業 績
The following is a list of some recent publications from the lab, along with the abstract and download links.
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Gfrα3 receptor in enteroendocrine cells
Simanjaya S, Kinoshita M, Sunardi M, Ito K, Shinohara M, Abe T, Kodama Y, Uesaka T, Enomoto H. (2026) |
The receptor tyrosine kinase RET regulates the development of multiple neuronal populations through GFRα co-receptors and their cognate ligands. Recent work has shown that RET is also required for enteroendocrine cell (EEC) function; however, the specific GFRα receptor operating in EECs has remained unclear. Here, we identify GFRα3 as the predominant GFRα receptor expressed in EECs. To determine its cell-autonomous role, we generated a Gfrα3 conditional allele and inactivated the Gfrα3 gene specifically in the intestinal epithelium. These Gfrα3 IEcKO mice exhibited a selective reduction in the number of CCK+ EECs among multiple EEC subtypes. A comparative decrease in CCK+ EECs in Ret IEcKO mice suggests that RET/GFRα3 signaling is required for the development and/or maintenance of this EEC lineage. Although CCK+ EECs can indirectly influence glucose metabolism, Gfrα3 IEcKO mice showed no significant change in glucose tolerance under high-fat diet (HFD) conditions. In contrast, they exhibited modest lipid malabsorption on HFD. Together, these findings demonstrate that GFRα3 is implicated in the structure and function of CCK EECs and highlight the utility of the Gfrα3 conditional allele for dissecting the biology of GFRα3-expressing cells.
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Neurogenic potential of enteric glia in aganglionic colon
Mueller JL, Han C, Leavitt A, Chauhan V, Ott L, Guyer RA, Uesaka T, Enomoto H, Cheng L, Hotta R, Burns AJ, Stavely R, Goldstein AM. (2025) |
Hirschsprung disease (HSCR) is a neurocristopathy, yet paradoxically, neural crest-derived EGCs are present within the muscle of the affected region. This study investigates the molecular identity, origins, and neurogenic potential of EGCs in the aganglionic mouse and human colon. We utilized single-cell RNA sequencing (scRNA-seq), immunohistochemistry, and in vitro culture of EGCs from aganglionic and ganglionated segments of Ednrb-null mice (Plp1-GFP;Baf-tdT;Ednrb-/-) and human HSCR tissues. Neurogenic potential and network formation were assessed, and the effects of glial cell line-derived neurotrophic factor (GDNF) on neurogenesis were evaluated. scRNA-seq and immunohistochemistry revealed the absence of GFAP+ intraganglionic (IG) glia in aganglionic colon, while CAMK2b+ extraganglionic (EG) glia and Schwann-like cells (SLCs) were present. EG glia exhibited a transcriptional profile similar to SLCs, suggesting a possible shared embryonic origin. EGCs in the aganglionic segment (comprising EGs and SLCs) exhibited reduced neurogenic potential and network complexity compared to EGCs from the ganglionated region (comprising EGs and IGs). GDNF partially restored neurogenic capacity and enhanced network complexity of EGCs isolated from the aganglionic segment, acting through a non-canonical NCAM1-dependent pathway independent of RET signaling.
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The long life span of enteroendocrine cells
Sesotyosari SL, Kinoshita M, Sunardi M, Lihan M, Orii A, Abe T, Kiyonari H, Nakai T, Uesaka T, Kodama Y, Enomoto H. (2025) |
Enteroendocrine cells (EECs) are sensory epithelial cells that sense the gut luminal environment and convey sensory information to the brain via the visceral afferent pathway. Although EECs are a part of gut epithelial cells, which generally undergo rapid turnover, some EECs have been reported to be long-lived. EECs consist of multiple subtypes, each of which displays distinct hormone production and distribution patterns. It remains unknown whether a long lifespan is a characteristic shared by all EEC subtypes. To address this issue, we conducted genetic pulse labeling of three EEC subtypes expressing serotonin (5-HT), peptide YY (PYY), and gastric inhibitory polypeptide (GIP) in mice and tracked their survival. In the proximal small intestine, all labeled GIP+ EECs disappeared completely within 5 days, whereas some PYY+ EECs survived for more than 7 days. In the proximal colon, some labeled 5-HT+ EECs lived for more than 28 days, whereas no PYY+ cells survived beyond 14 days. These long-lived 5-HT+ EECs were almost exclusively found in the upper half of the crypt in the mucosal fold, where visceral sensory fibers were enriched. This study reveals subtype- and region-dependent survival of EECs and suggests that EEC-nerve communication may underlie the long lifespan of certain EECs.
