Studies on DOCK family protein Sponge during Drosophila development ショウジョウバエ発生過程におけるDOCKファミリータンパク質Spongeの研究

The Sponge (Spg) belongs to DOCK family that is widely-conserved among species, and DOCK family members are known as DOCK1?DOCK11 in mammals. DOCK1 involves neurite elongation, DOCK2 involves immunocyte differentiation. However function of other DOCK family member is yet unknown. To carry out further functional analysis of DOCK family, I searched a novel DOCK gene in genome database of Drosophila, and identified the spg gene that is similar to DOCK3 and DOCK4. I focused on this spg gene and established UAS-spgIR transgenic fly strains for knockdown of spg. Specific knockdown of spg by GMR-GAL4 driver in eye imaginal discs induced abnormal compound eye in adults. To examine development of photoreceptor cells and localization of Spg, I crossed this knockdown strains with a variety of enhancer trap strains that specifically expresses lacZ in various set of photoreceptor cells. Immunostaining with anti-Spg and anti-lacZ antibodies revealed high expression of Spg in R7 photoreceptor cells that was specifically reduced by its knockdown with the GMR-GAL4 driver. Furthermore, immunostaining with anti-dpERK antibodies revealed that the level of activated ERK signal was extensively reduced by knockdown of spg with the GMR-GAL4 driver in eye discs. In other tissues, specific knockdown of spg by pannir-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK antibodies revealed the strong reduction of the JNK activity in the spg knockdown clones. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in the eye discs and those between Spg and Rac1 in the wing discs, respectively. Thus Spg that interact Rap1 or Rac1 appears to play a key role in differentiation of tissues by activating ERK or JNK pathway.