Maternal obesity modulates sexually dimorphic epigenetic regulation and expression of leptin receptor in offspring hippocampus.

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.

Developing neurites from mouse basal forebrain gonadotropin-releasing hormone neurons use Sonic hedgehog to modulate their growth.

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.

Obesity during pregnancy in the mouse alters the Netrin-1 responsiveness of foetal arcuate nucleus neuropeptide Y neurones.

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.

A novel role for the DNA repair gene Rad51 in Netrin-1 signalling.

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.

Interactions between mitochondrial and nuclear DNA in mammalian cells are non-random.

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.

The establishment of DOHaD working groups in Australia and New Zealand.

The evidence underpinning the developmental origins of health and disease (DOHaD) is overwhelming. As the emphasis shifts more towards interventions and the translational strategies for disease prevention, it is important to capitalize on collaboration and knowledge sharing to maximize opportunities for discovery and replication. DOHaD meetings are facilitating this interaction. However, strategies to perpetuate focussed discussions and collaborations around and between conferences are more likely to facilitate the development of DOHaD research. For this reason, the DOHaD Society of Australia and New Zealand (DOHaD ANZ) has initiated themed Working Groups, which convened at the 2014-2015 conferences. This report introduces the DOHaD ANZ Working Groups and summarizes their plans and activities. One of the first Working Groups to form was the ActEarly birth cohort group, which is moving towards more translational goals. Reflecting growing emphasis on the impact of early life biodiversity - even before birth - we also have a Working Group titled Infection, inflammation and the microbiome. We have several Working Groups exploring other major non-cancerous disease outcomes over the lifespan, including Brain, behaviour and development and Obesity, cardiovascular and metabolic health. The Epigenetics and Animal Models Working Groups cut across all these areas and seeks to ensure interaction between researchers. Finally, we have a group focussed on 'Translation, policy and communication' which focusses on how we can best take the evidence we produce into the community to effect change. By coordinating and perpetuating DOHaD discussions in this way we aim to enhance DOHaD research in our region.

A novel developmental role for kisspeptin in the growth of gonadotrophin-releasing hormone neurites to the median eminence in the mouse.

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.

Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius.

The transcriptional effects of deregulated myc gene overexpression are implicated in tumorigenesis in a spectrum of experimental and naturally occurring neoplasms. In follicles of the chicken bursa of Fabricius, myc induction of B-cell neoplasia requires a target cell population present during early bursal development and progresses through preneoplastic transformed follicles to metastatic lymphomas. We developed a chicken immune system cDNA microarray to analyze broad changes in gene expression that occur during normal embryonic B-cell development and during myc-induced neoplastic transformation in the bursa. The number of mRNAs showing at least 3-fold change was greater during myc-induced lymphomagenesis than during normal development, and hierarchical cluster analysis of expression patterns revealed that levels of several hundred mRNAs varied in concert with levels of myc overexpression. A set of 41 mRNAs were most consistently elevated in myc-overexpressing preneoplastic and neoplastic cells, most involved in processes thought to be subject to regulation by Myc. The mRNAs for another cluster of genes were overexpressed in neoplasia independent of myc expression level, including a small subset with the expression signature of embryonic bursal lymphocytes. Overexpression of myc, and some of the genes overexpressed with myc, may be important for generation of preneoplastic transformed follicles. However, expression profiles of late metastatic tumors showed a large variation in concert with myc expression levels, and some showed minimal myc overexpression. Therefore, high-level myc overexpression may be more important in the early induction of these lymphomas than in maintenance of late-stage metastases.

Analysis of chicken Wnt-13 expression demonstrates coincidence with cell division in the developing eye and is consistent with a role in induction.

