Genetic Background Influences Severity of Colonic Aganglionosis and Response to GDNF Enemas in the Holstein Mouse Model of Hirschsprung Disease.

Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon.

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.