Distinct responses to rare codons in select Drosophila tissues

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index, to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in restricting protein expression of an evolutionarily young gene, RpL10Aa, to the Drosophila testis. Rare codon-mediated restriction of this testis-specific protein is critical for female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

Germ cells commit somatic stem cells to differentiation following priming by PI3K/Tor activity in the Drosophila testis

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. We find that CySCs are maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, we find that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. We propose that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.

Chigno/CG11180 and SUMO are Chinmo-Interacting Proteins with a Role in Drosophila Testes Stem Cells

Maintenance of sexual identity on the cellular level ensures the proper function of sexually dimorphic genes expressed in the brain and gonads. Disruption of genes that regulate sex maintenance alters the cellular structure of these tissues and leads to infertility and diseases, such as diabetes, obesity, and gonadal cancers. Sex maintenance in the testis of Drosophila melanogaster depends on the previously identified gene chinmo (Chronologically inappropriate morphogenesis). Chinmo's effect on testis differentiation has been investigated in detail, but there is still much to be elucidated about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (Ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work indicates that SUMO functionally links Chinmo and CG11180 in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Childless Gambino (Chigno) for CG11180.

Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

SummaryTissue homeostasis often requires a properly placed niche to support stem cells. The morphogenetic processes that position a niche are just being described. We recently showed that Drosophila testis pro niche cells, specified at disparate positions during early gonadogenesis, must assemble in one collective at the gonad anterior. Here, we identify Slit and FGF signals emanating from adjacent visceral mesoderm (Vm) that regulate assembly. In response to signaling, niche cells express islet, which we find is also required for positioning the niche. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of the dispersed niche is severely disrupted, with pro-niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

Stem cells commit to differentiation following multiple induction events in the Drosophila testis.

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. We find that CySCs are maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, we find that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. We propose that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.

α-Tubulin Regulates the Fate of Germline Stem Cells in Drosophila Testis

The Drosophila testis provides an exemplary model for analyzing the extrinsic and intrinsic factors that regulate the fate of stem cell in vivo. Using this model, we show that the Drosophila αTub67C gene (full name αTubulin at 67C), which encodes α4-Tubulin (a type of α-Tubulin), plays a new role in controlling the fate of male germline stem cells (GSC). In this study, we have found that Drosophila α4-Tubulin is required intrinsically and extrinsically for GSCs maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that the gene αTub67C is not required for Dpp/Gbb signaling silencing of bam expression, suggesting that αTub67C functions downstream of or parallel to bam, and is independent of Gbb/Dpp-bam signaling pathway. Furthermore, overexpression of αTub67C fails to obviously increase the number of GSC/Gonialblast (GB). Given that the α-tubulin genes are evolutionarily conserved from yeast to human, which triggers us to study the more roles of the gene α-tubulin in other animals in the future.

A burst of transposon expression accompanies the activation of Y chromosome fertility genes during Drosophila spermatogenesis

Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of the Drosophila melanogaster testis are not well understood. We reanalyzed publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We developed a novel method for identification of TE and host gene expression programs and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are enriched on the Y chromosome and depleted on the X chromosome relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y-chromosome-specific transcriptional programs.