View Program - 63rd Annual Drosophila Research Conference

Broad (Br, CG11491, FBgn0283451) is a BTB domain (Broad-Complex, Tramtrack and Bric a brac) containing protein that has a homodimerization domain at the N terminus and multiple copies of either zinc fingers of the C2H2 type or Kelch repeats. Br is best known as a modulator of the ecdysone-response and has been attributed to cause chromatin changes by puffing of chromosomes to affect gene expression. We examined Br expression and function in third instar larval gonads when ecdysone levels are high.
In our whole gonad RNA-seq experiments, we detected 11 out of 15 Br transcripts encoding 4 different protein isoforms of Br (Z1-Z4), on the basis of distinct Zinc-fingers at C-terminus. Further, Single Cell RNA sequencing (scSeq) and immunostaining with antibody show that, Br, was expressed only in somatic cells of testis and ovary. In the testis, Br expression was enriched in cyst cells that enclose the spermatogonia, which are mitotic germline cells near the apex, but not the cyst cells surrounding spermatocytes. Br was also expressed in terminal epithelium, which ultimately attaches to the reproductive tract derived from the genital disc, and the pigment cells that ensheath the testis. In ovary, we found Br expression in all somatic cells (Sheath cells, Terminal Filament, Cap cells, Intermingled cells, follicle cell and Swarm cells). To determine the function of Br in these cell types we knocked down of all of its isoforms (by targeting common BTB domain) in the enclosing the germ cells (male cyst cells and female intermingled cells) using a traffic jam driver. This led to female-specific sterility due to germline defects including loss of germ cells. Br expression was lost from the spermatogonia cyst cells, but did not result in an overt phenotype. However, knockdown of Br using a doublesex driver resulted in sterility in both sexes. Knockdown of individual isoforms (RNAi against C-terminus) using doublesex driver results in testes that failed to elongate and attach to the reproductive tract, while in females egg retention phenotype was observed. Both males and females from these crosses were sterile. This indicates that Br is required in terminal epithelium to mediate attachment to the reproductive tract as well as egg laying in females and leads to sterility when Br is knocked down. Interestingly we found an enhancer that drives Br expression in both sexes, indicating dual role of enhancer in regulating Br function in sex and cell type specific way. In summary, we show that Br is required in both sexes, but in fundamentally different somatic cell types. This highlights the context-dependency of gene expression in sexual development and fertility.

Broad is sex and cell type specifically required in the Drosophila gonads for gametogenesis and fertility.