Regulation Of Drosophila Wingless And Hedgehog Signal Transduction Pathways

By using a vestigial-ga14 to deliver the tissue specific flipase to the wing, a F1 genetic screen was performed. A number of mutant alleles of known or unknown genes were isolated from this Fl screen. This study focused on three groups of mutant alleles from mutant alleles of unknown genes. All these three groups of alleles related to either Wingless (Wg) signaling pathway or Hedgehog (Hh) signaling pathway (each group composed of 3-4 mutant alleles). By analyzing the wing phenotype, cuticle phenotype of germline clone embryo, deficiency kit mapping, direct sequencing and searching the genome sequence database, three genes: sll, oxt and pygo were identified (each gene corresponding to one group of mutant alleles).1) The first group of the mutant alleles included B158, B140 and B173. These alleles showed wg-like phenotype (notch et al.), the germline clone embryo showed cuticle fusion phenotype. This group of mutant alleles turned out to be a gene encoding a Golgi adenosine 3'-phosphate 5'-phosphsulfate transporter (adenosine PAPS transporter). Kyte-Doolittle hydrophilicity plots showed that Sll is a very hydrophobic protein, especially in C terminus. The nature of the mutant alleles is either nonsense mutation or the mutation which disrupted the splicing of the primary transcripts.Anti-En antibody staining and in situ hybridization on sll Germline clone (GLC) embryo using wg antisense probe showed defective En protein bands and defective wg transcripts bands. Furthermore, the wing imaginal disc clonal analysis showed that in the homozygous sll mutant clones, the extracellular Wg was dramatically reduced. The reduction of extracellular Wg indicated that the Wg signaling in the wing imaginal disc was disrupted. The reason leaded to this phenomenon is that sll encodes a PAPS transporter, so the disruption of the sll gene would generate the unsulfated Heparan Sulfate Proteoglycan (HSPG), which most likely would lose normal function.2) The second group of mutant alleles included 15-108. A164, B104 and L5. These alleles showed wing and cuticle phenotypes related to both Wg and Hh signaling pathways (notch and vein defect et al.). This group of mutant alleles turned out to be a gene encoding a xylosyltransferase, which is an enzyme transfers the xylose from UDP-xylose to the serine of the protein core. The nature of all the three mutant alleles of this group is nonsense mutation disrupting the coding region of oxt gene. By tagging the oxt gene with a GFP tag, it was found that this gene mainly expressed in the cytosol. Comparison of conserved domains (Core-2/ I-Branching and Wsc domain) of Oxt with mammalian homologues showed that both of domains are conserved throughout the invertebrate and vertebrate.Anti-En antibody staining and in situ hybridization on oxt GLC embryo using wg antisense probe showed defective En protein and defective wg transcripts bands. The embryonic analysis results indicated that the disruption of oxt gene would lead to the disruption of wg transcription and the expression of engrailed gene, as the wgtranscription is dependant on the Engrailed. Clonal analysis in this study on the eye imaginal disc showed that in the oxt mutant clones, the Ci expression was completely deleted. On the Hh signal receiving cells, the function of HSPG in HhN movement might be that it competes with Ptc for binding of HhN, following the releasing from the Disp, HhN binds to the HSPG to prevent the HhN from being captured by the Ptc, hence facilitate it's transfer to the more distantly located cells. Also, HSPG might also be required for secreting cells: as a co-factor for secreting cells to displace the HhN from the Disp.3) The 3rd group of mutant alleles included F66, F15-28, F126 and F107. These alleles showed wing and cuticle phenotypes related to Wg signaling pathway. This group of mutant alleles turned out to be a new component of Wg singaling pathway. The sequence result showed that the nature of pygo mutant alleles F66, F15-108 and F126 were nonsense mutation, which probably led to tru...