Investigating the transcriptional heterogeneity of the beta cells experiencing Ca2+ excitotoxicity

Beta cells are remarkably heterogeneous. However, beta cells response to chronic stress at a single cell level remains largely unknown. Here, we used a chemo-genetic model of beta cell excitotoxcity coupled with single cell RNA sequencing to understand beta cells response to chronic stress. Remarkably, we found that a subset of stressed beta cells progressed towards death while the other group evaded the destruction by dedifferentiation. Vulnerable beta cells that progressed towards death showed upregulation of genes implicated in apoptosis, inflammation and immune system activation. The resilient beta cells that evaded the stress showed increased expression aldh1a3, a canonical marker of mammalian beta cell dedifferentiation. We used three different treatment groups for this study. Tg(ins:Kaede) larvae (n=20) treated with capsaicin (csn) for 48 hours i.e. from 3 to 5 days post fertilization (dpf) were used as control. Tg(ins:TRPV);Tg(ins:Kaede) larvae (n=20) treated with csn for 24 hours i.e. from 4 to 5 dpf were used to identify genes involved in immediate stress response and Tg(ins:TRPV);Tg(ins:Kaede) larvae (n=20) treated with csn for 48 hours i.e. from 3 to 5 dpf were used to identify the genes involved in chronic stress response. Post csn treatment, pancreatic islets from the larvae were manually dissected and then subsequently dissociated via enzymatic digestion. Post dissociation the beta cells were stained with calcein (viability marker dye) and FACS sorted. Individual beta cells were collected in a 384 well plate and their transcriptomes were profiled using smart seq2. RNA_01_H19, RNA_02_G11, RNA_03_P18 do not correspond to the other samples (eg, Ctrl, CNS24, CNS48-2).