The Homeodomain Transcription Factor NKX2.1 Is Essential for the Early Specification of Melanocortin Neuron Identity and ActivatesPomcExpression in the Developing Hypothalamus

Abstract

Food intake is tightly regulated by a group of neurons present in the arcuate nucleus of the hypothalamus, which release Pomc-encoded melanocortins, the absence of which induces marked hyperphagia and early-onset obesity. Although the relevance of hypothalamic POMC neurons in the regulation of body weight and energy balance is well appreciated, little is known about the transcription factors that establish the melanocortin neuron identity during brain development and its phenotypic maintenance in postnatal life. Here, we report that the transcription factor NKX2.1 is present in mouse hypothalamic POMC neurons from early development to adulthood. Electromobility shift assays showed that NKX2.1 binds in vitro to NKX binding motifs present in the neuronal Pomc enhancers nPE1 and nPE2 and chromatin immunoprecipitation assays detected in vivo binding of NKX2.1 to nPE1 and nPE2 in mouse hypothalamic extracts. Transgenic and mutant studies performed in mouse embryos of either sex and adult males showed that the NKX motifs present in nPE1 and nPE2 are essential for their transcriptional enhancer activity. The conditional early inactivation of Nkx2.1 in the ventral hypothalamus prevented the onset of Pomc expression. Selective Nkx2.1 ablation from POMC neurons decreased Pomc expression in adult males and mildly increased their body weight and adiposity. Our results demonstrate that NKX2.1 is necessary to activate Pomc expression by binding to conserved canonical NKX motifs present in nPE1 and nPE2. Therefore, NKX2.1 plays a critical role in the early establishment of hypothalamic melanocortin neuron identity and participates in the maintenance of Pomc expression levels during adulthood.Food intake and body weight regulation depend on hypothalamic neurons that release satiety-inducing neuropeptides, known as melanocortins. Central melanocortins are encoded by Pomc, and Pomc mutations may lead to hyperphagia and severe obesity. Although the importance of central melanocortins is well appreciated, the genetic program that establishes and maintains fully functional POMC neurons remains to be explored. Here, we combined molecular, genetic, developmental, and functional studies that led to the discovery of NKX2.1, a transcription factor that participates in the early morphogenesis of the developing hypothalamus, as a key player in establishing the early identity of melanocortin neurons by activating Pomc expression. Thus, Nkx2.1 adds to the growing list of genes that participate in body weight regulation and adiposity.

Comparative Transcriptomic Analyses of Developing Melanocortin Neurons Reveal New Regulators for the Anorexigenic Neuron Identity

“…Together, these findings support a recently proposed threshold of Pomc transcription (;30%-40% of normal values) on physiology, below which hyperphagia and obesity become evident (Lam et al, 2015). On the other hand, we believe the role of Prdm12 in POMC neurons may be different from that of Isl1, Nkx2.1, and Tbx3 Orquera et al, 2019;Quarta et al, 2019). Unlike Isl1, Prdm12 is not required for cell survival.…”

“…It is conceivable that both factors may continue to play a role in the maintenance of POMC neuron identity. However, while early deletion of Nkx2.1 led to a complete loss of POMC neurons, its selective deletion in POMC-Cre neurons did not change the number of Pomc-immunoreactive cells and only caused a mild reduction in Pomc levels (Orquera et al, 2019; and unpublished observation by our group). On the other hand, Isl1 is expressed by other ARH neurons and is necessary for the expression of multiple ARH markers, including Npy, Agrp, GHRH, and Sst.…”

“…By identifying TFs that bind these sequences, these investigators recently reported that two homeodomain TFs Isl1 and Nkx2.1 transactivate Pomc transcription in the developing hypothalamus. Loss of either factor in hypothalamic progenitors prevents the onset of Pomc expression in the developing ARH Orquera et al, 2019). As a result, these findings establish a critical role for both genes in specifying the POMC neuron identity.…”

Transcriptomic Profiling of Developing Melanocortin Neurons Reveals a Role for Prdm12 in Energy Balance

“…2a-c). These findings are consistent with recent reports that these factors continue to play a role in POMC or NPY/AgRP neurons after neurogenesis [21][22][23] .…”

Identification of candidate genes and regulatory factors related to growth rate through hypothalamus transcriptome analyses in broiler chickens

“…The efficiency of PCR reactions was estimated based on the standard curve method. The gene expression levels were calculated using the delta-delta CT method [20], and the significant differences in gene expression levels between HGR and LGR individuals within each age group were determined by ANOVA (Duncan's post hoc test; SAS Enterprise v. 7.1 with default settings; SAS Institute, Cary, USA) element-homeobox protein NK-2 homolog A (Nkx2-1) was found to be overexpressed, and Nkx2-1 ablation from POMC neurons leads to decreased POMC expression in adult males and mildly increased their body weight and adiposity [27,28] (Fig. 3).…”