טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAmit Lihi
SubjectThe Role of the Extracellular Matrix in the Morphogenesis
of Chordotonal Organs
DepartmentDepartment of Medicine
Supervisor Professor Adi Salzberg


Abstract

Proprioception, the ability to perceive the body position in regard to itself and to the environment, is based on stretch receptors that translate mechanical forces into neuronal signals.

In the fruit fly Drosophila melanogaster, chordotonal organs (ChO) are a type of stretch receptors located close to the muscles of the body wall. Each unit contains a bipolar neuron and a scolopale cell which is stretched between the accessory cells and anchored to the cuticle by specialized attachment cells. Our lab focuses on identifying the different components required for proper morphogenesis of the ChO, one of them is the extracellular matrix (ECM); previous work demonstrated that the lack of several ECM-related proteins led to abnormal phenotypes in the ChO.

The first goal of my research is to conduct an RNAi screen focused on ECM-related proteins, in order to identify additional key component of ChO morphogenesis. The second goal is to visualize the ChO in higher resolutions using a scanning electron microscope.

I began my work by conducting an RNAi screen using the UAS/Gal4 system that allowed me to scan through ~80 different strains; each strain targeted an ECM protein. Since ECM components are often secreted from different tissues, I had to use the dautherless-gal4 driver for an early and ubiquitous expression during embryogenesis. The knockdown of five genes led to obvious defects in the cap attachment cells, cap cells and/ or to the ligament attachment cell: plod, clos, cad99c, dpy and muc55b. Although each one of the five genes has a known role during embryogenesis, their role in morphogenesis of the ChO is currently unclear.

In order to visualize the ChO of the embryo and the larva we use confocal microscope, combined with expression of fluorescent markers and immunostaining. A major downside of the confocal microscope is its resolution limits (in the scale of 0.6- 1 micron). The growing need to inspect the ChO and surrounding tissues in three dimensions and in higher resolutions led us to investigate the use of scanning electron microscope. To find the proper conditions we had to develop a new protocol for dissection and fixation of 3rd instar larvae. Due to the location of the ChOs between the body wall muscles and the cuticle we had to remove several layers of muscles, a delicate process that can tear the ChO itself, while the dehydration process can cause further damage to the organ. Nevertheless, we were able to visualize the ChO structure and the tissues surrounding it in three dimensions. After establishing the protocol in wildtype larvae, I was able to compare the wildtype ChO to the ChO of prc mutant larvae, and identify differences in the shape of the ChO cells and the ECM wrapping them.