In order to characterize these cells, we took advantage of a Hes5-CreERT2.R26tdTomato reporter mouse line to carry out inducible genetic lineage tracing of Hes5+ cells in the ENS. Administration of a single dose of tamoxifen (TM) to adult Hes5-CreERT2.R26tdTomato animals resulted in appearance shortly afterward (3 days) of tdTomato+ intra-ganglionic glial cells also known as type I glial cells. The Hes5+ cells are part of the Enteric Glial Cell (EGC) population as they co-express the neural crest (NC) lineage marker Sox10 and the canonical glial cell marker S100b. Following a 4-week chase was observed an increase in the average size of the tdTomato+ cell clusters, which also included cells expressing the pan-neuronal marker HuC/D. These findings suggested that under physiological conditions the ganglia of the mammalian myenteric plexus include a cell population that undergoes proliferation at a low rate and is capable of generating enteric neurons. Nevertheless, same experiments shown highly variability in the cluster size between time points whereas neuronal-like tdTomato+ numbers appeared to be consistently higher over time.
Following these set of experiments these animals were treated with EdU in the drinking water in order to address proliferation. EdU is a thymidine analogue that is incorporated in the cells while they are synthetizing their DNA. Surprisingly, a low number of Hes5+ cells were EdU positive meaning that not all of them were undergoing cell division. These results showed a low proliferative capacity of the entire Sox10+ glial cells not only restricted to the Hes5+ population. Furthermore, we failed to find any EdU+ tdTomato+ neuron.
To test proliferation under injury conditions, mice were treated with Dextran Sodium Sulfate (DSS) to induce colitis. DSS is a chemical agent that induces intestinal inflammation by damaging the epithelial layer mostly of the large intestine allowing the dissemination of bacteria into other tissues. Colon tissues were isolated after approximately 3 weeks of DSS treatment. After exhaustive counting of Sox10+ S100+ EdU+ glial cells, we can conclude that there are no significative differences in terms of cell proliferation after inflammation.
To address the importance of Notch signaling pathway in glial cell proliferation and neuronal regeneration our next aim was to study the behavior of the ENS in the absence of Notch signaling. As hes5 is a target of Notch we deleted RBPJk (a key downstream component of the canonical Notch signaling pathway) specifically in the Sox10+ glial cells. For this purpose, we generated the following mouse line: Sox10ERT2.tdTomato x RBPjk flox. In line with previous experiments, EdU was administrated in the drinking water, however, no differences in terms of proliferation or neuronal generation were observed compared with their littermate controls. All analysis was done using confocal imaging.
We were able to identify Hes5 gene expression patterns in existing data from the lab. Our work was completely disrupted in terms of animals and The Francis Crick facilities were not available during the pandemic. All of these together generated a delay in the implementation of IDENSTEM as it was originally planned. All necessary approvals in terms of animal experimentation were obtained, however, meeting all deadlines was not possible.
