Rax Expression Identifies a Novel Cell Type in the Adult Mouse Hypothalamus

Abstract

Abstract Hypothalamic tanycytes are radial glia-like ependymal cells lining the ventrolateral walls and floor of the third ventricle. Recent data show that tanycytes are adult neural stem/progenitor cells, capable of generating neurons that populate the adjacent hypothalamic nuclei involved in the regulation of feeding and energy balance. Thus, the genetic fate mapping of tanycytes has become an invaluable tool to identify and study tanycyte-derived adult-born hypothalamic neurons. Perhaps the most selective tanycyte marker identified to date is the retina and anterior neural fold homeobox (Rax), that has been used as a tanycyte marker in multiple single-cell transcriptomic studies. By using in situ hybridization and immunofluorescence, we show that Rax mRNA and RAX protein are also expressed in a minor but significant population of parenchymal cells that are concentrated in the caudal arcuate nucleus. RAX-positive nuclei in the parenchyma were often observed in pairs, suggesting recent cell divisions. The morphology of these cells was studied in tamoxifen-treated Rax-CreERT2; Ai34(RCL-Syp/tdT)-D mice, in which the synaptophysin-tdTomato fusion protein permanently labels Rax-expressing cells and their progeny. While some parenchymal RAX-positive cells had tanycyte-like morphology indicative of tanycyte migration into the parenchyma, the majority had a very different morphology with extensive local processes that often encircled adjacent neurons (termed “frizzy cells”). The tdTomato labeling also revealed numerous frizzy cells that were negative for RAX, indicating downregulation of endogenous Rax expression subsequent to the induction of synaptophysin-tdTomato reporter expression. Many of these cells were distributed outside the caudal arcuate nucleus, including the rostral lateral arcuate nucleus, ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamus. RAX-negative frizzy cells were also conspicuous in the paraventricular nucleus, and occasionally observed in the preoptic region and bed nucleus of the stria terminalis. Frizzy cells were negative for the tanycyte-enriched proteins vimentin, monocarboxylate transporter 8 (MCT8) or glial fibrillary acidic protein (GFAP). These results identify a novel Rax-expressing cell type in the adult hypothalamus that differs from tanycytes in location, morphology and gene expression characteristics. Future studies are required to determine whether frizzy cells are derived from tanycytes or constitute a separate cell lineage, and whether they represent a migratory form of neural precursor cells in the adult hypothalamus.