Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy.

Midbrain, hindbrain and vagal neural crest (NC) produced abundant enteric nervous system (ENS) in co-grafted aneural hindgut and midgut, using chick-quail chorio-allantoic membrane grafts, forming complete myenteric and submucosal plexuses. This ability dropped suddenly in cervical and thoracic NC levels, furnishing an incomplete ENS in one or both plexuses. Typically, one plexus was favoured over the other. This deficiency was not caused by lower initial trunk NC number, yet overloading the initial number decreased the deficiency. No qualitative difference in neuronal and glial differentiation between cranial and trunk levels was observed. All levels formed HuC/D+ve, NOS+ve, ChAT+ve, and TH-ve enteric neurons with SoxE+ve, GFAP+ve, and BFABP+ve glial cells. We mathematically modelled a proliferative difference between NC populations, with a plexus preference hierarchy, in the context of intestinal growth. High proliferation achieved an outcome similar to cranial NC, while low proliferation described the trunk NC outcome of incomplete primary plexus and even more deficient secondary plexus. We conclude that cranial NC, relative to trunk NC, has a positionally-determined proliferation advantage favouring ENS formation. This has important implications for proposed NC stem cell therapy for Hirschsprung's disease, since such cells may need to be optimised for positional identity.

Role of Dlx genes in craniofacial morphogenesis: Dlx2 influences skeletal patterning by inducing ectomesenchymal aggregation in ovo.

Dlx homeodomain transcription factors are expressed in neural crest-derived mesenchyme of the pharyngeal arches and are required for patterning of the craniofacial skeleton. However, the cellular and molecular mechanisms by which Dlx factors control skeletogenesis in the facial primordia are unclear. We have investigated the function of Dlx2 and Dlx5 by sustained misexpression in ovo. We find that RCAS-Dlx2- and RCAS-Dlx5-infected avian embryos exhibit very similar patterns of local, stereotypical changes in skeletal development in the upper jaw. The changes include ectopic dermal bone along the jugal arch, and ectopic cartilages that develop between the quadrate and the trabecula. The ectopic cartilage associated with the trabecula is reminiscent of a normally occurring element in this region in some bird taxa. Analysis of the distribution of RCAS-Dlx2-infected cells suggests that Dlx2 induces aggregation of undifferentiated mesenchyme, which subsequently develops into the ectopic skeletal elements. Comparison of infected embryos with restricted or widespread misexpression, and of embryos in which Dlx genes were delivered to migratory or postmigratory neural crest, indicate that there are limited regions of competence in which the ectopic elements can arise. The site-specific differentiation program that the aggregates undergo may be dependent on local environmental signals. Our results suggest that Dlx factors mediate localization of ectomesenchymal subpopulations within the pharyngeal arches and in doing so define where skeletogenic condensations will arise. Consequently, variation in Dlx expression or activity may have influenced the morphology of jaw elements during vertebrate evolution.