Cellular mechanisms that spatiotemporally direct neural crest cell migration and enteric nerve system formation

Abstract

The enteric nervous system (ENS) in the gastrointestinal tract is a complex nervous network. It is essential for gut secretion, absorption and peristalsis. Most of the ENS arises from vagal neural crest cells (NCCs) which migrate from the neural tube (level somite s1 to s7) into the foregut then colonize the rest of the intestine as a rostro-caudal wave and form the ENS. If this cell migration fails to be completed, the distal intestine lacks ENS, this results in Hirschsprung Disease (HSCR). My research project is going to answer: Do all levels of vagal NCC contribute to ENS equally? What is the enteric NCCs population expansion potential and how do individual initial enteric NCCs contribute to the final ENS? Does the ENS population retain its colonization capacity during development? How do enteric NC/glia cells and neurons interact in directing ENS cell invasion and axon extension? What is the mechanism that control ENS ganglion formation, especially the role of differential adhesion of enteric neurons and NCCs on gangliogenesis?

This thesis using avian models combined with multiple techniques demonstrated that: The vagal NCCs of single somite levels origins migrate separately and converge at the foregut, where they all mixed. Along the whole vagal length, the mid-vagal region NC-derived cells -- that is s3 and s4 level -- arrive earliest at the foregut, and contribute the greatest numbers. During colonization along the gut, all levels of vagal NC-derived cells are mixed throughout the entire gut, but mid-vagal NC-derived cells contribute to all region of gut from foregut to distal hindgut in greatest numbers. Regarding the ENS forming potential, all levels of vagal NCC could form ENS, but mid-vagal NCCs (s3 and s4) have greatest competency.

The temporal expansion of the enteric NC population in developing quail gut was investigated from E2.5 to E12, and the expansion potential was challenged by drastically reducing the starting enteric NCC numbers. By this means, the enteric NCCs showed an extremely high capacity to regulate their proliferation while forming the ENS. However, single cell lineage tracing technique developed in this thesis demonstrated that the contribution of individual enteric NCC to final ENS was unequal and unpredictabe, a few dominant ENS “superstar” cell clones emerge but most clones are small. These “superstar” clones are not predetermined, instead they achieve this status stochastically.

The extremely high gut-colonization capacity of enteric NCCs from the early embryo gut was found to rapidly decline with embryonic age, and this declines was more rapid in the distal intestine-derived enteric NCCs than in more proximal enteric NCCs. This age-related loss in colonization capacity involves changes in the ENS cell population, rather than the maturity of the mesoderm. However, it is not caused by reducing the “undifferentiated” ENS cell number, since this capacity can neither be rescued with more time to catch up, nor mimicked by reducing ENS cell number from young donor. Actually, unlike their proportion in the ENS, the absolute number of apparently undifferentiated enteric NCCs does not decline with age. Therefore, this age-related loss in colonization capacity is caused by changes in qualitative aspects with age.

The early development of the ENS shows two invasive events: rostral-to-caudal invasion by enteric NCCs and extension of ENS axons. Experimentally, the gut mesoderm supports these two invasive events for longer than the normal time-window. Both these events can occur bidirectionally when permitted, although normally they are unidirectional. However, a fresh invasion wave of enteric NCCs is prevent by pre-existing ENS cells. This prevention of invasion does not occur in gut with pre-existing ENS neurons and axons but without enteric NCCs. Therefore the conclusion is that this prevention is caused by the pre-existing enteric NCCs and not by other ENS components. In the other hand, pre-existing ENS neurons and axons inhibit neuronal differentiation of the invading enteric NCCs. In contrast, ENS axon invasion is not prevented by pre-existing ENS cells and axons. These invasive axons do not use pre-existing ENS axon tracts or ENS neurons to facilitate invasion of the gut, but have an absolute requirement for enteric NCC (either in situ or co-invading) in order to advance into gut mesoderm.

Follow these colonizing aspects of ENS development, the ratio of enteric NCC to neuron is stabilized and ENS ganglia are formed with a core of neurons and a shell of enteric NC/glia cells. Using cell-cell aggregation assays this thesis revealed that during aggregation, both Ca+2-dependent and independent adhesion mechanisms are required. Neurons sorted to the core of aggregates, surrounded by outer enteric NCCs, showing that neurons had higher adhesion than enteric NCCs. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM (Ca+2–independent) and N-cadherin (Ca+2–dependent) on this surface of the outer non-neuronal enteric NCCs. In addition, the ganglion size is intrinsically regulated by the ratio of enteric NCCs to neurons likely by generation of an outer non-adhesive surface.

Overall, my research covers almost the entire process of the ENS development from initiating of NC on its origin site to the colonization of enteric NCC along the entire gut, and the final steps of ENS ganglion formation. The results from this thesis revealed many critical issues in understanding of the cellular mechanisms that control these processes. These basic researches have important implications for understanding not only ENS development but also enteric neuropathologies and for designing NC stem cell therapies.