The Nf2 tumor suppressor regulates cell-cell adhesion during tissue fusion.

Tissue fusion, the morphogenic process by which epithelial sheets are drawn together and sealed, has been extensively studied in Drosophila. However, there are unique features of mammalian tissue fusion that remain poorly understood. Notably, detachment and apoptosis occur at the leading front in mammals but not in invertebrates. We found that in the mouse embryo, expression of the Nf2 tumor suppressor, merlin, is dynamically regulated during tissue fusion: Nf2 expression is low at the leading front before fusion and high across the fused tissue bridge. Mosaic Nf2 mutants exhibit a global defect in tissue fusion characterized by ectopic detachment and increased detachment-induced apoptosis (anoikis). By contrast with core components of the junctional complex, we find that merlin is required specifically for the assembly but not the maintenance of the junctional complex. Our work reveals that regulation of Nf2 expression is a previously unrecognized means of controlling adhesion at the leading front, thereby ensuring successful tissue fusion.

Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system.

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells.

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.

Hirschsprung disease is linked to defects in neural crest stem cell function.

Genes associated with Hirschsprung disease, a failure to form enteric ganglia in the hindgut, were highly up-regulated in gut neural crest stem cells relative to whole-fetus RNA. One of these genes, the glial cell line-derived neurotrophic factor (GDNF) receptor Ret, was necessary for neural crest stem cell migration in the gut. GDNF promoted the migration of neural crest stem cells in culture but did not affect their survival or proliferation. Gene expression profiling, combined with reverse genetics and analyses of stem cell function, suggests that Hirschsprung disease is caused by defects in neural crest stem cell function.

Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity.

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.

Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness.

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.

Brain repair by endogenous progenitors.

Stem cells within the adult brain can be stimulated by injury and growth factor treatment to replace damaged neurons, even neurons that are not normally generated in adults. Coupled with recent insights into the mechanism by which Nogo inhibits axonal regeneration, this discovery may inspire new treatments for central nervous system injuries and neurodegenerative diseases.