The transcriptional regulator PRDM12 is critical for Pomc expression in the mouse hypothalamus and controlling food intake, adiposity, and body weight.

OBJECTIVE: Regulation of food intake and energy balance depends on a group of hypothalamic neurons that release anorexigenic melanocortins encoded by the Pomc gene. Although the physiological importance of central melanocortins is well appreciated, the genetic program that defines the functional identity of melanocortin neurons and assures high levels of hypothalamic Pomc expression is only beginning to be understood. This study assessed whether the transcriptional regulator PRDM12, identified as a highly expressed gene in adult mouse POMC neurons, plays an important role in the identity and function of melanocortin neurons. METHODS: We first determined the cellular distribution of PRDM12 in the developing hypothalamus. Then we studied mutant mice with constitutively inactivated Prdm12 to evaluate possible changes in hypothalamic Pomc expression. In addition, we characterized conditional mutant mice specifically lacking Prdm12 in ISL1-positive or POMC neurons during development. Finally, we measured food intake, body weight progression up to 16 weeks of age, adiposity, and glucose tolerance in adult mice lacking Prdm12 selectively from POMC neurons. RESULTS: PRDM12 co-expressed with POMC in mouse hypothalamic neurons from early development to adulthood. Mice lacking Prdm12 displayed greatly reduced Pomc expression in the developing hypothalamus. Selective ablation of Prdm12 from ISL1 neurons prevented hypothalamic Pomc expression. The conditional ablation of Prdm12 limited to POMC neurons greatly reduced Pomc expression in the developing hypothalamus and in adult mice led to increased food intake, adiposity, and obesity. CONCLUSIONS: Altogether, our results demonstrate that PRDM12 plays an essential role in the early establishment of hypothalamic melanocortin neuron identity and the maintenance of high expression levels of Pomc. Its absence in adult mice greatly impairs Pomc expression and leads to increased food intake, adiposity, and obesity.

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

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.SIGNIFICANCE STATEMENT Food intake and body weight regulation depend on hypothalamic neurons that release satiety-inducing neuropeptides, known as melanocortins. Central melanocortins are encoded byPomc, 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.

Evolution of the Rax family of developmental transcription factors in vertebrates.

Rax proteins comprise a small family of paired-type, homeodomain-containing transcription factors with essential functions in eye and forebrain development. While invertebrates possess only one Rax gene, vertebrates can have several Rax paralogue genes, but the evolutionary history of the members of the family has not been studied in detail. Here, we present a thorough analysis of the evolutionary relationships between vertebrate Rax genes and proteins available in diverse genomic databases. Phylogenetic and synteny analyses indicate that Rax genes went through a duplication in an ancestor of all jawed vertebrates (Gnathostomata), giving rise to the ancestral vertebrate Rax1 and Rax2 genes. This duplication event is likely related to the proposed polyploidisations that occurred during early vertebrate evolution. Subsequent genome-wide duplications in the lineage of ray-finned fish (Actinopterygii) originated new Rax2 paralogues in the genomes of teleosts. In the lobe-finned fish lineage (Sarcopterygii), the N-terminal octapeptide domain of Rax2 was lost in a common ancestor of tetrapods, giving rise to a shorter version of Rax2 in this lineage. Within placental mammals, the Rax2 gene was lost altogether in an ancestor of rodents and lagomorphs (Glires). Finally, we discuss the scientific literature in the light of Rax gene evolution and propose new avenues of research on the function of this important family of transcriptional regulators.

Essential function of the transcription factor Rax in the early patterning of the mammalian hypothalamus.

The hypothalamus is a region of the anterior forebrain that controls basic aspects of vertebrate physiology, but the genes involved in its development are still poorly understood. Here, we investigate the function of the homeobox gene Rax/Rx in early hypothalamic development using a conditional targeted inactivation strategy in the mouse. We found that lack of Rax expression prior to embryonic day 8.5 (E8.5) caused a general underdevelopment of the hypothalamic neuroepithelium, while inactivation at later timepoints had little effect. The early absence of Rax impaired neurogenesis and prevented the expression of molecular markers of the dorsomedial hypothalamus, including neuropeptides Proopiomelanocortin and Somatostatin. Interestingly, the expression domains of genes expressed in the ventromedial hypothalamus and infundibulum invaded dorsal hypothalamic territory, showing that Rax is needed for the proper dorsoventral patterning of the developing medial hypothalamus. The phenotypes caused by the early loss of Rax are similar to those of eliminating the expression of the morphogen Sonic hedgehog (Shh) specifically from the hypothalamus. Consistent with this similarity in phenotypes, we observed that Shh and Rax are coexpressed in the rostral forebrain at late head fold stages and that loss of Rax caused a downregulation of Shh expression in the dorsomedial portion of the hypothalamus.

Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood.

Food intake and body weight regulation depend on proper expression of the proopiomelanocortin gene (Pomc) in a group of neurons located in the mediobasal hypothalamus of all vertebrates. These neurons release POMC-encoded melanocortins, which are potent anorexigenic neuropeptides, and their absence from mice or humans leads to hyperphagia and severe obesity. Although the pathophysiology of hypothalamic POMC neurons is well understood, the genetic program that establishes the neuronal melanocortinergic phenotype and maintains a fully functional neuronal POMC phenotype throughout adulthood remains unknown. Here, we report that the early expression of the LIM-homeodomain transcription factor Islet 1 (ISL1) in the developing hypothalamus promotes the terminal differentiation of melanocortinergic neurons and is essential for hypothalamic Pomc expression since its initial onset and throughout the entire lifetime. We detected ISL1 in the prospective hypothalamus just before the onset of Pomc expression and, from then on, Pomc and Isl1 coexpress. ISL1 binds in vitro and in vivo to critical homeodomain binding DNA motifs present in the neuronal Pomc enhancers nPE1 and nPE2, and mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to hypothalamic POMC neurons in transgenic mice and zebrafish. ISL1 is necessary for hypothalamic Pomc expression during mouse and zebrafish embryogenesis. Furthermore, conditional Isl1 inactivation from POMC neurons impairs Pomc expression, leading to hyperphagia and obesity. Our results demonstrate that ISL1 specifies the identity of hypothalamic melanocortin neurons and is required for melanocortin-induced satiety and normal adiposity throughout the entire lifespan.