Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A.

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

Glyphosate Toxicity: In Vivo, In Vitro, and Epidemiological Evidence.

Glyphosate is the most applied agricultural chemical worldwide and has become nearly ubiquitous throughout the environment. Glyphosate is an effective herbicide because it disrupts the shikimate pathway, which is responsible for the synthesis of essential amino acids in plants and microorganisms. Given that there is no known target for glyphosate in higher animals, its toxicity to humans and other animals is heavily debated, especially after the 2015 IARC ruling that glyphosate is carcinogenic. Today, a growing body of literature shows in vitro, in vivo, and epidemiological evidence for the toxicity of glyphosate across animal species. With the application of glyphosate increasing globally, it is important to discuss these reports to enable a broader conversation on glyphosate toxicity and its impact on human and environmental health. Here, we summarize the recent glyphosate literature and discuss its implications.

Gestational Bisphenol A Exposure Impacts Embryonic Hypothalamic Microglia Numbers, Ramification, and Phagocytic Cups.

Microglia are a resident population of phagocytic immune cells that reside within the central nervous system (CNS). During gestation, they are highly sensitive to their surrounding environment and can alter their physiology to respond to perceived neural insults, potentially leading to adverse influences on nearby neural progenitors. Given that bisphenol A (BPA) itself can impact developing brains, and that microglia express estrogen receptors to which BPA can bind, here we asked whether fetal microglia are responsive to gestational BPA exposure. Accordingly, we exposed pregnant dams to control or 50 mg of BPA per kg diet during gestation to investigate the impact of maternal BPA on embryonic hypothalamic microglia. Gestational BPA exposure from embryonic day 0.5 (E0.5) to E15.5 resulted in a significant increase in the number of microglia present in the hypothalamus of both male and female embryos. Staining for microglial activation using CD68 showed no change between control and prenatal BPA-exposed microglia, regardless of sex. Similarly, analysis of cultured embryonic brains demonstrated that gestational BPA exposure failed to change the secretion of cytokines or chemokines, regardless of embryo sex or the dose (50 µg of BPA per kg or 50 mg of BPA per kg maternal diet) of BPA treatment. In contrast, live-cell imaging of microglia dynamics in E15.5 control and gestationally-exposed BPA hypothalamic slices showed increased ramification of microglia exposed to BPA. Moreover, live-cell imaging also revealed a significant increase in the number of microglial phagocytic cups visible following exposure to gestational BPA. Together, these results suggest that gestational BPA exposure impacts embryonic hypothalamic microglia, perhaps leading them to alter their interactions with developing neural programs.

Live-cell imaging of microglial interactions with radial glia in transgenic embryonic mouse brains using slice culture.

Microglial dynamics and interactions with nearby radial glia can be visualized in real time in embryonic mouse brain tissue using time-lapse imaging in slice culture. This live-cell imaging protocol can be used to study the morphology and activities of a number of cell types across a variety of brain regions and developmental time points. The advantage of this brain slice culture model is that it allows for the visualization of cellular interactions and movements in real time, especially across embryogenesis. For complete details on the use and execution of this protocol, please refer to Rosin et al. (2021).

Gestational low-dose BPA exposure impacts suprachiasmatic nucleus neurogenesis and circadian activity with transgenerational effects.

Critical physiological processes such as sleep and stress that underscore health are regulated by an intimate interplay between the endocrine and nervous systems. Here, we asked how fetal exposure to the endocrine disruptor found in common plastics, bisphenol A (BPA), causes lasting effects on adult animal behaviors. Adult mice exposed to low-dose BPA during gestation displayed notable disruption in circadian activity, social interactions, and associated neural hyperactivity, with some phenotypes maintained transgenerationally. Gestational BPA exposure increased vasopressin+ neurons in the suprachiasmatic nucleus (SCN), the region that regulates circadian rhythms, of F1 and F3 generations. Mechanistically, BPA increased proliferation of hypothalamic neural progenitors ex vivo and caused precocious neurogenesis in vivo. Co-antagonism of both estrogen and androgen receptors was necessary to block BPA's effects on hypothalamic neural progenitors, illustrating a dual role for these endocrine targets. Together, gestational BPA exposure affects development of circadian centers, with lasting consequences across generations.

Embryonic Microglia Interact with Hypothalamic Radial Glia during Development and Upregulate the TAM Receptors MERTK and AXL following an Insult.

Despite a growing appreciation for microglial influences on the developing brain, the responsiveness of microglia to insults during gestation remains less well characterized, especially in the embryo when microglia themselves are still maturing. Here, we asked if fetal microglia could coordinate an innate immune response to an exogenous insult. Using time-lapse imaging, we showed that hypothalamic microglia actively surveyed their environment by near-constant "touching" of radial glia projections. However, following an insult (i.e., IUE or AAV transduction), this seemingly passive touching became more intimate and long lasting, ultimately resulting in the retraction of radial glial projections and degeneration into small pieces. Mechanistically, the TAM receptors MERTK and AXL were upregulated in microglia following the insult, and Annexin V treatment inhibited radial glia breakage and engulfment by microglia. These data demonstrate a remarkable responsiveness of embryonic microglia to insults during gestation, a critical window for neurodevelopment.

Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has adverse sex-specific effects on mice, including accelerated weight gain, hyperactivity and anxiolytic-like behaviour.

