Deborah M. Kurrasch

Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish

Significance Here we demonstrate that bisphenol A (BPA) exposure during a time point analogous to the second trimester in humans has real and measurable effects on brain development and behavior. Furthermore, our study is the first, to our knowledge, to show that bisphenol S, a replacement used in BPA-free products, equally affects neurodevelopment. These findings suggest that BPA-free products are not necessarily safe and support a societal push to remove all structurally similar bisphenol analogues and other compounds with endocrine-disruptive activity from consumer goods. Our data here, combined with over a dozen physiological and behavioral human studies that begin to point to the prenatal period as a BPA window of vulnerability, suggest that pregnant mothers limit exposure to plastics and receipts. Bisphenol A (BPA), a ubiquitous endocrine disruptor that is present in many household products, has been linked to obesity, cancer, and, most relevant here, childhood neurological disorders such as anxiety and hyperactivity. However, how BPA exposure translates into these neurodevelopmental disorders remains poorly understood. Here, we used zebrafish to link BPA mechanistically to disease etiology. Strikingly, treatment of embryonic zebrafish with very low-dose BPA (0.0068 μM, 1,000-fold lower than the accepted human daily exposure) and bisphenol S (BPS), a common analog used in BPA-free products, resulted in 180% and 240% increases, respectively, in neuronal birth (neurogenesis) within the hypothalamus, a highly conserved brain region involved in hyperactivity. Furthermore, restricted BPA/BPS exposure specifically during the neurogenic window caused later hyperactive behaviors in zebrafish larvae. Unexpectedly, we show that BPA-mediated precocious neurogenesis and the concomitant behavioral phenotype were not dependent on predicted estrogen receptors but relied on androgen receptor-mediated up-regulation of aromatase. Although human epidemiological results are still emerging, an association between high maternal urinary BPA during gestation and hyperactivity and other behavioral disturbances in the child has been suggested. Our studies here provide mechanistic support that the neurogenic period indeed may be a window of vulnerability and uncovers previously unexplored avenues of research into how endocrine disruptors might perturb early brain development. Furthermore, our results show that BPA-free products are not necessarily safer and support the removal of all bisphenols from consumer merchandise.

The Neonatal Ventromedial Hypothalamus Transcriptome Reveals Novel Markers with Spatially Distinct Patterning

The ventromedial hypothalamus (VMH) is a distinct morphological nucleus involved in feeding, fear, thermoregulation, and sexual activity. It is essentially unknown how VMH circuits underlying these innate responses develop, in part because the VMH remains poorly defined at a cellular and molecular level. Specifically, there is a paucity of cell-type-specific genetic markers with which to identify neuronal subgroups and manipulate development and signaling in vivo. Using gene profiling, we now identify ∼200 genes highly enriched in neonatal (postnatal day 0) mouse VMH tissue. Analyses of these VMH markers by real or virtual (Allen Brain Atlas; http://www.brain-map.org) experiments revealed distinct regional patterning within the newly formed VMH. Top neonatal markers include transcriptional regulators such as Vgll2, SF-1, Sox14, Satb2, Fezf1, Dax1, Nkx2-2, and COUP-TFII, but interestingly, the highest expressed VMH transcript, the transcriptional coregulator Vgll2, is completely absent in older animals. Collective results from zebrafish knockdown experiments and from cellular studies suggest that a subset of these VMH markers will be important for hypothalamic development and will be downstream of SF-1, a critical factor for normal VMH differentiation. We show that at least one VMH marker, the AT-rich binding protein Satb2, was responsive to the loss of leptin signaling (Lepob/ob) at postnatal day 0 but not in the adult, suggesting that some VMH transcriptional programs might be influenced by fetal or early postnatal environments. Our study describing this comprehensive “VMH transcriptome” provides a novel molecular toolkit to probe further the genetic basis of innate neuroendocrine behavioral responses.

Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues.

Regulators of G-protein signaling (RGS) play a critical role in G-protein-coupled receptor signaling in mammalian cells. RGS proteins are GTPase-accelerating proteins (GAPs) for alpha subunits of heterotrimeric G proteins of the Gi and Gq class. RGS GAPs can modulate the frequency and duration of G-protein signaling and may constitute a new family of therapeutic targets. Identifying the tissue distribution and cellular localization of RGS proteins has been hindered by the lack of effective antibodies for immunodetection. The measurement of mRNA levels for RGS proteins, however, can provide insight into their tissue specificity and regulation. This article describes the use of a highly sensitive and rapid method for measuring RGS mRNA in mouse tissues. This quantitative real-time polymerase chain reaction method is established for the 19 reported mouse RGS genes and is used to study the tissue distribution of the R4 family of RGS genes and the diurnal regulation of RGS16 in mouse liver.

