The Jing zinc finger protein: its transcriptional regulation by bHLH-PAS, POU, and ETS-domain transcription factors and its role in tracheal cell migration during Drosophila embryogenesis

Abstract

In this research project we investigated a regulatory region upstream of the jing open reading frame containing bHLH-PAS consensus DNA binding sites in order to identify control elements conferring tracheal specificity. Three transgenic fly strains were generated that carried LacZ fusions with genomic sequences from the jing5' regulatory region. Two overlapping fragments (1.5 kb and 2.8 kb) from the jing 5'cis-regulatory region were fused to a lacZ reporter and in vivo expression patterns studied. The 1.5 kb fragment was chosen because it contains three CMEs DNA binding sites for Trh::Tgo and consensus binding sites for the Pou domain transcription factor Drifter and ETS domain transcription factor Pointed. Transgenic embryos carrying the jing1.5-lacZ reporter show expression in the CNS midline, lateral CNS, and in the fusion cells of the trachea. During the early stages of tracheal development (stage 11), jing1.5-lacZ is expressed in the cells adjacent to the tracheal pits. However, despite the widespread distribution of Trh in trachea, jing1.5-lacZ expression was restricted to the fusion cells as confirmed by double labeling of the embryos with anti-beta-gal and the fusion cell-specific antibody, anti-Dysfusion (anti-Dys) and was dependent on trh. trh ectopically activated jing1.5-lacZ in Drosophila embryos in combination with Drifter. Furthermore, the CME sites were required for tracheal jing1.5-lacZ activation. A larger 2.8 kb fragment, encompassing jing1.5 and with no additional CMEs, drove lacZ expression in a larger subset of tracheal cells. jing2.8-lacZ is expressed in all tracheal branches, as early as stage 11. These results suggest that activation of jing in the trachea requires regulators in addition to Trh::Tgo. jing2.8-lacZ reporter is also expressed in the CNS midline, tracheal fusion cells, segmental ectodermal stripes, and posterior spiracles consistent with endogenous jing expression. Previous studies showed that the Jing zinc finger protein is required for both EGFR- and FGFR-dependent tyrosine kinase signaling during tracheal development. Consistent with this, Jing protein is present in the nuclei of most tracheal cells throughout development. We studied its relationship with bHLH-PAS, POD and ETS-domain transcription factors which control FGFR expression during branching in the embryonic Drosophila trachea. The influence of tyrosine kinase signaling on jing1.5-lacZ expression was determined by ectopic expression and mutant analysis. EGFR-mediated signaling activates jing1.5-lacZ expression in surviving midline glia and was required for jing1.5-lacZ tracheal fusion cell expression in rhomboid7M (rho7M) mutant embryos. In addition, genes known to regulate tracheal FGFR expression were required for jing1.5-lacZ activation in the fusion cells. Loss of pointed function and deletion of putative Pnt DNA binding sites in the jing1.5-lacZ reporter was associated with significant reductions in jing1.5-lacZ expression in fusion cells. In combination with reduced jing mRNA in pointed and drifter mutants, these results reveal that jing is regulated by Pnt and Dfr. This is consistent with a reduction in jing1.5-lacZ expression in breathless and branchless mutants which are known to regulate pointed. Furthermore, we show that jing is required for maximal levels of btl expression. In situ hybridization on whole mount embryos revealed that btl transcript levels are significantly reduced in jing loss-of-function backgrounds. Reduction of jing mRNA by RNA interference, specifically in tracheal cells, was associated with reductions in btl mRNA. Finally, we show that expression of ling protein with truncated N-terminal transactivation domains specifically in tracheal cells was associated with reduced btl mRNA. Together, these results suggest that jing is an integral component of the positive feedback mechanism that maintains expression of itself and btl downstream of Trh/Tgo during primary branching. (Abstract shortened by UMI.)