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The biological function of Polycomb group RING finger protein 1 (PCGF1)
Putra BP, Ito K, Cirillo C, Sunardi M, Koseki H, Uesaka T, Enomoto H. (2023) |
The enteric nervous system (ENS) regulates gut functions independently from the central nervous system (CNS) by its highly autonomic neural circuit that integrates diverse neuronal subtypes. Although several transcription factors are shown to be necessary for the generation of some enteric neuron subtypes, the mechanisms underlying neuronal subtype specification in the ENS remain elusive. In this study, we examined the biological function of Polycomb group RING finger protein 1 (PCGF1), one of the epigenetic modifiers, in the development and differentiation of the ENS by disrupting the Pcgf1 gene selectively in the autonomic-lineage cells. Although ENS precursor migration and enteric neurogenesis were largely unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the numbers of neurons expressing somatostatin (Sst+ ) decreased in multiple gut regions. Notably, the decrease in Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the proximal colon. These findings suggest that neuronal subtype conversion may occur in the absence of PCGF1, and that epigenetic mechanism is primarily involved in specification of some enteric neuron subtypes.
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HSCR mouse model with a RET missense mutation
Sunardi M, Ito K, Sato Y, Uesaka T, Iwasaki M, Enomoto H. (2023) |
Background & aims: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance in vivo has remained largely unclear.
Methods: We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. Results: RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. Conclusions: This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations. |
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Uts2b is expressed in a microbiota-dependent manner
Yoshioka Y, Tachibana Y, Uesaka T, Hioki H, Sato Y, Fukumoto T, Enomoto H. (2022) |
Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.
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SCPs are a cellular source to compensate for missing enteric neurons in HSCR
Uesaka T, Okamoto M, Nagashimada M, Tsuda Y, Kihara M, Kiyonari H, Enomoto H. (2021) |
Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.
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Increased RET activity coupled with a decreased gene dosage can cause intestinal aganglionosis
Okamoto M, Uesaka T, Ito K, Enomoto H. (2004) |
Mutations of the gene encoding the RET tyrosine kinase causes Hirschsprung's disease (HSCR) and medullary thyroid carcinoma (MTC). Current consensus holds that HSCR and MTC are induced by inactivating and activating RET mutations, respectively. However, it remains unknown whether activating mutations in the RET gene have adverse effects on ENS development in vivo We addressed this issue by examining mice engineered to express RET51(C618F), an activating mutation identified in MTC patients. Although Ret51(C618F)/51(C618F) mice displayed hyperganglionosis of the ENS, Ret51(C618F)/- mice exhibited severe intestinal aganglionosis because of premature neuronal differentiation. Reduced levels of glial cell-derived neurotrophic factor (GDNF), a RET-activating neurotrophic factor, ameliorated the ENS phenotype of Ret51(C618F)/- mice, demonstrating that GDNF-mediated activation of RET51(C618F) is responsible for severe aganglionic phenotype. The RET51(C618F) allele showed genetic interaction with Ednrb gene, one of modifier genes for HSCR. These data reveal that proliferation and differentiation of ENS precursors are exquisitely controlled by both the activation levels and total dose of RET. Increased RET activity coupled with a decreased gene dosage can cause intestinal aganglionosis, a finding that provides novel insight into HSCR pathogenesis.
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REVIEW: Development of the intrinsic and extrinsic innervation of the gut
Uesaka T, Young HM, Pachnis V, Enomoto H. (2016) |
The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract.
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