Diverse wMel variants of Wolbachia pipientis differentially rescue fertility and cytological defects of the bag of marbles partial loss of function mutation in Drosophila melanogaster

In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F ) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

Functional divergence of the bag of marbles gene in the Drosophila melanogaster species group

The combination of recent advances in both genomic and gene editing technologies have opened up new possibilities for assessing the functional consequences and drivers of positive selection. In Drosophila melanogaster, a key germline stem cell differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between its D. melanogaster and its sibling species D. simulans, but not in the outgroup species D. ananassae. We previously hypothesized that a genetic conflict with the maternally inherited, intracellular bacteria W. pipientis could be driving the adaptive evolution of bam as W. pipientis increases the fertility of a bam partial loss of function mutant. However, we have not been able to further test this hypothesis by assessing bam variation in other Drosophila lineages and their interactions with W. pipientis because bam function has not been examined in non-melanogaster Drosophila species. Since bam is rapidly evolving at the protein level, its function may not be conserved between species, and therefore different evolutionary pressures may be shaping bam in individual lineages. Here, we ask if bam is necessary for GSC daughter differentiation in five Drosophila species in the melanogaster species group that span approximately 15 million years of divergence and show different patterns of nucleotide sequence evolution at bam. We find that bam function is not fully conserved across these species, and that bam function may change on a relatively short time scale. Ultimately, we conclude that a simple gain in function as the germline stem cell differentiation factor alone does not explain our population genetic and functional genetic results we have observed. Our findings provide a foundation on which to explore the evolution of bam as a GSC differentiation factor and its interactions with W. pipientis in specific lineages.

Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster

Wolbachia is an intracellular, maternally inherited endosymbiotic bacteria that infects over 65% of insects and manipulates their reproduction for its own transmission. In Drosophila melanogaster, Wolbachia genetically interacts with the adaptively evolving germline stem cell gene bag of marbles (bam). Since Wolbachia must enter the host female germline to propagate, one hypothesis is that Wolbachia and bam are in a genetic conflict for control of oogenesis. In order to understand if Wolbachia could be driving the adaptive evolution of bam, we must understand the nature of the genetic interaction between bam and Wolbachia. Previously, we documented that the wMel strain of Wolbachia rescued the fertility and cytological ovarian defect of a bam hypomorphic mutant. However, this mutant was generated in a variable genetic background, and thus we have not been able to perform controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation and a bam null mutation into the w1118 isogenic background. We assess the female fertility of wildtype bam, a bam hypomorph/null mutant, and a homozygous bam hypomorphic mutant, each infected individually with 10 diverse Wolbachia variants. Overall, we find that the Wolbachia variants tested here do not generally increase bam+ female fertility, but they do rescue bam hypomorphic defects with variation in the effect size of some wMel variants on female fertility. Therefore, both bam and Wolbachia interact in genotype-specific ways to modulate a critical fitness phenotype

A transitory signaling center controls timing of primordial germ cell differentiation

Organogenesis requires exquisite spatio-temporal coordination of cell morphogenesis, migration, proliferation and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis primordial germ cells (PGCs) are either sequestered in stem cell niches and maintained in an undifferentiated, germline stem cell state, or transition directly towards differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning them close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso RTK signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinate ovarian morphogenesis with stem cell self-renewal and differentiation programs, a concept applicable broadly to the integration of stem cells and their niches.

The target specificity of the RNA binding protein Pumilio is determined by distinct co-factors

Puf family proteins are translational regulators essential to a wide range of biological processes, including cell fate specification, stem cell self-renewal, and neural function. Yet, despite being associated with hundreds of RNAs, the underlying mechanisms of Puf target specification remain to be fully elucidated. In Drosophila, Pumilio – a sole Puf family protein – is known to collaborate with cofactors Nanos (Nos) and Brain Tumor (Brat); however, their roles in target specification are not clearly defined. Here, we identify Bag-of-marbles (Bam) as a new Pum cofactor in repression of Mothers against dpp (mad) mRNAs, for which Nos is known to be dispensable. Notably, our data show that Nos (but not Bam) was required for Pum association with hunchback (hb) mRNAs, a well-known target of Pum and Nos. In contrast, Bam (but not Nos) was required for Pum association with mad mRNAs. These findings show for the first time that Pum target specificity is determined not independently but in collaboration with cofactors.

Tip60 complex promotes expression of a differentiation factor to regulate germline differentiation in female Drosophila

Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles ( bam) and benign gonial cell neoplasm ( bgcn), work in concert in the stem cell daughter to promote the generation of eggs. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. Here we find that the conserved protein Nipped-A acts through the Tat interactive protein 60-kDa (Tip60) histone acetyl transferase complex in the germ line to promote GSC daughter differentiation. We find that Nipped-A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of a differentiation factor, bgcn. Loss of Nipped-A results in accumulation of GSC daughters . Forced expression of bgcn in Nipped-A germline-depleted ovaries rescues this differentiation defect. Together, our results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.

JNK signaling triggers spermatogonial dedifferentiation during chronic stress to maintain the germline stem cell pool in the Drosophila testis

Exhaustion of stem cells is a hallmark of aging. In the Drosophila testis, dedifferentiated germline stem cells (GSCs) derived from spermatogonia increase during lifespan, leading to the model that dedifferentiation counteracts the decline of GSCs in aged males. To test this, we blocked dedifferentiation by mis-expressing the differentiation factor bag of marbles (bam) in spermatogonia while lineage-labeling these cells. Strikingly, blocking bam-lineage dedifferentiation under normal conditions in virgin males has no impact on the GSC pool. However, in mated males or challenging conditions, inhibiting bam-lineage dedifferentiation markedly reduces the number of GSCs and their ability to proliferate and differentiate. We find that bam-lineage derived GSCs have significantly higher proliferation rates than sibling GSCs in the same testis. We determined that Jun N-terminal kinase (JNK) activity is autonomously required for bam-lineage dedifferentiation. Overall, we show that dedifferentiation provides a mechanism to maintain the germline and ensure fertility under chronically stressful conditions.