Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS.Bevan L. Cheeseman, Dongcheng Zhang, Benjamin J. Binder, Donald F. Newgreen and Kerry A. Landma

Divergent roles for Eph and Ephrin in Avian Cranial Neural Crest

<p>Abstract</p> <p>Background</p> <p>As in other vertebrates, avian hindbrain neural crest migrates in streams to specific branchial arches. Signalling from Eph receptors and ephrins has been proposed to provide a molecular mechanism that guides the cells restricting them to streams. In mice and frogs, cranial neural crest express a combination of Eph receptors and ephrins that appear to exclude cells from adjacent tissues by forward and reverse signalling. The objective of this study was to provide comparative data on the distribution and function of Eph receptors and ephrins in avian embryos.</p> <p>Results</p> <p>To distinguish neural crest from bordering ectoderm and head mesenchyme, we have co-labelled embryos for Eph or ephrin RNA and a neural crest marker protein. Throughout their migration avian cranial neural crest cells express EphA3, EphA4, EphA7, EphB1, and EphB3 and move along pathways bordered by non-neural crest cells expressing ephrin-B1. In addition, avian cranial neural crest cells express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Thus, the distribution of avian Eph receptors and ephrins differs from those reported in other vertebrates. In stripe assays when explanted cranial neural crest were given the choice between FN or FN plus clustered ephrin-B1 or EphB2 fusion protein, the cells strongly localize to lanes containing only FN. This preference is mitigated in the presence of soluble ephrin-B1 or EphB2 fusion protein.</p> <p>Conclusion</p> <p>These findings show that avian cranial neural crest use Eph and ephrin receptors as other vertebrates in guiding migration. However, the Eph receptors are expressed in different combinations by neural crest destined for each branchial arch and ephrin-B1 and ephrin-B2 appear to have opposite roles to those reported to guide cranial neural crest migration in mice. Unlike many of the signalling, specification, and effector pathways of neural crest, the roles of Eph receptors and ephrins have not been rigorously conserved. This suggests diversification of receptor and ligand expression is less constrained, possibly by promiscuous binding and use of common downstream pathways.</p

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

<p>Abstract</p> <p>Background</p> <p>A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.</p> <p>Methods</p> <p>PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.</p> <p>Results</p> <p>When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.</p> <p>Conclusion</p> <p>ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.</p

Genetic Background Strongly Modifies the Severity of Symptoms of Hirschsprung Disease, but Not Hearing Loss in Rats Carrying Ednrbsl Mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrbsl mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome

Chemotactic Cellular Migration: Smooth and Discontinuous Travelling Wave Solutions

A simple model of chemotactic cell migration gives rise to travelling wave solutions. By varying the cellular growth rate and chemoattractant production rate, travelling waves with both smooth and discontinuous fronts are found using phase plane analysis. The phase plane exhibits a curve of singularities whose position relative to the equilibrium points in the phase plane determines the nature of the heteroclinic orbits, where they exist. Smooth solutions have trajectories connecting the steady states lying to one side of the singular curve. Travelling shock waves arise by connecting trajectories passing through a special point in the singular curve and recrossing the singular curve, by way of a iscontinuity. Hyperbolic partial differential equation theory gives the necessary shock condition. Conditions on the parameter values determine when the solutions are smooth travelling waves versus discontinuous travelling wave solutions. These conditions provide bounds on the travelling wave speeds, corresponding to bounds on the chemotactic velocity or bounds on cellular growth rate. This analysis gives rise to the possibility of representing sharp fronts to waves of invading cells through a simple chemotactic term, without introducing a nonlinear diffusion term. This is more appropriate when cell populations are sufficiently dense

Data for: Stochastic clonal expansion of “superstars” enhances the reserve capacity of enteric nervous system precursor cells.

Files of counts of cells in the enteric nervous system in normal growth and in CAM grafts

Physical influences on neural crest cell migration in avian embryos: contact guidance and spatial restriction

Several ideas on how neural crest (NC) cell migration in bird embryos might be dependent on the physical qualities of the internal embryonic environment were studied. Contact guidance has been suggested to direct NC cells ventrally in the trunk, but this has been subject to doubt (see Newgreen and Erickson, 1986, Int. Rev. Cytol. 103, 118-119). On reexamination, in situ extracellular matrix (ECM) and cell processes on the medial face of the somites were found appropriately oriented for this function. In addition, tissue culture models of oriented ECM could induce orientation of NC cells which mimicked that observed in the embryo. It is concluded that in this situation, oriented structures contribute to directed migration of NC cells in vivo, but the mechanism of contact guidance (i.e., steric or adhesive guidance) could not be ascertained. Contact guidance, in the form of steric guidance, has also been suggested as limiting ventrad NC cell movement at the midbrain level due to an insurmountable ridge on the side of the midbrain. The presence of this ridge was confirmed but it is unlikely to be responsible for prevention of ventrad migration, because, although it subsides very rapidly, the cells still refuse to move ventrad, and because models of this ridge in vitro proved to be no obstacle to NC cells. NC cell migration is also described as being limited by gross space between other organs or tissues. In vitro, NC cells could penetrate Nucleopore filters with pore diameters of 0.86 micron or greater. Observation of cell-free spaces in embryos showed that these were almost all much larger than the minimum pore size established experimentally. It is therefore concluded that in general the dimensions of gross tissue spaces probably do not set important limits for NC cell migration, but that the dimensions of transiently distensible microspaces between ECM fibrils may be a critical physical parameter

Control of the onset of migration of neural crest cells in avian embryos

To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of CA++ and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3-8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca++-and Mg++-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca++-free medium duplicated the effects of CMF, but neither Mg++-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane Ca++-channel antagonists D600 and Nifedipine, without any exernal Ca++-depletion. The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca++-depletion inactivates Ca++-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca++-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca++-channels suggest that Ca++ levels may be involved in regulating this system