Functional Analysis Of Apontic During Organs Development In Drosophila

Transcription factors have the ability to bind DNA, which play crucial role during biological development by regulating the expression of target genes. Identification of target genes of transcription factor would clarify its function. Apontic(Apt) has been identified as a bZIP transcription factor and is required for the development of tracheae, head, heart, border cell, and central nervous system in the embryo of Drosophila. However, the mechanism of Apt during development is unclear. The human homolog of Apt(FSBP), is also a cancer-related factor and is also expressed in many tissues, including heart, brain, lung, liver, skeletal muscle, kidney, and pancreas. The mechanism of FSBP during human development is also unclear.Cyclin E is an important regulator which regulates S phase entry. In addition, due to Cyclin E is involved in cell cycle regulation, mis-regulation of cyclin E leading to cancer. However, Cyclin E transcription is controlled by a large and complex cis-regulatory region containing tissue- and stage-specific components. Therefore, the regulation of cyclin E is temporal and spatial specificity and tissue-specific, many regulatory factors need to be further identified.Hh signaling as one of the five conserved signaling pathway, which is crucial for organism development. However, the current studies focused on studying the downstream genes regulation of Hh signaling pathway, the regulation of hh remains to be clarified.In this study, we found that Apt was an evolutionarily conserved DNA binding protein from Drosophila to humans, and the C- terminus DNA binding domain of Apt had the ability to bind DNA. In addition, Apt regulated the expression of two evolutionarily conserved cancer-related genes, cyclin E and hh. Furthermore, Apt and Ubx co-regulate salivary gland endocycle. The functional analysis of Apt could provide a theoretical basis for understanding the mechanism of Apt human homologous gene, FSBP, and the new mechanism for understanding endocycle. The main results presented in this thesis are as follows:(1) Apt controls the G1/S progression by inducing cyclin E during eye developmentapt mutant clone cells affected the development of photoreceptor cells during Drosophila eye development, resulting in the disruption of the adult eye. Overexpressed apt leading to the absent of adult eye. We found Apt was evolutionarily conserved from Drosophila to humans through evolution analysis and Apt could bind DNA by EMSA. Next we identified the target of Apt by microarray analysis and qRT-PCR. We found cyclin E was the target of Apt. In the eye disc of wild type, Apt and Cyclin E were co-localization. Furthermore, the expression of cyclin E is significantly reduced in the apt mutant clones whereas it is increased by overexpression of Apt. Moreover, we analyzed cyclin E promoter element, we identified one potential Apt binding sequence within the region. We conclude that Apt controls S phase entry during eye development through directly binding to the Apt site in cyclin E regulatory region by transgenic reporter assays.(2) Apt regulates cell proliferation and development by coordinating expression of hedgehog and cyclin EThe blistered and smaller wing was induced in apt mutant clones during our research. Overexpressed apt resulting in diminished and blistered wing and the pattern of wing was disrupted. Ectopic expressed apt leading to no wing phenotype. Hh has been reported as the organizer to control wing pattern by inducing and regulating a series of downstream genes which expressed temporal specificity. We found regulatory relationships existed between apt and hh by genetic interactions between apt and hh. Immunofluorescence staining showing that Apt and Hh were co-localization in the wing disc. Hh-lac Z and Hh protein were decreased in the apt mutant clones. Overexpression of Apt could induce the expression of hh. We found Apt binding sites in the promoter region of hh. We mutated binding sequences by CRISPR / Cas9 methods. The mRNA and protein of hh were significantly decreased in the binding site mutation flies. In addition, binding site mutation flies enhance the defect phenotype of hh mutant. Previous research suggested that Hh signaling could regulate the transcription of Cyclin E by transcription factor. Based on observing the expression pattern of Ci and Cyclin E and altering the Hh signaling, we detected the expression of Cyclin E. The results showing that the expression of Cyclin E was not induced by Hh signaling in the wing disc. Apt could directly regulated the expression of hh and cyclin E by binding the promoter regions of hh and cyclin E. The regulation of cyclin E by Apt is independent from Hh and Hh signaling could not induce the expression of cyclin E in the wing disc.(3) Apt and Ubx control salivary cells endocycleNormal cell cycles were consist with G1 phase, S phase, G2 phase and M phase. Endocycle was a variant cell cycles, such cycles were observed widely throughout both the plants and animals, and were so ubiquitous that they maight account for up to half the world’s biomass. Endocycle cells undergo endoreplication but without mitosis. It means this cell cycle comprised of DNA synthesis(S)- and gap(G)-phases but lacking mitosis. The Cyclin E was required when S phase entry, however, consistent Cyclin E activity blocking the assembly of pre-Replication Complexes(preRCs) for the next S-phase. In order to continue endocycling, Cyclin E oscillation was required. In our research we found Apt participated in cell endocycle by inducing the expression of cyclin E. While Apt induced the expression of cyclin E, a Hox gene ubx also be induced. Ubx could affected protein localization of Apt by impacting its nuclear localization signal. When Apt transported from nucleus to cytoplasm the dose of cyclin E decreased to ensure the next G/S transition. Besides, S phase cells could also induce the expression of Ubx to change the localization of Apt. Previous studies showed that the transcription factor E2f1 and ubiquitin ligase CRL4CDT2 controlled the salivary glands endocycle. In our data we found the E2f1-dependent endocycle had a marginal impact on the expression of Apt and this effect by Ubx. We speculated the endocycle control by Apt and Ubx might dominate in salivary gland development.