Lineage analysis and identification of novel genes involved in differentiation processes in the zebrafish enteric nervous system
Developmental Biology
Final Report Abstract
The enteric nervous system (ENS) is the most complex part of the peripheral nervous system. It regulates essential gut functions such as motility, secretion, and homeostasis, and is composed of diverse neuronal subtypes and glia. Abnormal ENS development causes well-studied human diseases, for example Hirschsprung disease (HSCR), in which caudal gut is uninnervated and nonmotile. We have a relatively good understanding of early ENS development, including how ENS progenitors navigate to the gut. However, little is known about later events of ENS development, particularly the coordinated differentiation of enteric neurons and glia and the generation of appropriate numbers of different neuronal subtypes and glia, all of which are necessary to ensure proper ENS functionality. The overall goal of this project was to gain new insights into the developmental potential of ENS progenitors, including underlying genetic mechanisms. We generated several new tools for lineage tracing of specific enteric progenitor subpopulations using BAC-recombineering. We used a transgenic line we generated to characterize glial development in zebrafish for the first time and found that enteric glia develop at about the same time as enteric neurons. We showed that glia have dynamic behavior with respect to developing a network of processes and have diverse morphologies similar to those found in mammals. To find new regulators of ENS development, we identified zebrafish mutants "b1074" and "b1088" from a genetic screen based on a reduction in ENS neurons. We mapped both mutants to chromosomal intervals that do not contain genes known to be involved in ENS development. For “b1074” mutants, we found that, similar to human HSCR patients, there is a variable reduction of ENS neurons caudally and a milder reduction rostrally. They also have fewer enteric glia. Our "in vivo" imaging and analysis of enteric progenitor position along the gut at different developmental time points revealed that there is a significant delay of "phox2b+" ENS progenitor migration in "b1074" mutants compared to wildtype siblings. For "b1088" mutants, we found a strong reduction in the number of ENS neurons caudally, similar to human HSCR patients. Two main neuronal subtypes, nNOS+ neurons and 5HT+ neurons are both significantly affected in this mutant. Both zebrafish mutants will provide exciting new information about genes involved in regulating development of the ENS and should reveal novel genes involved in HSCR.
