intracellular trafficking, cytoplasmic endoplasmic reticulum-like membranes accumulate in <it>nsf</it>^-/-hypothalamic neurons similar to that observed for <it>SEC18 </it>(<it>nsf ortholog</it>) yeast mutants. Our data support a model in which unspent neuropeptide cargo feedbacks to extinguish transcription in neuropeptidergic cells just as they become functionally required. In support of this model we found that <it>gnrh3 </it>transcripts remained unchanged in pre-migratory, non-functional gonadotropin-releasing hormone (GnRH) neurons in <it>nsf</it>^-/-zebrafish. Furthermore, <it>oxytocin-like </it>(<it>oxtl</it>, <it>intp</it>) transcripts, which are found in osmoreceptive neurons and persist in mutant zebrafish, drop precipitously after mutant zebrafish are acutely challenged with high salt.</p> <p>Conclusion</p> <p>Our analyses of <it>nsf </it>mutant zebrafish reveal an unexpected role for NSF in hypothalamic development, with mutant 5 days post-fertilization larvae exhibiting a stage-dependent loss of neuroendocrine transcripts and a corresponding accumulation of neuropeptides in the soma. Based on our collective findings, we speculate that neuroendocrine transcriptional programs adapt dynamically to both the supply and demand for neuropeptides to ensure adequate homeostatic responses.</p

Feeding and fasting controls liver expression of a regulator of G protein signaling (Rgs16) in periportal hepatocytes

BACKGROUND: Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism. RESULTS: Rgs16 is the only RGS gene that is diurnally regulated in liver of ad libitum fed mice. Rgs16 transcription, mRNA and protein are up regulated during fasting and rapidly down regulated after refeeding. Rgs16 is expressed in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominates. Restricting feeding to 4 hr of the light phase entrained Rgs16 expression in liver but did not affect circadian regulation of Rgs16 expression in the suprachiasmatic nuclei (SCN). CONCLUSION: Rgs16 is one of a subset of genes that is circadian regulated both in SCN and liver. Rgs16 mRNA expression in liver responds rapidly to changes in feeding schedule, coincident with key transcription factors controlling the circadian clock. Rgs16 expression can be used as a marker to identify and investigate novel G-protein mediated metabolic and circadian pathways, in specific zones within the liver

Rnd2 and Rnd3 expression in the ventromedial hypothalamus is compromised during embryonic development in absence of proneural genes Neurog2 and Ascl1

The ventromedial hypothalamus (VMH) is a hypothalamic nucleus with essential roles in homeostatic functions such as satiety signaling and reproductive behaviors. Despite the functional importance of the VMH, mechanisms driving VMH development are just starting to be explored. Among the most important unanswered questions regarding to VMH development is: what are the mechanisms responsible for VMH neurons migrate from their birthplace to their final positions? Here, we investigated whether the absence of the proneural genes Neurogenin-2 (Neurog2) and Achaete-scute homolog 1 (Ascl1) compromises the expression of downstream Rho family GTPase 2 and 3 (Rnd2 and Rnd3), respectively, in migrating VMH neurons. In migrating cortical neurons, Neurog2 has been shown to directly induce Rnd2 expression, and Rnd3 expression is likewise induced by Ascl1. When expressed normally, Rnd2 and Rnd3 both facilitate critical aspects of radial migration, and Rnd3 further maintains the correct timing and direction of migration. We used Ascl1GFP/KI and Neurog2GFP/KI mouse embryos as model organisms. Rnd2 and Rnd3 expression throughout critical development periods was examined by individually immunostaining for anti-Rnd2 and anti-Rnd3 at E12.5, E15.5, and E19.5 slices and imaging slices using compound fluorescence microscopy. VMH neurons expressing Rnd2 and Rnd3 were visually counted and compared with counts of control slices. Up until now, we have found that Ascl1 null embryos had fewer cells expressing Rnd3 at E15.5 and E19.5, and Neurog2 null embryos had fewer cells expressing Rnd2 at E19.5. Preliminary immunostaining results shows a reduction in transcription of both Ascl1 and Neurog 2. Results from this work will shed important insight into the mechanisms employed by hypothalamic neurons during migration. Abnormalities in neuronal migration to the VMH may result in obesity disorders and early puberty; thus, understanding migration mechanisms is an essential first step in developing tools to treat and prevent potentially debilitating conditions

Genetic labeling of steroidogenic factor‐1 (SF‐1) neurons in mice reveals ventromedial nucleus of the hypothalamus (VMH) circuitry beginning at neurogenesis and development of a separate non‐SF‐1 neuronal cluster in the ventrolateral VMH