We used a degenerate polymerase chain reaction (PCR) strategy to search for Wnt RNA in developing ocular tissues. We isolated a Macaca monkey Wnt-13 PCR fragment, orthologous to the human and murine Wnt-13 and Xenopus Wnt-2b, and a chick Wnt13 cDNA. Wnt-13 is a member of the Wnt-1 class of transforming Wnt molecules. In situ RNA hybridization revealed a dynamic Wnt-13 expression pattern in numerous developing tissues. Within the eye, Wnt-13 is expressed in the proliferative epithelium of the lens and both pigmented and non-pigmented layers of the ciliary margin. In vitro BrdU incorporation studies coupled with in situ hybridization showed that cWnt-13 expression domains in the lens were coincident with cell division. In addition to the eye, cWnt-13 was expressed in head ectoderm, prospective forelimb mesenchyme, lung bud, pharyngeal arches, the brain, as well as the otic vesicle. Our data are consistent with previous observations linking transforming Wnts with cell division and implicate a cascade of events involving cWnt-13 first in dorsoventral patterning and later in cell proliferation regulation associated with lens development. Dev Dyn 1999;215:215-224.

Mapping HLA for single nucleotide polymorphisms.

Knowledge of DNA sequence variation may help us understand how genetic variability gives rise to functional variability and, in so doing, revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1,000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human leukocyte antigen (HLA) complex. Interestingly, the precise molecular defects in the HLA genes are unknown and the notion that non-HLA genes, within the same chromosomal region, are involved remains a formal possibility. We have determined the nucleotide sequence of a contiguous 2.2 Mbp segment of chromosome six that includes all of the HLA class I region, and have identified over 10,000 SNPs therein. Because of the derivative knowledge of gene and SNP content and position, the scientific community is now uniquely poised to identify disease-contributory SNPs that lie within the MHC.

The homeobox gene cut interacts genetically with the homeotic genes proboscipedia and Antennapedia.

The cut locus (ct) codes for a homeodomain protein (Cut) and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify ct-interacting genes, we observed that flies containing a hypomorphic ct mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different ct mutant backgrounds. We show that this phenotype is accompanied by pronounced changes in the expression patterns of both ct and pb in labial discs. Furthermore, a significant proportion of ct mutant flies that are heterozygous for certain Antennapedia (Antp) alleles have thoracic defects that mimic loss-of-function Antp phenotypes, and ectopic expression of Cut in antennal discs results in ectopic Antp expression and a dominant Antp-like phenotype. Our results implicate ct in the regulation of expression and/or function of two homeotic genes and document a new role of ct in the control of segmental identity.

Spatial and temporal expression of cone opsins during monkey retinal development.

The primate retina requires a coordinated series of developmental events to form its specialized photoreceptor topography. In this study, the temporal expression of cone photoreceptor opsin was determined in Macaca monkey retina. Markers for mRNA and protein that recognize short wavelength (S) and long/medium wavelength (L/M) opsin were used to determine (1) the temporal and spatial patterns of opsin expression, (2) the spatial relationship between S and L/M cones at the time of initial opsin expression, and (3) the relative time of cone and rod opsin expression (Dorn et al. [1995] Invest. Ophthalmol. Vis. Sci. 36:2634-2651). Adult cone outer segments were recognized by either L/M or S opsin antiserum. Of all adult cone inner segments, 88-90% contained L/M opsin mRNA, whereas 10-12% contained S opsin mRNA. Fetal cones initially showed cell membrane as well as outer segment labeling for opsin protein, but cell membrane labeling disappeared by birth. No cones at any age contained markers for both S and L/M opsin mRNA or protein. S and L/M opsin protein appeared in the fovea at fetal day 75. Once opsin expression progressed beyond the fovea, both mRNA and protein for S opsin were consistently detected more peripherally than L/M opsin. Cones at the peripheral edge of S opsin expression had basal telodendria that appeared to reach toward neighboring cones. Because interactions between cone populations could organize the cone mosaic, the spatial relationship between S cones and the first cones to express L/M protein was analyzed quantitatively by using double-label immunocytochemistry. No consistent relationship was found between these two cone populations. Cones are generated at least 1 week before rods across monkey retina. However, rod opsin protein appears in and around the fovea at fetal day 66, 1 week before cone opsin protein. This suggests that independent local factors control differentiation in these two photoreceptor populations.

Temporal and spatial pattern of MASH-1 expression in the developing rat retina demonstrates progenitor cell heterogeneity.

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.

A chicken achaete-scute homolog (CASH-1) is expressed in a temporally and spatially discrete manner in the developing nervous system.

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.