GSK3 temporally regulates neurogenin 2 proneural activity in the neocortex.

The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2–Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2–E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2–Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2–E47 heterodimerization by GSK3 is a central component of the neuronal differentiation “clock” that coordinates the timing and tempo of neocortical neurogenesis in mouse.

RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis.

Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1s fate specification properties in murine cortical progenitors–at higher RAS/ERK levels, Ascl1+ progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1s GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

Rax is a selector gene for mediobasal hypothalamic cell types.

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

Neurodevelopmental consequences of maternal distress: what do we really know?

Schuurmans C, Kurrasch DM. Neurodevelopmental consequences of maternal distress: what do we really know?

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‐1TauGFP) or by crossing the transgenic Sf1:Cre driver to a GFP reporter strain (Z/EGSf1: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 (VMHvl) persist in Z/EGSf1:Cre embryos but are virtually absent in Sf‐1TauGFP. Therefore, although the entire VMH including the VMHvl shares a common lineage, the VMHvl 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 VMHvl adopt a distinct fate early in development, which might underlie the unique physiological functions associated with this VMH subregion. J. Comp. Neurol., 521:1268–1288, 2013.

Neurog1 and Neurog2 coordinately regulate development of the olfactory system

BackgroundProneural genes encode basic helix–loop–helix transcription factors that specify distinct neuronal identities in different regions of the nervous system. In the embryonic telencephalon, the proneural genes Neurog1 and Neurog2 specify a dorsal regional identity and glutamatergic projection neuron phenotype in the presumptive neocortex, but their roles in cell fate specification in the olfactory bulb, which is also partly derived from dorsal telencephalic progenitors, have yet to be assessed. Given that olfactory bulb development is guided by interactions with the olfactory epithelium in the periphery, where proneural genes are also expressed, we investigated the roles of Neurog1 and Neurog2 in the coordinated development of these two olfactory structures.ResultsNeurog1/2 are co-expressed in olfactory bulb progenitors, while only Neurog1 is widely expressed in progenitors for olfactory sensory neurons in the olfactory epithelium. Strikingly, only a remnant of an olfactory bulb forms in Neurog1−/−;Neurog2−/− double mutants, while this structure is smaller but distinguishable in Neurog1−/− single mutants and morphologically normal in Neurog2−/− single mutants. At the cellular level, fewer glutamatergic mitral and juxtaglomerular cells differentiate in Neurog1−/−;Neurog2−/− double-mutant olfactory bulbs. Instead, ectopic olfactory bulb interneurons are derived from dorsal telencephalic lineages in Neurog1−/−;Neurog2−/− double mutants and to a lesser extent in Neurog2−/− single mutants. Conversely, cell fate specification is normal in Neurog1−/− olfactory bulbs, but aberrant patterns of cell proliferation and neuronal migration are observed in Neurog1−/− single and Neurog1−/−;Neurog2−/− double mutants, probably contributing to their altered morphologies. Finally, in Neurog1−/− and Neurog1−/−;Neurog2−/− embryos, olfactory sensory neurons in the epithelium, which normally project to the olfactory bulb to guide its morphogenesis, fail to innervate the olfactory bulb.ConclusionsWe have identified a cell autonomous role for Neurog1/ 2 in specifying the glutamatergic identity of olfactory bulb neurons. Furthermore, Neurog1 (and not Neurog2) is required to guide olfactory sensory neuron innervation of the olfactory bulb, the loss of which results in defects in olfactory bulb proliferation and tissue morphogenesis. We thus conclude that Neurog1/2 together coordinate development of the olfactory system, which depends on tissue interactions between the olfactory bulb and epithelium.

Neurog1 and Neurog2 Control Two Waves of Neuronal Differentiation in the Piriform Cortex

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genes Neurog1 and Neurog2 are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find that Neurog1 has a unique early role in reducing CR cell neurogenesis by tempering Neurog2s proneural activity. In addition, Neurog1 and Neurog2 have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase, Neurog1 and Neurog2 are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation of Trp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement. Neurog1 and Neurog2 thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.