Cell Polarity Protein Miranda Precisely Determines The Intestinal Stem Cell Number By Controlling Threshold Of Prospero

Objective:Control of stem cell number is central to understand tissue homeostasis,cancer and aging during individual development.Drosophila pupal intestinal stem cell asymmetrically divides first,then symmetrically divides again,generating one pair of stem cells and one pair of enteroendocrine?ee?cells,forming 1200 stem cells before eclosion.We found that the cell polarity protein Mira segregates ee transcription factor Pros into the basal daughter cell during asymmetric divisions.However,Mira expression is absent during symmetric division and as a consequence Pros symmetrically distributes into two daughter cells.Therefore,Mira is the key factor to determine stem cell number.We studied the molecular mechanisms of stem cell number control by overexpressing or knocking out mira in the intestinal stem cell lineage.Methods:?1?We used esg^ts to overexpress Mira in the intestinal stem cell lineage during pupal stage and counted the number of stem cells and ee cells.?2?The molecular mechanism of overexpressing Mira was revealed by the immunostaining of Mira-GFP,Pros and Bazooka?Baz?.?3?We used esg^ts to knock down mira in the intestinal stem cell lineage during pupae stage and counted the number of stem cells and ee cells.?4?We used the MARCM clone technique to tracking the intestinal stem cell lineage when overexpressing or knocking out mira.?5?The molecular mechanism of knocking out mira was revealed by analysing the fluorescence intensity of Pros in each type of cell and the staining of Baz.?6?We used esg^ts to drive different RNAi to block cell cycle in different checkpoints to confirm the expression period of Pros and Mira.?7?We used esg^ts to overexpress or knock down pros and to overexpress Asense in intestinal stem cells to study the regulatory mechanism of Mira and Pros.Results:?1?Overexpression of Mira in the intestinal stem cell lineage results in a doubled number of stem cells and ee cell number is not affected.?2?Overexpression of Mira in the intestinal stem cell lineage results in loss of polarity of stem cells.?3?Knockout of mira in the intestinal stem cell lineage results in a doubled number of stem cells and ee cell number is not affected.?4?The differentiation of intestinal stem cell progeny cells into ee cells depends on the threshold distribution of Pros.?5?Pros and Mira are expressed in the G2 phase,and Pros is expressed earlier than Mira.?6?Ac/Sc regulates the expression of Pros and Mira,and Pros inhibits Mira.Conclusion:Cell polarity protein Mira affects the distribution of Pros in progeny cells and precisely regulates the number of intestinal stem cells together with the top polarity protein Baz.Overexpression or knockout of mira in the intestinal stem cell lineage results in a doubled number of stem cells and ee cell number is not affected.