A Single RET Mutation in Hirschsprung Disease Induces Intestinal Aganglionosis Via a Dominant-Negative Mechanism.

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.

Uts2b is a microbiota-regulated gene expressed in vagal afferent neurons connected to enteroendocrine cells producing cholecystokinin.

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.

Live visualization of a functional RET-EGFP chimeric receptor in homozygous knock-in mice.

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET-dependent developmental processes involve long-distance cell-cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs.

Enhanced enteric neurogenesis by Schwann cell precursors in mouse models of Hirschsprung disease.

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.

Increased RET Activity Coupled with a Reduction in the Gene Dosage Causes Intestinal Aganglionosis in Mice.

Mutations of the gene encoding the 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 gene have adverse effects on ENS development We addressed this issue by examining mice engineered to express RET51(C618F), an activating mutation identified in MTC patients.

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene.

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated.

Generation of conditional ALK F1174L mutant mouse models for the study of neuroblastoma pathogenesis.

Neuroblastoma, an embryonal tumor arising from the sympathetic ganglia and adrenal medulla, is among the most intractable pediatric cancers. Although a variety of genetic changes have been identified in neuroblastoma, how they contribute to its pathogenesis remains largely unclear. Recent studies have identified alterations of the anaplastic lymphoma kinase (ALK) gene in neuroblastoma; ALK F1174L (a phenylalanine-to-leucine substitution at codon 1174) represents one of the most frequent of these somatic mutations, and is associated with amplification of the MYCN gene, the most reliable marker for the poor survival.

Technologies for Live Imaging of Enteric Neural Crest-Derived Cells.

Time-lapse imaging of gut explants from embryonic mice in which neural crest-derived cells express fluorescent proteins allows the behavior of enteric neural crest cells to be observed and analyzed. Explants of embryonic gut are dissected, mounted on filter paper supports so the gut retains its tubular three-dimensional structure, and then placed in coverglass bottom culture dishes in tissue culture medium. A stainless steel ring is placed on top of the filter support to prevent movement.

Mice conditionally expressing RET(C618F) mutation display C cell hyperplasia and hyperganglionosis of the enteric nervous system.

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET.

Catenins Steer Cell Migration via Stabilization of Front-Rear Polarity.

Cell migration plays a pivotal role in morphogenetic and pathogenetic processes. To achieve directional migration, cells must establish a front-to-rear axis of polarity. Here we show that components of the cadherin-catenin complex function to stabilize this front-rear polarity.

Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest.

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations.

Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on the brain dopamine system are controversial and not fully defined. To address this question, we analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control of the glial Gfap promoter.

Retrograde transport of neurotrophic factor signaling: implications in neuronal development and pathogenesis.

Neurotrophic factors and their receptors play a central role in neuronal survival. Since neurons have a highly polarized morphology, target-derived neurotrophic factor signaling is transported retrogradely along the axon to the cell body. A body of evidence suggests that retrograde transport of the neurotrophic factors and their receptors is required for signal propagation.

Development of the intrinsic and extrinsic innervation of the gut.

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.

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System.

Unlabelled: Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear.

The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function.

Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand. However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown.

Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states.

The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing "As model," stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis.

A promoter-level mammalian expression atlas.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles.

Colonizing while migrating: how do individual enteric neural crest cells behave?

Background: Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance.

GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors.

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development; however, the mechanisms by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop by radial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting neurotrophic factor for ENS development in a mouse model.

Tissue interactions in neural crest cell development and disease.

The neural crest is a transient population of migratory cells in the embryo that gives rise to a wide variety of different cell types, including those of the peripheral nervous system. Dysfunction of neural crest cells (NCCs) is associated with multiple diseases, such as neuroblastoma and Hirschsprung disease. Recent studies have identified NCC behaviors during their migration and differentiation, with implications for their contributions to development and disease.

Proliferation and cell cycle dynamics in the developing stellate ganglion.

Cell proliferation during nervous system development is poorly understood outside the mouse neocortex. We measured cell cycle dynamics in the embryonic mouse sympathetic stellate ganglion, where neuroblasts continue to proliferate following neuronal differentiation. At embryonic day (E) 9.

GDNF and endothelin 3 regulate migration of enteric neural crest-derived cells via protein kinase A and Rac1.

Enteric neural crest-derived cells (ENCCs) migrate from the anterior foregut in a rostrocaudal direction to colonize the entire gastrointestinal tract and to form the enteric nervous system. Genetic approaches have identified many signaling molecules regulating the migration of ENCCs; however, it remains elusive how the activities of the signaling molecules are regulated spatiotemporally during migration. In this study, transgenic mice expressing biosensors based on Förster resonance energy transfer were generated to video the activity changes of the signaling molecules in migrating ENCCs.