RESUMEN
Maternal obesity during pregnancy is associated with a greater risk for obesity and neurodevelopmental deficits in offspring. This developmental programming of disease is proposed to involve neuroendocrine, inflammatory, and epigenetic factors during gestation that disrupt normal fetal brain development. The hormones leptin and insulin are each intrinsically linked to metabolism, inflammation, and neurodevelopment, which led us to hypothesise that maternal obesity may disrupt leptin or insulin receptor signalling in the developing brain of offspring. Using a C57BL/6 mouse model of high fat diet-induced maternal obesity (mHFD), we performed qPCR to examine leptin receptor (Lepr) and insulin receptor (Insr) gene expression in gestational day (GD) 17.5 fetal brain. We found a significant effect of maternal diet and offspring sex on Lepr regulation in the developing hippocampus, with increased Lepr expression in female mHFD offspring (p < 0.05) compared to controls. Maternal diet did not alter hippocampal Insr in the fetal brain, or Lepr or Insr in prefrontal cortex, amygdala, or hypothalamus of female or male offspring. Chromatin immunoprecipitation revealed decreased binding of histones possessing the repressive histone mark H3K9me3 at the Lepr promoter (p < 0.05) in hippocampus of female mHFD offspring compared to controls, but not in males. Sex-specific deregulation of Lepr could be reproduced in vitro by exposing female hippocampal neurons to the obesity related proinflammatory cytokine IL-6, but not IL-17a or IFNG. Our findings indicate that the obesity-related proinflammatory cytokine IL-6 during pregnancy leads to sexually dimorphic changes in the modifications of histones binding at the Lepr gene promoter, and concomitant changes to Lepr transcription in the developing hippocampus. This suggests that exposure of the fetus to metabolic inflammatory molecules can impact epigenetic regulation of gene expression in the developing hippocampus.
Asunto(s)
Obesidad Materna , Efectos Tardíos de la Exposición Prenatal , Animales , Dieta Alta en Grasa , Epigénesis Genética , Femenino , Hipocampo , Leptina , Masculino , Ratones , Ratones Endogámicos C57BL , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Receptores de Leptina/genéticaRESUMEN
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. GnRH neuron cell bodies reside in the basal forebrain, and most extend long neurites in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. Using in vitro neurite growth assays, histological methods, and genetic deletion strategies in mice we have analysed the role of the morphogen and neurite growth and guidance molecule, Sonic hedgehog (Shh), in the growth of GnRH neurites to their target. Immunohistochemistry revealed that Shh was present in the basal forebrain, the preoptic area (POA) and mediobasal hypothalamus (MBH) at gestational day 14.5 (GD 14.5), a time when GnRH neurites grow towards the ME. Furthermore, in situ hybridization revealed that mRNA encoding the Shh receptor, Smoothened (Smo), was present in GnRH neurons from GD 15.5, when the first GnRH neurites are extending towards the MBH. In vitro neurite growth assays using hypothalamic explants from GD 15.5 fetuses in 3-D collagen gels showed that Shh was able to significantly stimulate GnRH neurite outgrowth. Finally, genetic deletion of Smo specifically from GnRH neurons in vivo, using Cre-loxP technology, resulted in a significant decrease in GnRH neurites innervating the ME. These experiments demonstrate that GnRH neurites use Shh for their neurite development, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional hypothalamic neuronal population for which Shh/Smo signaling is developmentally important.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Hormona Liberadora de Gonadotropina/metabolismo , Proteínas Hedgehog/metabolismo , Neuritas/fisiología , Neuronas/ultraestructura , Prosencéfalo/citología , Factores de Edad , Animales , Femenino , Hormona Liberadora de Gonadotropina/genética , Proteínas Hedgehog/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Prosencéfalo/embriología , Prosencéfalo/crecimiento & desarrolloRESUMEN
When individuals undergo gestation in an obese dam, they are at increased risk for impairments in the ability of the brain to regulate body weight. In rodents, gestation in an obese dam leads to a number of changes to the development of the hypothalamic neurones that regulate body weight, including reduced neuronal connectivity at birth. In the present study, we aimed to clarify how this neural circuitry develops normally, as well as to explore the mechanism underpinning the deficiency in connectivity seen in foetuses developing in obese dams. First, we developed an in vitro model for observing and manipulating the axonal growth of foetal arcuate nucleus (ARN) neuropeptide (NPY) neurones. We then used this model to test 2 hypotheses: (i) ARN NPY neurones respond to Netrin-1, one of a small number of axon growth and guidance factors that regulate neural circuit formation throughout the developing brain; and (ii) Netrin-1 responsiveness would be lost upon exposure to the inflammatory cytokine interleukin (IL)-6, which is elevated in foetuses developing in obese dams. We observed that ARN NPY neurones responded to Netrin-1 with a significant expansion of their growth cones, comprising the terminal apparatus that neurones use to navigate. Unexpectedly, we found further that NPY neurones from obese pregnancies had a reduced responsiveness to Netrin-1, raising the possibility that ARN NPY neurones from foetuses developing in obese dams were phenotypically different from normal NPY neurones. Finally, we observed that IL-6 treatment of normal NPY neurones in vitro led to a reduced growth cone responsiveness to Netrin-1, essentially causing them to behave similarly to NPY neurones from obese pregnancies. These results support the hypothesis that IL-6 can disrupt the normal process of axon growth from NPY neurones, and suggest one possible mechanism for how the body weight regulating circuitry fails to develop properly in the offspring of obese dams.
