Highlights d Hypothalamic-pituitary functional units can be generated from 3D-cultured human iPS cells d Maturation of induced pituitary through simultaneous culture with hypothalamic neurons d ACTH secretion ability of induced pituitary is comparable with adult mice d Induced pituitary cells respond to low-glucose stimulation through the CRH-ACTH pathway SUMMARYThe pituitary is a major hormone center that secretes systemic hormones responding to hypothalamusderived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs).Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a lifethreatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

Arginine-vasopressin (AVP) neurons exist in the hypothalamus, a major region of the diencephalon, and play an essential role in water balance. Here, we established the differentiation method for AVP-secreting neurons from human embryonic stem cells (hESCs) by recapitulating in vitro the in vivo embryonic developmental processes of AVP neurons. At first, the differentiation efficiency was improved. That was achieved through the optimization of the culture condition for obtaining dorsal hypothalamic progenitors. Secondly, the induced AVP neurons were identified by immunohistochemistry and these neurons secreted AVP after potassium chloride stimulation. Additionally, other hypothalamic neuropeptides were also detected, such as oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone, pro-opiomelanocortin, agouti-related peptide, orexin, and melanin-concentrating hormone. This is the first report describing the generation of secretory AVP neurons derived from hESCs. This method will be applicable to research using disease models and, potentially, for regenerative medicine of the hypothalamus.

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FMRFamide-gated Na(+) channel (FaNaC) is a peptide-gated sodium channel in the epithelial Na(+) channel/degenerin family. Although there are some data on the location of the putative peptide binding site, there is no structural information on the activation gating of FaNaC. Here, we addressed the function of a conserved aspartate residue in the second transmembrane domain of FaNaC. We used Aplysia kurodai FaNaC (AkFaNaC) and examined the function of the aspartate (D552) by site-directed mutagenesis and electrophysiological recording in Xenopus oocytes. We found that the macroscopic activation, desensitization, and potency of FMRFamide and its modification by external Ca(2+) and Mg(2+) are greatly affected by physicochemical properties of the amino acid at position 552. We conclude that D552 is situated in a key position that affects the gating properties of FaNaC.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability of TH determined by the degradation remains unknown; although the TH molecule phosphorylated at its Ser19 was observed in the nucleus, and the phosphorylation suspected to trigger its proteasome-mediated degradation. Computer-assisted analysis using the cNLS Mapper program predicted that two sequences of nuclear localization signals (NLS) exist in the N-terminus of TH molecule containing the phosphorylation sites Ser19, Ser31, and Ser40 (Pro-Arg and Lys-Ile): the NLS scores indicated that TH could become localized in both nucleus and cytoplasm. Moreover, inhibition of the importin α/β-mediated nuclear import pathway increased the level of TH phosphorylated at its Ser19 in PC12D cells. The results suggest that TH might be imported to nucleus from cytoplasm to be degraded. Recent studies revealed that proteasomes predominantly exist in the nucleus rather than in the cytoplasm to degrade the nuclear proteins related to cell-cycle progression, gene expression, DNA damage, and DNA repair. Therefore, these studies suggest that the relationship between the phosphorylation and the nuclear localization of the TH molecule should be a matter of focus to understand the mechanism of proteasome-mediated degradation of the enzyme as a first priority.