Expression and function of cell adhesion molecules during neural crest migration.

Neural crest cells are highly migratory cells that give rise to many derivatives including peripheral ganglia, craniofacial structures and melanocytes. Neural crest cells migrate along defined pathways to their target sites, interacting with each other and their environment as they migrate. Cell adhesion molecules are critical during this process. In this review we discuss the expression and function of cell adhesion molecules during the process of neural crest migration, in particular cadherins, integrins, members of the immunoglobulin superfamily of cell adhesion molecules, and the proteolytic enzymes that cleave these cell adhesion molecules. The expression and function of these cell adhesion molecules and proteases are compared across neural crest emigrating from different axial levels, and across different species of vertebrates.

Genetic interactions and modifier genes in Hirschsprung's disease.

Hirschsprung's disease is a congenital disorder that occurs in 1:5000 live births. It is characterised by an absence of enteric neurons along a variable region of the gastrointestinal tract. Hirschsprung's disease is classified as a multigenic disorder, because the same phenotype is associated with mutations in multiple distinct genes. Furthermore, the genetics of Hirschsprung's disease are highly complex and not strictly Mendelian. The phenotypic variability and incomplete penetrance observed in Hirschsprung's disease also suggests the involvement of modifier genes. Here, we summarise the current knowledge of the genetics underlying Hirschsprung's disease based on human and animal studies, focusing on the principal causative genes, their interactions, and the role of modifier genes.

L1cam acts as a modifier gene during enteric nervous system development.

The enteric nervous system is derived from neural crest cells that migrate from the caudal hindbrain and colonise the gut. Failure of neural crest cells to fully colonise the gut results in an "aganglionic zone" that lacks a functional enteric nervous system over a variable length of the distal bowel, a condition in human infants known as Hirschsprung's disease. The variability observed in the penetrance and severity of Hirschsprung's disease suggests a role for modifier genes. Clinical studies have identified a population of Hirschsprung's patients with mutations in L1CAM that also have a common polymorphism in RET, suggesting a possible interaction between L1CAM and RET. Therefore, we examined whether L1cam could interact with Ret, its ligand Gdnf, and a known transcriptional activator of Ret, Sox10. Using a two-locus complementation approach, we show that loss of L1cam in conjunction with a heterozygous loss of Ret or Gdnf did not result in aganglionosis. However, L1cam did interact with Sox10 to significantly increase the incidence of aganglionosis. We show that an interaction between L1cam and Sox10 significantly perturbs neural crest migration within the developing gut, and that neural crest cells undergo excessive cell death prior to gut entry. Finally, we show that Sox10 can regulate the expression of L1cam. Thus, L1cam can act as a modifier gene for the HSCR associated gene, Sox10, and is likely to play a role in the etiology of Hirschsprung's disease.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: time course and molecular characterization.

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin(+) and calcitonin gene-related peptide (CGRP)(+) sprouts peaking at day 14, matching both large, neuropeptide(+) subpopulations of axotomized facial motoneurons, but with CGRP(+) sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin(+) sprouts revealed a stronger response following cut compared with crush (day 7-14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12-48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting.

Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation.

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.

The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells.

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.

Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract.

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4-14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2-3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of alpha-smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of alpha-smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.