Mechanism Of Signal Regulation Of Intestinal Development And Cell Lineage Pattern In The Hindgut Of Drosophila Melanogaster

Model organism Drosophila melanogaster is an important model for studying the mechanism of disease and organ development.Drosophila intestine is highly similar to human intestine.Taking Drosophila intestine as the research object can provide an important reference for revealing the mechanism of human intestinal disease occurrence and damage repair.Up to now,there have been many in-depth studies on the development mechanism of the midgut of Drosophila melanogaster,but relatively few studies on the hindgut of Drosophila melanogaster.What important signals regulate the formation of the hindgut of Drosophila melanogaster? In the process of pupal organ dissociation and adult tissue remodeling,what is the fate of the hindgut cells of the larvae,whether they are apoptotic or surviving and participate in the reconstruction of the hindgut of adult? At present,these studies on the functional mechanism of hindgut are little known.We all know that stem cells are responsible for regulating the fate of tissues and organ maintenance at the site.How will the hindgut stem cells repair the damage when the tissue is damaged? Gal4/UAS system is a common transgenic system in Drosophila melanogaster.It can be used to achieve regionalized and flexible target gene expression.In order to more clearly track the proliferation and differentiation pattern of cell lineages and the migration and distribution of cell communities,a new genetic cell lineage tracing technique,G-TRACE(Gal4 technology for real-time and clonal expression),which combines FLP(flipase)-out technology and fluorescent protein labeling technology on the basis of Gal4/UAS system,which can clearly analyze the cell lineage pattern during development.In this study,G-TRACE technology will be used to explore the developmental lineage of intestinal cells in the hindgut,the signal regulation mechanism of their formation,and the repair mode of stem cells in the damaged hindgut.The main research contents are as follows:1: Tracking the developmental pattern of posterior intestinal cell lineage in Drosophila melanogaster by G-TRACEEngrailed-Gal4(en-Gal4)line and lineage-tracing line(G-TRACE)of Drosophila melanogaster were hybridized,and tub-Gal80 ts was introduced to temporally control Gal4 activity.The cell lineage was traced at the larval and pupal stages,respectively.For larval stage tracing,eggs were cultured at 30℃ after egg-laying by the parental generation,and the mid 3rd instar larvae were shifted to 18℃.The adult guts were detected within 1d after eclosion.For pupal stage tracing,eggs were cultured at 18℃after egg-laying by the parental generation,pupae were shifted to 30℃ at different stages,and adult guts were detected after eclosion.For larval stage tracing of Drosophila,green intestinal cells appeared in the posterior section of midgut,which is adjacent to the hindgut-midgut boundary and Malpighian tubules.For pupal stage tracing of Drosophila,green intestinal cells appeared at different sections in the midgut and Malpighian tubules,and engrailed gene was expressed in Drosophila intestine at the pupal stage.These results suggest that during pupa formation,the hindgut cells from larval stage did not apoptosis completely,a part of the hindgut cells migrate into the midgut or Malpighian tubules.They are involved in adult midgut or Malpighian tubules reformation.2: Exploring the signal regulation of hindgut formation by G-TRACESheng-An Yang et al.considered that the imaginal ring of larvae(the imaginal ring,located in the pylorus of the hindgut)was the progenitor cell region of larval tissues,which affected the formation of the hindgut tissues of adult.Some Gal4 were hybridized with G-TRACE strains,and tub-Gal80 ts were introduced to control the opening time of Gal4.The larval and adult patterns of hybrid offspring and the contribution of GFP cells(gene expression cells)activated in larval stage to the intestinal tissue of adult were analyzed.Finally,we conclude that the ci expression region in larval stage is located in the imaginal ring,that is,the progenitor cell region of larvae.The Hh signal detected in the pylorus adjacent to the "ci progenitor cell region" in larval stage.The Wg and Dpp(Ptc)is located in the hindgut proliferation zone(HPZ)of adult.Wg and Dpp(Ptc)signals may be related to each other and affect the hindgut proliferation zone(HPZ)of adult.3: Repair mechanism of the injury of the hindgut tissueAfter analyzing the fate of the hindgut intestinal cells and the signal regulation mechanism of their formation,we then explore the repair mechanism of the damaged tissues by the hindgut stem cells of Drosophila melanogaster.bam-Gal4,vasa-Gal4,wg-Gal4 strain was used to drive G-TRACE strain and tub-Gal80 ts was introduced to control the opening and closing of Gal4 activity.20%-40% concentration gradient of Nacl solution was added to the culture medium to damage the adult of Drosophila.The region of "wg ring" was detected by the antibody of germline cells markers,which is located in the HPZ region of adult.The final results showed that no germline cells markers were detected in the wg ring region of the hindgut of Drosophila melanogaster fed on normal medium without Nacl solution,and no wg cells expressing EGFP were found in the hindgut tissue of adults,because the stem cells in the hindgut tissue were silent under normal conditions.After Nacl solution injury,not only germline cells markers were detected,but also the migration of wg cells expressing EGFP was found in the posterior intestinal tissue.EGFP cells flow moved to the posterior part of the hindgut in a special trajectory.It can be seen that intestinal stem cells repair and replenish damaged parts along a certain trajectory when they perform their functions.