Asunto(s)
Núcleo Arqueado del Hipotálamo/embriología , Netrina-1/fisiología , Neuronas/fisiología , Neuropéptido Y/metabolismo , Obesidad/fisiopatología , Complicaciones del Embarazo/fisiopatología , Animales , Núcleo Arqueado del Hipotálamo/citología , Células Cultivadas , Femenino , Conos de Crecimiento/fisiología , Interleucina-6/administración & dosificación , Interleucina-6/fisiología , Masculino , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Obesidad/complicaciones , EmbarazoRESUMEN
Mutations in RAD51 have recently been linked to human Congenital Mirror Movements (CMM), a developmental disorder of the motor system. The only gene previously linked to CMM encodes the Netrin-1 receptor DCC, which is important for formation of corticospinal and callosal axon tracts. Thus, we hypothesised that Rad51 has a novel role in Netrin-1-mediated axon development. In mouse primary motor cortex neurons, Rad51 protein was redistributed distally down the axon in response to Netrin-1, further suggesting a functional link between the two. We next manipulated Rad51 expression, and assessed Netrin-1 responsiveness. Rad51 siRNA knockdown exaggerated Netrin-1-mediated neurite branching and filopodia formation. RAD51 overexpression inhibited these responses, whereas overexpression of the CMM-linked R250Q mutation, a predicted loss-of-function, had no effect. Thus, Rad51 appears to negatively regulate Netrin-1 signalling. Finally, we examined whether Rad51 might operate by modulating the expression of the Unc5 family, known negative regulators of Netrin-1-responsiveness. Unc5b and Unc5c transcripts were downregulated in response to Rad51 knockdown, and upregulated with RAD51 overexpression, but not R250Q. Thus, Rad51 negatively regulates Netrin-1 signalling, at least in part, by modulating the expression of Unc5s. Imbalance of positive and negative influences is likely to lead to aberrant motor system development resulting in CMMs.
Asunto(s)
Corteza Motora/metabolismo , Netrina-1/metabolismo , Recombinasa Rad51/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Corteza Motora/citología , Corteza Motora/crecimiento & desarrollo , Mutación , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Netrina-1/genética , Proyección Neuronal , Recombinasa Rad51/genética , Transducción de SeñalRESUMEN
Chromosome Conformation Capture techniques regularly detect physical interactions between mitochondrial and nuclear DNA (i.e. mito-nDNA interactions) in mammalian cells. We have evaluated mito-nDNA interactions captured by HiC and Circular Chromosome Conformation Capture (4C). We show that these mito-nDNA interactions are statistically significant and shared between biological and technical replicates. The most frequent interactions occur with repetitive DNA sequences, including centromeres in human cell lines and the 18S rDNA in mouse cortical astrocytes. Our results demonstrate a degree of selective regulation in the identity of the interacting mitochondrial partners confirming that mito-nDNA interactions in mammalian cells are not random.