Microglia are the resident immune cells in the central nervous system (CNS). Originally thought to be primarily responsible for disposing of cellular debris and responding to neural insults, emerging research now shows that microglia are highly dynamic cells involved in a variety of neurodevelopmental processes. The hypothalamus is a brain region critical for maintaining homeostatic processes such as energy balance, thirst, food intake, reproduction, and circadian rhythms. Given that microglia colonize the embryonic brain alongside key steps of hypothalamic development, here we tested whether microglia are required for the proper establishment of this brain region. The Colony-stimulating factor-1 receptor (Csf1r) is expressed by microglia, macrophages and osteoclasts, and is required for their proliferation, differentiation, and survival. Therefore, to eliminate microglia from the fetal brain, we treated pregnant dams with the CSF1R inhibitor PLX5622. We showed that approximately 99% of microglia were eliminated by embryonic day 15.5 (E15.5) after pregnant dams were placed on a PLX5622 diet starting at E3.5. Following microglia depletion, we observed elevated numbers of apoptotic cells accumulating throughout the developing hypothalamus. Once the PLX5622 diet was removed, microglia repopulated the postnatal brain within 7 days and did not appear to repopulate from Nestin+ precursors. Embryonic microglia depletion also resulted in a decreased litter size, as well as an increase in the number of pups that died within the first two postnatal days of life. In pups that survived, the elimination of microglia in the fetal brain resulted in a decrease in the number of Pro-opiomelanocortin (POMC) neurons and a concomitant accelerated weight gain starting at postnatal day 5 (P5), suggesting that microglia could be important for the development of cell types key to hypothalamic satiety centers. Moreover, surviving PLX5622 exposed animals displayed craniofacial and dental abnormalities, perhaps due to non-CNS effects of PLX5622 on macrophages and/or osteoclasts. Finally, depletion of microglia during embryogenesis had long-term sex-specific effects on behaviour, including the development of hyperactivity and anxiolytic-like behaviour in juvenile and adult female mice, respectively. Together, these data demonstrate an important role for microglia during the development of the embryonic hypothalamus, and perhaps the CNS more broadly.

Bisphenol A and microglia: could microglia be responsive to this environmental contaminant during neural development?

There is a growing interest in the functional role of microglia in the developing brain. In our laboratory, we have become particularly intrigued as to whether fetal microglia in the embryonic brain are susceptible to maternal challenges in utero (e.g., maternal infection, stress) and, if so, whether their precocious activation could then adversely influence brain development. One such challenge that is newly arising in this field is whether microglia might be downstream targets to endocrine-disrupting chemicals, such as the plasticizer bisphenol A (BPA), which functions in part by mimicking estrogen structure and function. A growing body of evidence demonstrates that gestational exposure to BPA has adverse effects on brain development, although the exact mechanisms are still emerging. Given that microglia express estrogen receptors and steroid-producing enzymes, microglia might be an unappreciated target of BPA. Mechanistically, we propose that BPA binding to estrogen receptors within microglia initiates transcription of downstream target genes, which then leads to activation of microglia that can then perhaps adversely influence brain development. Here, we first briefly outline the current understanding of how microglia may influence brain development and then describe how this literature overlaps with our understanding of BPA's effects during similar time points. We also outline the current literature demonstrating that BPA exposure affects microglia. We conclude by discussing our thoughts on the mechanisms through which exposure to BPA could disrupt normal microglia functions, ultimately affecting brain development that could potentially lead to lasting behavioral effects and perhaps even neuroendocrine diseases such as obesity.

Granulocyte-colony-stimulating factor (G-CSF) signaling in spinal microglia drives visceral sensitization following colitis.

Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.

Adverse morphological development in embryonic zebrafish exposed to environmental concentrations of contaminants individually and in mixture.

Exposure to environmental contaminants has been linked to developmental and reproductive abnormalities leading to infertility, spontaneous abortion, reduced number of offspring, and metabolic disorders. In addition, there is evidence linking environmental contaminants and endocrine disruption to abnormal developmental rate, defects in heart and eye morphology, and alterations in behavior. Notably, these effects could not be explained by interaction with a single hormone receptor. Here, using a whole-organism approach, we investigated morphological changes to developing zebrafish caused by exposure to a number of environmental contaminants, including bisphenol A (BPA), di(2-ethylhexyl)phthalate (DEHP), nonylphenol, and fucosterol at concentrations measured in a local water body (Oldman River, AB), individually and in mixture. Exposure to nanomolar contaminant concentrations resulted in abnormal morphological development, including changes to body length, pericardia (heart), and the head. We also characterize the spatiotemporal expression profiles of estrogen, androgen, and thyroid hormone receptors to demonstrate that localization of these receptors might be mediating contaminant effects on development. Finally, we examined the effects of contaminants singly and in mixture. Combined, our results support the hypothesis that adverse effects of contaminants are not mediated by single hormone receptor signaling, and adversity of contaminants in mixture could not be predicted by simple additive effect of contaminants. The findings provide a framework for better understanding of developmental toxicity of environmental contaminants in zebrafish and other vertebrate species.

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

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.

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 Ascl1's 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 Ascl1's 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.

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

BACKGROUND: 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. RESULTS: We 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. CONCLUSION: Our 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.