|Abstract:||The hypothalamus is an ancient structure of the brain in many orders of vertebrates and invertebrates involved in nearly all homeostatic regulation in the body. These processes include, but are not limited to reproductive development and function, energy balance, thermoregulation, water balance, and circadian cycles. These functions are regulated by the collaborative efforts of neurons expressing well-defined neuropeptide content as well as other cellular subtypes of the brain such as glia, ependymal cells, and hypothalamic tanycytes. It is hypothesized that each of these cell types arise from a common progenitor early in development. Disruption during the developmental window of any of these cellular types can result in persistent health conditions related to the processes of the hypothalamus they control, including infertility and obesity. Within the hypothalamus, these processes are controlled in two nuclei: the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). Although the function of a large number of these functional cell types has been studied in great detail, the developmental cues associated with fate decisions of a common progenitor remain highly elusive. We explored the effects that the Notch signaling pathway had on development of neurons related to these processes; first the effect that Notch had on the development on kisspeptin expressing neurons of the ARC involved in control of reproductive function. We observed that both in an early genetic ablation of canonical Notch signaling (Rbpj cKO) as well as a persistent Notch signaling expressing (NICD tg) mouse model, kisspeptin neurons of the ARC do not develop. This is in stark contrast to neurons involved in energy homeostasis, as while NICD tg mice showed no neuronal differentiation, Rbpj cKO mice showed significantly more of both Pomc or NPY neurons involved in feeding. Follow-up studies established that this observation was not due to differences in the timing of development of neurons involved in these processes, as each subtype were born between embryonic day (E)11.5 and 13.5. Additionally, neurons involved in these processes appeared to arise from a common immature neuronal lineage, providing further evidence for the hypothesis that Notch signaling is necessary for kisspeptin neuron development. These observations persist into adulthood and have profound reproductive consequences on female and male mice.
Next we aimed to characterize development of the AVPV, a previously understudied area of the brain in regards to its development. Utilizing an identical Rbpj cKO mouse model, we chose to explore the effects that loss of Notch signaling would have on AVPV formation, cellular dynamics, and fate decisions. By E13.5 we noticed a dramatic expansion of the tissue underlying the AVPV which was accompanied by a clear increase in cellular proliferation as marked by Ki-67. Interestingly, SOX2-positive progenitors of the area were not affected by our genetic manipulation; however, we noted an obvious increase in neurogenesis as marked by the general neuronal marker HuC/D and a significant increase in mature neurons as marked by the tyrosine hydroxylase (TH) enzyme, a common marker of the AVPV. Additionally, we noted a significant increase in OLIG2-positive oligodendrocytic precursor (OPC) cells of the AVPV at postnatal day (P)0 (day of birth). These findings imply that Notch signaling may be involved in restricting processes such as cellular proliferation, neurogenesis, and gliogenesis in the developing AVPV. In order to better understand the direct effects Notch signaling has on early fate decisions of progenitors of the hypothalamus, we next utilized a primary progenitor culture system consisting of late progenitor cells of the early neonatal hypothalamic ventricular zone (HVZ). These cells expand and spontaneously form neurospheres which can be manipulated and studied at the level of mRNA or protein expression. We found that acute chemical inhibition of Notch signaling was sufficient to significantly reduce direct downstream Notch signaling Hes1, Hes5, and Hey1. This coincided with a significant increase in the proneural gene Mash1, suggesting that some of the machinery required for progenitor differentiation was present in cultured progenitors. In vitro results recapitulated some in vivo findings, as we noted significant increases in mKi67 and Olig2 transcript. However, the neuronal precursor cell (NPC) marker Nestin (Nes) was not changed, suggesting neurogenesis was not yet affected. To this end, we inhibited Notch signaling an additional 72 hours and assessed neuronal quality via immunocytochemistry (ICC). We noted that 144 hours of Notch inhibition resulted in significant increases in TH expression in both cultured cell soma and neurite extensions, as well as a significant increase in neurite outgrowth, suggesting that progenitors began to adopt a more neuronal identity.
Finally, we chose to use our developed in vitro neurosphere assay for other applications of progenitor fate decisions within the hypothalamus. Conditions such as gestational diabetes result in aberrant signaling in developing tissue of the fetus and have implications with Notch signaling. To this end, we cultured neurospheres in varying concentrations of insulin and reported that a 100-fold increase in insulin signaling resulted in increased number of spheres in a random field of view as well as an increase in the average sphere size. Interestingly, in high concentrations of insulin, chemical inhibition of Notch reversed this observation, suggesting that Notch signaling may be responsible for expansion of these neurospheres in this assay. In order to expedite understanding of multiple signaling pathways at once and the effects they have on neurosphere progenitor-quality and proliferation, we adopted a novel assay developed by the Department of Engineering. To this end, neurospheres were plated on microenvironments of varying extracellular matrix protein (ECMs) contexts in the presence of absence of Notch signaling. We report that chemical inhibition of Notch on specific ECMs reduced cellular proliferation and progenitor-quality and that neurospheres preferred growing on laminin- or fibronectin-based ECMs. Future studies will be able to combine several of these novel techniques to better understand the mechanism underlying hypothalamic development and early fate decisions.