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Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

ABSTRACT: Background: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. Results: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3rd ventricle, while β-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were, unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. Conclusion: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections.

ORGANISM(S): Mus musculus

PROVIDER: GSE75878 | GEO | 2016/04/14

SECONDARY ACCESSION(S): PRJNA305587

REPOSITORIES: GEO

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Project description:Background: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein GÎ±s, termed XLÎ±s, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. Results: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive Î±-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3rd ventricle, while Î²-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were, unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. Conclusion: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since Î±-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections. 6 wildtype and 6 Gnasxlm+/p- RNA isolates were used, which were pooled in pairs in equal concentrations prior to rRNA removal using the RiboMinus kit (Life Technologies)

Project description:Purpose: We aimed to reconstruct the development formation of cellular identities' heterogeneity in the hypothalamus with single-cell RNA sequencing. We planned to track branching process of neurogenesis and follow the formation of cell identities. Results: We integrate single-cell RNA-seq data on 51,199 cells of ectodermal origin, representing critical stages of pre- and postnatal hypothalamus development, supported by anatomical lineage reconstruction to show that bulk neurogenesis in the hypothalamus, dominated by Notch signalling and its interaction with Ascl1, persists until the neonatal period to produce molecularly distinct astrocytes, ependyma, tanycytes and 30 protoclusters of neurons. Alike in adults, the assignment of neurotransmitters and neuropeptides together with transcription factors as positional marks for each neuronal cluster is sufficient to establish the spatially-resolved taxonomy of hypothalamic neurons. By deducing gene regulatory networks, exemplified by hierarchical gene clusters driven by Onecut2/Onecut3 transcription factors for dopamine neurons, we resolve how neuronal lineages diverge into functionally non-overlapping endocrine subtypes. Dopamine neurons are used to illustrate molecular convergence with their shared neurotransmitter identity representing a mismatch to their molecular distances on multidimensional vector spaces. We demonstrate the existence of non-canonical chemotropic axes in the hypothalamus, particularly sequential ligand-receptor switching in the unexpectedly abundant Slit/Robo cassette. Slit2 is identified in ventricular ependymocytes and implicated in repulsing new-born neurons that sequentially express Robo1 and Robo2 receptors for migration and differentiation. Conclusions: Overall, our unbiased map of hypothalamic cell types and extracellular guidance cues uncovers molecular principles shaping the developmental architecture of the mammalian hypothalamus and rationalise how hypothalamic neuronal heterogeneity is transformed into a multimodal neural unit that retains a virtually infinite adaptive potential throughout life.

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Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

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