Study On The Fate Decision Mechanism Of Drosophila Intestinal Stem Cells During Differentiation

The adult stem cells are responsible for maintenance of many adult tissues.These multipotent cells constantly divide and produce daughter cells,which,through a process called differentiation,become multiple types of mature cells.Scientifically,it's unclear how these diverse distinct cell types are specified from common stem cell pools.To study this question,we investigated lineage commitment of adult intestinal stem cell(ISC)in Drosophila,in which the Delta-Notch-signaling-guided progenitor cell differentiation into enterocytes is the default mode,while the process of differentiation of enteroendocrine cells(EE),which usually produces EE pairs,is less understood.Previous studies showed acheate-scute complex(As-c)genes promote EE production by inducing the transcription factor Prospero(Pros),which acts as an EE-fate-determination factor.Among As-c genes,scute(sc)is both necessary and sufficient for EE specification.Nevertheless,important questions remain about both the molecular and cellular mechanisms through which Sc functions in EE fate decision,and how Sc is regulated in ISCs to control EE specification,remain to be addressed.In this study,we found specification of EEs is initiated by the transient upregulation of Sc expression in ISCs.Based on the finding that Sc is required for EE generation from ISCs,we temporally knocked down sc starting from the pupal stage,and this process produced flies with midguts lacking EE cells.We used these EE-less midguts to examine the process of EE production by using temperature shift to re-introduce Sc expression in the midgut.With this assay,we discovered that(i)ISCs actually undergo an initial division to generate a new EE progenitor cell reffered as EEP,and(ii)the EEP then undergoes one final round of cell division prior to its terminal differentiation,yielding an EE pair..To further analyze this two-step cell division process,weconditionally induced sc expression in ISCs and monitored the cellular events in a time lapse.This transient sc induction caused a rapid cell division response,revealing that Sc is a potent mitogenic factor.Sc also induced expression of the EE-marker gene Pros,which is,however,a potent cell-cycle inhibitor.These properties enable us to precisely define the regulatory circuitry that directs the formation of a pair of EEs from each ISC via two stepwise cell divisions:an asymmetric division of ISC to generate an EEP,followed by EEP division to produce the EE pair.Next,to visualize the expression of Sc in midgut,we generated a green fluorescent protein(GFP)knock-in line for Sc with CRISPR-Cas9 technique.Sc-GFP is dynamic expression in ISCs,with a weak expression level in most ISCs,and increased expression levels in?15%of the ISCs(Figure 2).We also identified one GMR enhancer-GAL4 lines generated for sc,which drives GFP expression in small cell nests of 2-3 cells that contains ISCs and newly formed EEs.Follow-up cell lineage tracing studies with this Sc-Gal4 line revealed that the immediate daughter cells of Sc-GAL4+ ISCs were mainly EEs;however,these ISCs ressume their default EC fate once Sc expression is downregulated.Mechanistically,it is intriguing how such transient upregulation of Sc in ISCs occurs.By combining genetic assays,RNA-seq analysis,ChIP-seq,and targeted DamID analysis,we established two feedback regulatory loops control the transient upregulation of Sc in ISCs prior to EE fate commitment:i)a transcriptional self-stimulation loop that allows Sc to gradually build up and eventually reach a high level to induce EEP specification;ii)a negative feedback regulation loop between Sc and enhancer of split complex(E(spl))genes that returns sc expression back to the baseline level.Given that negative feedback is a common mechanism underlying biochemical oscillations,these feedback interplays could plausibly drive oscillatory expression pattern for Sc in ISCs,which could potentially serve as an internal timer for periodic production of EEs from ISCs.Future cellular and molecular analysis,in combination with in vivo live imaging work will allow further testing and refining of this oscillation model,and to determine whether and how this internal timer is regulated by certain endogenous and/or environmental cues.