Identification of factors regulating individualization.
Authors: Sepideh Dadkhah; Douglas Harrison
Affiliation: University of Kentucky, Lexington, KY
Keywords: a. spermatogenesis; e. JAK/STAT
Sperm development in Drosophila has many similarities to spermiogenesis in other organisms. Individualization is one of the later stages of spermiogenesis in which the 64 interconnected spermatids in a cyst are separated and excess cytoplasm eliminated. The synchronous differentiation of spermatids from interconnected spermatocytes is evolutionarily conserved. While JAK/STAT pathway activation in the somatic cyst cells is required for individualization, the mechanism by which it controls the process is not known. The aim of this project is to identify effectors regulated by JAK/STAT signaling that initiate individualization.
Using RNA-seq, we have examined expression profiles from testes in which JAK/STAT signaling has been impaired prior to individualization by expressing the negative regulator Eye-transformer/Latran (ET/Lat) and compared to testes dissected from wild type flies. Estimates of transcript abundance of the control and experiment profiles were calculated. Differential expression analysis identified more than 400 genes that were differentially expressed upon the arrest of JAK/STAT signaling at elongation. The differentially expressed genes identified in the RNA-Seq analysis were prioritized based on being associated with relevant GO terms and relation to the JAK/STAT pathway. Knockdown of 38 prioritized genes was carried out by utilizing the UAS-Gal4 system to express corresponding RNAi constructs with somatic and germline drivers. Functional analysis was done by quantitative analysis of elongated spermatid nuclei bundles and investment cones, both hallmark events of progression of individualization. From this functional analysis, genes involved in extracellular matrix and cell signaling were found to influence individualization.