Young Lab ~ Current Projects

Project 1 - The dynamic behaviour of neural crest-derived cells as they colonize the developing gut.

Investigators: Heather Young, Annette Bergner, Richard Anderson, Don Newgreen, Paul Whitington.

Neural crest cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastroin test inal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest cell migration in segments of embryonic gut in organ culture. These mice were generated by Drs Hideki Enomoto and Jeff Milbrandt at the Washington University School of Medicine, St Louis, USA.

Time-lapse imaging revealed that GFP+ neural crest cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells formed a scaffold along which later cells migrated, which is similar to axon tract formation. When a small number of the leading cells were isolated from the population behind them, they migrated slower, suggesting that population pressure in an important driving force for migration. The phenotype of migrating cells was examined. The migrating cells we examined expressed the neural crest cell marker, Sox10, but not neuronal markers.

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Project 2 - The phenotype and location of proliferating neural crest cells in the developing gut.

Investigators: Heather Young, Kirsty Turner, Annette Bergner

While they are migrating, neural crest-derived cells are also proliferating. However, it is unknown whether there are neural proliferative zones along the developing gut as there are in the CNS. We used a fluorescent nucleic acid stain to identify dividing cells or BrdU labeling (two hours after administration of BrdU to the mother), combined with neural crest-cell specific antibodies to determine the percentage of proliferating crest-derived cells in different gut regions of E11.5 and E12.5 mice.

The rate of proliferation of crest-derived cells did not vary significantly in different regions of the gut (including the caecum), or at different distances from the migratory wavefront of vagal crest-derived cells.

The phenotype of mitotic enteric crest-derived cells was also examined. Cells expressing the pan-neuronal markers, neurofilament-M and Hu, or the glial marker, S100b, were observed undergoing mitosis.

However, no evidence was found for proliferation of cells expressing neuron type-specific markers such as nitric oxide synthase (at E12.5) or calcitonin gene-related peptide (at E18.5). Thus for enteric neurons, exit from the cell cycle appears to occur after the expression of pan-neuronal proteins, but prior to the expression of markers of terminally differentiated neurons.

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Project 3 - Phenotype of pre-enteric vagal and sacral neural crest cells.

Investigators: Richard Anderson, Ashley Stewart, Heather Young

Cells derived from the vagal level neural crest migrate into the foregut.

The phenotype of neural crest cells destined to colonize the gut changes as they are migrating presumably due to intrinsic changes in the cells and response to cues they encounter in the environment. We investigated the order of expression of a number of markers expressed by enteric neural crest-derived cells, and found that Sox10 was expressed first, then p75, Phox2b, Ret and then Hu. Some cells derived from the sacral neural crest also enter the gut, but they do not appear to enter the hindgut until after the arrival of vagal cells. Our study showed that sacral cells must enter the hindgut during a narrow developmental window (E13.5-E15.5).

Many sacral cells outside of the distal hindgut had a different phenotype from crest cells within the hindgut.

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Project 4 - Factors regulating the migrating of cells through the hindgut.

Investigators: Heather Young, Annette Bergner, Richard Anderson

To examine whether the colonization of the hindgut might be influenced by gradients of molecules affecting migration along the hindgut, we set up co-cultures in which caeca from transgenic mice in which the neural crest cells express reporter genes (GFP or lacZ) were placed at both the rostral and caudal ends of segments of hindgut that had been removed prior to being colonized by neural crest cells. We then compared the rate that neural crest cells migrated rostrally or caudally through the hindgut explant.

Crest cells migrated caudally significantly faster than they migrated rostrally. This suggests that gradients do exist along the hindgut and that population pressure alone does not account for the colonization of the hindgut by neural crest cells.