Neurotrophin-3 Is Required for the Survival–Differentiation of Subsets of Developing Enteric Neurons

Abstract

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest-and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of 125 I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of 125 I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine ␤-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Bone Morphogenetic Protein-2 and -4 Limit the Number of Enteric Neurons But Promote Development of a TrkC-Expressing Neurotrophin-3-Dependent Subset

“…Immunoreactivity was used to detect markers that enabled cultured cells to be identified. Procedures used for the immunocytochemical detection of antigens in cultured cells were similar to those described in detail previously (Chalazonitis et al, 1994(Chalazonitis et al, , 1997(Chalazonitis et al, , 1998a(Chalazonitis et al, , 2001. Briefly, cultures were fixed for 1 hr with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 mM PBS.…”

“…Apoptotic TrkC-immunoreactive cells were identified by using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated UTP nick end labeling (TUNEL) method, which was applied after the immunocytochemical demonstration of TrkC (Chalazonitis et al, 2001). A commercial kit (TACS; TdT in situ apoptosis detection kit; R & D Systems) was used for TUNEL staining according to the directions of the manufacturer.…”

“…Methods are similar to those described previously (Chalazonitis et al, 2001). Briefly, fetal mouse gut (E18) and bowel from 4-week-old mice were fixed with 4% formaldehyde (from paraformaldehyde), pH 7.4, and dissected to obtain laminar preparations of the gut wall.…”

“…Neurotrophin-3 (NT-3) also enhances the development of enteric neurons in vitro (Chalazonitis et al, 1994) and is required in vivo for the survival of the subset of enteric neurons that expresses TrkC (Chalazonitis et al, 2001); however, NT-3 responsiveness does not begin until E14 (Chalazonitis et al, 1998b). NT-3-dependent enteric neurons are important because they include the submucosal intrinsic primary afferent neurons (Chalazonitis et al, 2001) that enable the ENS to mediate peri-staltic and secretory reflexes (Cooke, 2000;Pan and Gershon, 2000). Both the requirements of enteric crest-derived cells for growth factors and their responsiveness to such factors are thus developmentally regulated.…”

Differential responses to the application of exogenous NT‐3 are observed for subpopulations of motor and sensory neurons depending on the presence of skeletal muscle

“…TrkC is the only Trk receptor expressed in the large proprioceptive neurons of the DRG by E9.5 and is expressed throughout the CNS by E11.5 (Barbacid, 1994) and, along with TrkB, is expressed in SC MNs by E13.5 (Yan et al, 1993). Importantly, retrograde labeling has enabled investigators to confirm that both adult and developing MNs transport NT-3 from the target muscle to the cell body of respective neurons, in vivo (Yan et al, 1993, Chalazonitis et al, 2001). The results of several studies suggest that both neurotrophin and Trk receptor are transported, by means of endocytosis, to the cell body and that chemical signals may in fact be exerting an effect before the neurotrophin (or NT/receptor complex) arrives to the cell body.…”