Asunto(s)
Núcleo Celular/genética , ADN/genética , ADN/metabolismo , Mitocondrias/genética , Animales , Humanos , RatonesRESUMEN
The puberty- and fertility-regulating neuropeptide kisspeptin (KISS1) exerts dramatic effects on the physiology of adult gonadotrophin-releasing hormone (GnRH) neurones as a master regulator of mammalian reproduction. Given the action of KISS1 directly on adult GnRH neurones, and that KISS1 activates a signal transduction cascade involved in neurite growth in other neurones, we investigated whether KISS1 may play a role in the normal growth of GnRH neurites to the median eminence. A reverse transcription-polymerase chain reaction demonstrated the expression of Kiss1 mRNA in the embryonic mediobasal hypothalamus, the target region for GnRH neurite termination, as early as embryonic day 13.5 (E13.5), a time when the first GnRH neurites are arriving. Complementary expression of the mRNA encoding the KISS1 receptor, Kiss1r, in the preoptic area (POA) at E13.5 was also observed, suggesting that POA-resident GnRH neurones can respond to KISS1 from an early age. To examine the effects of KISS1 on GnRH neurite growth in isolation, E15.5 POA explants, containing GnRH neurones actively extending neurites, were grown in three-dimensional collagen gels. In the presence of KISS1 (1 µm), both the number and length of GnRH neurites were increased significantly compared to controls without KISS1. The effects of KISS1 on GnRH neurite growth could be inhibited by pretreatment with the phospholipase C inhibitor U73122 (50 µm), indicating that embryonic and adult GnRH neurones respond to KISS1 with the same intracellular signalling pathway. KISS1 provided in a concentration gradient from a fixed source had no effect on GnRH neurite growth, indicating that KISS1 does not function as a long-range chemoattractant. Taken together, these results identify KISS1 as a stimulator of GnRH neurite growth, and suggest that it influences GnRH neurites at close-range to innervate the median eminence. These data add a novel developmental role to the repertoire of the functions of KISS1 in mammalian reproduction.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Eminencia Media/citología , Neuritas/fisiología , Proteínas Supresoras de Tumor/farmacología , Animales , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/anatomía & histología , Femenino , Humanos , Kisspeptinas , Eminencia Media/fisiología , Ratones , Ratones Transgénicos , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Embarazo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Transducción de Señal/fisiología , Técnicas de Cultivo de Tejidos , Proteínas Supresoras de Tumor/genética , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The temporal and spatial pattern of mammalian achaete-scute homolog 1 (MASH-1) expression in the developing rat retina was examined in an effort to correlate achaete-scute homolog expression with the generation of particular cell classes. The expression of MASH-1 was restricted to the latter portion of retinal neurogenesis and was most closely correlated with the appearance of bipolar cells and Müller glia, two cell classes that are generated late in retinogenesis. We also examined the proliferative nature of the MASH-1 -expressing cell type to confirm that MASH-1 is expressed by progenitor cells and to determine the proportion of the proliferating population that expresses MASH-1. MASH-1 was expressed by only 10-30% of the total proliferating population, depending on the age examined. Thus, MASH-1 expression provides a molecular marker of heterogeneity among retinal progenitor cells and may play a role in the commitment and/or differentiation of one or more of the late-appearing retinal phenotypes.
Asunto(s)
Retina/crecimiento & desarrollo , Células Madre/fisiología , Animales , Inmunohistoquímica , Ratas , Retina/fisiología , Timidina/farmacología , Factores de TiempoRESUMEN
We have identified a basic helix-loop-helix encoding cDNA from embryonic chicken retina which shares sequence similarity with the achaete-scute family of genes of Drosophila. The deduced amino acid sequence of this chicken achaete-scute homolog (CASH-1) is identical, over the region encoding the basic helix-loop-helix domain, to the recently identified mammalian achaete-scute homolog (MASH-1) and to the Xenopus homolog (XASH1), and 70% identical, over the same region, to Drosophila achaete-scute complex members. The expression of CASH-1 is restricted to subsets of neuronal progenitor cells in the developing chicken nervous system, similar in distribution to that reported for MASH-1 and XASH1. In addition, in situ localization in the retina reveals a dynamic character of expression of the gene in a particular region of the CNS, and suggests that the expression of CASH-1 may be important in defining a particular stage in the progenitor cell necessary for the differentiation of particular neuronal phenotypes.