The ventromedial nucleus of the hypothalamus (VMH) influences a wide variety of physiological responses. Here, using two distinct but complementary genetic tracing approaches in mice, we describe the development of VMH efferent projections, as marked by steroidogenic factor-1 (SF-1; NR5A1). SF-1 neurons were visualized by Tau-green fluorescent protein (GFP) expressed from the endogenous Sf-1 locus (Sf-1(TauGFP)) or by crossing the transgenic Sf1:Cre driver to a GFP reporter strain (Z/EG(Sf1:Cre)). Strikingly, VMH projections were visible early, at embryonic (E) 10.5, when few postmitotic SF1 neurons have been born, suggesting that formation of VMH circuitry begins at the onset of neurogenesis. At E14.5, comparison of these two reporter lines revealed that SF1-positive neurons in the ventrolateral VMH (VMH(vl)) persist in Z/EG(Sf1:Cre) embryos but are virtually absent in Sf-1(TauGFP). Therefore, although the entire VMH including the VMH(vl) shares a common lineage, the VMH(vl) further differentiates into a neuronal cluster devoid of SF-1. At birth, extensive VMH projections to broad regions of the brain were observed in both mouse reporter lines, matching well with those previously discovered by injection of axonal anterograde tracers in adult rats. In summary, our genetic tracing studies show that VMH efferent projections are highly conserved in rodents and are established far earlier than previously appreciated. Moreover, our results imply that neurons in the VMH(vl) adopt a distinct fate early in development, which might underlie the unique physiological functions associated with this VMH subregion

Neuroendocrine transcriptional programs adapt dynamically to the supply and demand for neuropeptides as revealed in NSF mutant zebrafish.

BackgroundRegulated secretion of specialized neuropeptides in the vertebrate neuroendocrine system is critical for ensuring physiological homeostasis. Expression of these cell-specific peptide markers in the differentiating hypothalamus commences prior to birth, often predating the physiological demand for secreted neuropeptides. The conserved function and spatial expression of hypothalamic peptides in vertebrates prompted us to search for critical neuroendocrine genes in newly hatched zebrafish larvae.ResultsWe screened mutant 5 days post-fertilization zebrafish larvae that fail to undergo visually mediated background adaptation for disruption in hypothalamic pomc expression. To our surprise, the ATPase N-ethylmaleimide sensitive factor (nsf) was identified as an essential gene for maintenance of neuroendocrine transcriptional programs during the embryo-to-larva transition. Despite normal hypothalamic development in nsf(st53) mutants, neuropeptidergic cells exhibited a dramatic loss of cell-specific markers by 5 days post-fertilization that is accompanied by elevated intracellular neuropeptide protein. Consistent with the role of NSF in vesicle-membrane fusion events and intracellular trafficking, cytoplasmic endoplasmic reticulum-like membranes accumulate in nsf(-/-) hypothalamic neurons similar to that observed for SEC18 (nsf ortholog) yeast mutants. Our data support a model in which unspent neuropeptide cargo feedbacks to extinguish transcription in neuropeptidergic cells just as they become functionally required. In support of this model we found that gnrh3 transcripts remained unchanged in pre-migratory, non-functional gonadotropin-releasing hormone (GnRH) neurons in nsf(-/-) zebrafish. Furthermore, oxytocin-like (oxtl, intp) transcripts, which are found in osmoreceptive neurons and persist in mutant zebrafish, drop precipitously after mutant zebrafish are acutely challenged with high salt.ConclusionOur analyses of nsf mutant zebrafish reveal an unexpected role for NSF in hypothalamic development, with mutant 5 days post-fertilization larvae exhibiting a stage-dependent loss of neuroendocrine transcripts and a corresponding accumulation of neuropeptides in the soma. Based on our collective findings, we speculate that neuroendocrine transcriptional programs adapt dynamically to both the supply and demand for neuropeptides to ensure adequate homeostatic responses

Embryonic microglia influence developing hypothalamic glial populations

Abstract Background Although historically microglia were thought to be immature in the fetal brain, evidence of purposeful interactions between these immune cells and nearby neural progenitors is becoming established. Here, we examined the influence of embryonic microglia on gliogenesis within the developing tuberal hypothalamus, a region later important for energy balance, reproduction, and thermoregulation. Methods We used immunohistochemistry to quantify the location and numbers of glial cells in the embryonic brain (E13.5–E17.5), as well as a pharmacological approach (i.e., PLX5622) to knock down fetal microglia. We also conducted cytokine and chemokine analyses on embryonic brains in the presence or absence of microglia, and a neurosphere assay to test the effects of the altered cytokines on hypothalamic progenitor behaviors. Results We identified a subpopulation of activated microglia that congregated adjacent to the third ventricle alongside embryonic Olig2+ neural progenitor cells (NPCs) that are destined to give rise to oligodendrocyte and astrocyte populations. In the absence of microglia, we observed an increase in Olig2+ glial progenitor cells that remained at the ventricle by E17.5 and a concomitant decrease of these Olig2+ cells in the mantle zone, indicative of a delay in migration of these precursor cells. A further examination of maturing oligodendrocytes in the hypothalamic grey and white matter area in the absence of microglia revealed migrating oligodendrocyte progenitor cells (OPCs) within the grey matter at E17.5, a time point when OPCs begin to slow their migration. Finally, quantification of cytokine and chemokine signaling in ex vivo E15.5 hypothalamic cultures +/− microglia revealed decreases in the protein levels of several cytokines in the absence of microglia. We assayed the influence of two downregulated cytokines (CCL2 and CXCL10) on neurosphere-forming capacity and lineage commitment of hypothalamic NPCs in culture and showed an increase in NPC proliferation as well as neuronal and oligodendrocyte differentiation. Conclusion These data demonstrate that microglia influence gliogenesis in the developing tuberal hypothalamus