Characterization of Hypothalamic MCH Neuron Development in a 3D Differentiation System of Mouse Embryonic Stem Cells

Abstract

Hypothalamic melanin-concentrating hormone (MCH) neurons are important regulators of multiple physiological processes, such as sleep, feeding, and memory. Despite the increasing interest in their neuronal functions, the molecular mechanism underlying MCH neuron development remains poorly understood. We report that a three-dimensional culture of mouse embryonic stem cells (mESCs) can generate hypothalamic-like tissues containing MCH-positive neurons, which reproduce morphologic maturation, neuronal connectivity, and neuropeptide/neurotransmitter phenotype of native MCH neurons. Using this in vitro system, we demonstrate that Hedgehog (Hh) signaling serves to produce major neurochemical subtypes of MCH neurons characterized by the presence or absence of cocaine- and amphetamine-regulated transcript (CART). Without exogenous Hh signals, mESCs initially differentiated into dorsal hypothalamic/prethalamic progenitors and finally into MCH + CART + neurons through a specific intermediate progenitor state. Conversely, activation of the Hh pathway specified ventral hypothalamic progenitors that generate both MCH + CART − and MCH + CART + neurons. These results suggest that in vivo MCH neurons may originate from multiple cell lineages that arise through early dorsoventral patterning of the hypothalamus. Additionally, we found that Hh signaling supports the differentiation of mESCs into orexin/hypocretin neurons, a well-defined cell group intermingled with MCH neurons in the lateral hypothalamic area (LHA). The present study highlights and improves the utility of mESC culture in the analysis of the developmental programs of specific hypothalamic cell types.

Hedgehog Activity Gradient in Combination with Transcription Network Confers Multiple Hypothalamic Identities

“…We did not observe any Nkx2.1- or Nkx2.2-positive cells in the absence of SAG, although it has been shown that such ventral hypothalamic cells appear regardless of the absence of SAG (Kodani et al, 2022; Wataya et al, 2008). This is presumably due to the differences of the cell lines used in each study.…”

“…For differentiation, three thousand ES cells were seeded onto a 96-well non-absorbable plate (Greiner, Cell-Repellent, #650970) and cultured in growth factor-free chemically defined medium We did not observe any Nkx2.1-or Nkx2.2-positive cells in the absence of SAG, although it has been shown that such ventral hypothalamic cells appear regardless of the absence of SAG (Kodani et al, 2022;Wataya et al, 2008). This is presumably due to the differences of the cell lines used in each study.…”

Discrete projections from MCH neurons mediate anti-parkinsonian effects of acupuncture

“…We further identified two subpopulations of MCH LH/ZI neurons and delineated their discrete functional relevance: motor behaviors (MCH LH/ZI→SNpc ) and non-motor behaviors (MCH LH→HPC ). This finding is noteworthy because it has not been studied whether the separate subpopulations of MCH LH/ZI neurons have distinct projections involving differential functions in the system level, despite recent highlights on the diversity of several hypothalamic neurons (43)(44)(45). Our findings strongly suggest that these two distinct circuits from MCH LH/ZI neurons are the cellular and circuit-level neuroanatomical basis of the multi-function of acupuncture in PD (Fig.…”