Fine scale differences within the vagal neural crest for enteric nervous system formation.

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1-7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions.

Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca (2+) -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface.

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.

BACKGROUND: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. RESULTS: Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. CONCLUSIONS: Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.

The quail anatomy portal.

The Japanese quail is a widely used model organism for the study of embryonic development; however, anatomical resources are lacking. The Quail Anatomy Portal (QAP) provides 22 detailed three-dimensional (3D) models of quail embryos during development from embryonic day (E)1 to E15 generated using optical projection tomography. The 3D models provided can be virtually sectioned to investigate anatomy. Furthermore, using the 3D nature of the models, we have generated a tool to assist in the staging of quail samples. Volume renderings of each stage are provided and can be rotated to allow visualization from multiple angles allowing easy comparison of features both between stages in the database and between images or samples in the laboratory. The use of JavaScript, PHP and HTML ensure the database is accessible to users across different operating systems, including mobile devices, facilitating its use in the laboratory.The QAP provides a unique resource for researchers using the quail model. The ability to virtually section anatomical models throughout development provides the opportunity for researchers to virtually dissect the quail and also provides a valuable tool for the education of students and researchers new to the field. DATABASE URL: http://quail.anatomyportal.org (For review username: demo, password: quail123).

Retinoic acid upregulates ret and induces chain migration and population expansion in vagal neural crest cells to colonise the embryonic gut.

Vagal neural crest cells (VNCCs) arise in the hindbrain, and at (avian) embryonic day (E) 1.5 commence migration through paraxial tissues to reach the foregut as chains of cells 1-2 days later. They then colonise the rest of the gut in a rostrocaudal wave. The chains of migrating cells later resolve into the ganglia of the enteric nervous system. In organ culture, E4.5 VNCCs resident in the gut (termed enteric or ENCC) which have previously encountered vagal paraxial tissues, rapidly colonised aneural gut tissue in large numbers as chains of cells. Within the same timeframe, E1.5 VNCCs not previously exposed to paraxial tissues provided very few cells that entered the gut mesenchyme, and these never formed chains, despite their ability to migrate in paraxial tissue and in conventional cell culture. Exposing VNCCs in vitro to paraxial tissue normally encountered en route to the foregut conferred enteric migratory ability. VNCC after passage through paraxial tissue developed elements of retinoic acid signalling such as Retinoic Acid Binding Protein 1 expression. The paraxial tissue's ability to promote gut colonisation was reproduced by the addition of retinoic acid, or the synthetic retinoid Am80, to VNCCs (but not to trunk NCCs) in organ culture. The retinoic acid receptor antagonist CD 2665 strongly reduced enteric colonisation by E1.5 VNCC and E4.5 ENCCs, at a concentration suggesting RARα signalling. By FACS analysis, retinoic acid application to vagal neural tube and NCCs in vitro upregulated Ret; a Glial-derived-neurotrophic-factor receptor expressed by ENCCs which is necessary for normal enteric colonisation. This shows that early VNCC, although migratory, are incapable of migrating in appropriate chains in gut mesenchyme, but can be primed for this by retinoic acid. This is the first instance of the characteristic form of NCC migration, chain migration, being attributed to the application of a morphogen.

Spatial analysis of multi-species exclusion processes: application to neural crest cell migration in the embryonic gut.

Hindbrain (vagal) neural crest cells become relatively uniformly distributed along the embryonic intestine during the rostral to caudal colonization wave which forms the enteric nervous system (ENS). When vagal neural crest cells are labeled before migration in avian embryos by in ovo electroporation, the distribution of labeled neural crest cells in the ENS varies vastly. In some cases, the labeled neural crest cells appear evenly distributed and interspersed with unlabeled neural crest cells along the entire intestine. However, in most specimens, labeled cells occur in relatively discrete patches of varying position, area, and cell number. To determine reasons for these differences, we use a discrete cellular automata (CA) model incorporating the underlying cellular processes of neural crest cell movement and proliferation on a growing domain, representing the elongation of the intestine during development. We use multi-species CA agents corresponding to labeled and unlabeled neural crest cells. The spatial distributions of the CA agents are quantified in terms of an index. This investigation suggests that (i) the percentage of the initial neural crest cell population that is labeled and (ii) the ratio of cell proliferation to motility are the two key parameters producing the extreme differences in spatial distributions observed in avian embryos.