טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTimerman Yael
SubjectStudying the Role of DPax2, a New Determinant of
Proprioceptor Morphogenesis Identified via a Large
Scale RNAi Screen
DepartmentDepartment of Medicine
Supervisor Professor Adi Salzberg
Full Thesis textFull thesis text - English Version


Abstract

_ The objective of this work was to identify novel genes required for cell-fate determination, differentiation and morphogenesis of the Drosophila melanogaster chordotonal organs (ChO), sensory organs that function as stretch receptive proprioceptors. Each ChO contains a neuron and a scolopale cell, which is stretched between two accessory cells termed cap and ligament. The cap and ligament cells are attached to the cuticle via two epidermal attachment cells, the cap attachment (CA) and the ligament attachment (LA) cells.

The transcription factor Delilah is expressed in the cap, ligament, CA and LA cells, but is excluded from the sensing unit. Analysis of the regulatory region of the dei locus identified two regulatory elements that were used to generate transgenic fly strains with color-coded ChO in which GFP is expressed in cap and ligament cells, and RFP is expressed in CA and LA cells. These transgenic flies made it possible to design a large-scale RNAi-based screen for new determinants of ChO development. We have screened a collection of 920 transgenic strains with RNAi constructs directed against specific genes in the fly genome. Among them we have identified 29 genes (represented by 52 RNAi strains) whose knockdown caused a ChO-specific phenotype. Those genes were classified on the basis of their knockdown phenotype into three phenotypic classes:  loss of cells, abnormal length proportions and abnormal cell morphology. 

One of the genes identified in this RNAi screen was DPax2, whose knockdown caused a loss of GFP and RFP expression in the cap and CA cells. Previous studies have described the function of DPax2 in the Drosophila eye and the external sensory organs. The known function of DPax2 during development, as well as the RNAi-induced phenotype, led us to hypothesize that DPax2 is a regulatory factor that directs cell fate decision and/or differentiation of the ChO cells. Since no previous studies have examined the role that DPax2 plays in the ChOs, we characterized its expression pattern throughout development and found that DPax2 is expressed in the cap and scolopale cells. Furthermore, we analyzed the phenotype of DPax2 loss-of-function in the ChOs. We first tested whether the “loss” of cap and CA cells caused by down-regulation of DPax2 represents cellular fate transformation. The loss of DPax2 expression may have caused the first ACD to occur abnormally, leading to an increase in the number of neurons, scolopale, and ligament cells at the expense of cap and CA. Our results however, showed no significant change in the number of these cells.  In order to test whether the "cell loss” represents an abnormal differentiation of the cap and CA cells, we examined the affected ChOs with a battery of cell-specific markers. This analysis revealed that loss of DPax2 expression disrupts the differentiation of the cap and CA cells. Thus we conclude that DPax2 is a regulatory factor that directs cell fate and differentiation of the ChO cells, specifically in the cap and CA cells.


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