Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.     

The drosophila angiotensin-converting enzyme homologue Ance is required for spermiogenesis

The Angiotensin-converting enzyme (Ance) gene of Drosophila melanogaster is a homologue of mammalian angiotensin-converting enzyme (ACE), a peptidyl dipeptidase implicated in regulation of blood pressure and male fertility. In Drosophila, Ance protein is present in vesicular structures within spermatocytes and immature spermatids. It is also present within the lumen of the testis and the waste bag, and is associated with the surface of elongated spermatid bundles. Ance mRNA is found mainly in large primary spermatocytes and is not detectable in cyst cells. Testes lacking germ cells have reduced levels of ACE activity, and no Ance protein is detectable by immunocytochemistry, indicating that the germ cells are the major site of Ance synthesis. Ance mutant testes lack individualised sperm and have very few actin-based individualisation complexes. Spermatid nuclei undergo scattering along the cyst and have abnormal morphology, similar to other individualisation mutants. Mutant spermatids also have abnormal ultrastructure with grossly defective mitochondrial derivatives. The failure of Ance mutant testes to form individualisation complexes may be due to a failure in correct spermatid differentiation. Taken together, the expression pattern and mutant phenotype suggest that Ance is required for spermatid differentiation, probably through the processing of a regulatory peptide synthesised within the developing cyst.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.     

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or "niches", which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. Here, we investigate the formation of the testis GSC niche, or "hub", during the late stages of embryogenesis. By morphological and molecular criteria, we identify and follow the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad.     

TGFbeta receptor saxophone non-autonomously regulates germline proliferation in a Smox/dSmad2-dependent manner in Drosophila testis

Elucidating the regulatory mechanism of cell proliferation is central to the understanding of cancer development or organ size control. Drosophila spermatogenesis provides an excellent model to study cell proliferation since the germline cells mitotically amplify in a precise manner. However, the underlying molecular mechanism remains elusive. Germ cells derived from each gonialblast develop synchronously as one unit encapsulated by two somatic support cells (called cyst cells). Components of TGFbeta pathway have previously been found to restrict germ cell proliferation via their functions in cyst cells. Here we report that saxophone (sax), a TGFbeta type I receptor, is required in somatic cells to prevent the mitotically dividing spermatogonia from over-amplifying. Using various approaches, we demonstrate that Mad (Mothers against Dpp), a receptor-Smad usually associated with Sax-mediated TGFbeta/BMP signaling, is dispensable in this process. Instead, Smox (Smad on X, Drosophila Smad2), the other receptor-Smad formerly characterized in TGFbeta/activin signaling, is necessary for the precise mitotic divisions of spermatogonia. Furthermore, over-expressing Smox in cyst cells can partially rescue the proliferation phenotype induced by sax mutation. We propose that Smox acts downstream of Sax to prevent spermatogonial over-proliferation in Drosophila.     

A new Drosophila gene wh (wuho) with WD40 repeats is essential for spermatogenesis and has maximal expression in hub cells

Through mutagenesis by P-element transposition, we identified a series of mutants with deletions in topoisomerase 3beta gene (top3beta) and an adjacent, previously uncharacterized gene CG15897, here named wuho (wh). Whereas top3beta truncation does not affect viability or fertility, wh null mutants display male sterile and female semi-sterile phenotypes. Furthermore, wh mutants can be fully rescued by wh transgenes, but not by top3beta transgenes, suggesting that the fertility phenotypes are caused by wh deletion. The alignment of WH protein sequence with other eukaryotic putative homologues shows they are evolutionarily conserved proteins with 5 WD40 repeats in the middle portion of the protein, and a bipartite nuclear localization signal at the carboxyl terminus. Yeast homologue with 5 WD40 repeats, Trm82, is the non-catalytic subunit of a tRNA methylase. Immunostaining shows that WH has the highest expression in hub cells, a niche for germline stem cells of testis. However, WH is not required for the maintenance of hub cells or the germline stem cells. In wh mutant males, spermatogenesis is arrested at the elongating stage of the developing spermatids, resulting in an absence of mature sperms in the seminal vesicles. The decreased fertility in wh mutant females is mostly due to defects in oogenesis. There are abnormal egg chambers present in the mutant females, in which the cystocytes fail to arrest their cell division at the fourth mitotic cycle, resulting in more than 16 cells in a single egg chamber. Additionally, these abnormal cystocytes do not undergo multiple rounds of endoreplication as the nurse cells do in a normal egg chamber. Therefore, the cytological analyses demonstrate that wh has a critical function in cellular differentiation for germline cells during gametogenesis.     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

A Temporal Signature of Epidermal Growth Factor Signaling Regulates the Differentiation of Germline Cells in Testes of Drosophila melanogaster

Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.