E3 ligase Cul2 mediates Drosophila early germ cell differentiation through targeting Bam.

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

Yorkie and Hedgehog independently restrict BMP production in escort cells to permit germline differentiation in the Drosophila ovary.

Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic follicle stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves escort cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs, but Hh also signals to ECs, which are quiescent. Here, we show that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of the Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs.

Control of germline stem cell differentiation by Polycomb and Trithorax group genes in the niche microenvironment.

Polycomb and Trithorax group (PcG and TrxG) genes function to regulate gene transcription by maintaining a repressive or active chromatin state, respectively. This antagonistic activity is important for body patterning during embryonic development, but whether this function module has a role in adult tissues is unclear. Here, we report that in the Drosophila ovary, disruption of the Polycomb repressive complex 1 (PRC1), specifically in the supporting escort cells, causes blockage of cystoblast differentiation and germline stem cell-like tumor formation. Tumors are caused by derepression of decapentaplegic (dpp), which prevents cystoblast differentiation. Interestingly, activation of dpp in escort cells requires the function of the TrxG gene brahma (brm), suggesting that loss of PRC1 in escort cells causes Brm-dependent dpp expression. Our study suggests a requirement for balanced activity between PcG and TrxG in an adult stem cell niche, and disruption of this balance could lead to the loss of tissue homeostasis and tumorigenesis.

MicroRNA-dependent roles of Drosha and Pasha in the Drosophila larval ovary morphogenesis.

The Drosophila larval ovary morphogenesis mainly involves coordinated development of somatic and germ cell lineages that is essential for forming a correct number of niche-germline stem cell (GSC) units (ovarioles) in the adult ovary. Ecdysone, Insulin, Activin, Dpp and EGFR signaling pathways form a regulatory network that orchestrates ovarian soma and germ line throughout larval development. Identification and characterization of additional genes or machineries involved in this process will provide more insights into the underlying mechanisms. Here, we show that the core microRNA (miRNA) pathway components Drosha and Pasha are required for coordinated development of somatic and germ cell precursors in the larval ovary. Drosha or pasha mutants display defective proliferation of primordial germ cells (PGCs), the precursors of GSCs prior to late third larval instar (LL3) and promoted PGC differentiation at LL3. In the mean time, loss of Drosha or Pasha function perturbs somatic precursor development, causing defects in formation of terminal filaments (TFs), a major composition of the GSC niche at LL3, as well as in TF precursor accumulation at early larval stages. Comparative analysis of the mutant phenotypes reveals that three other key miRNA pathway components, Dicer-1 (Dcr-1), Loquacious (Loqs) and Argonaute-1 (Ago-1) have similar effects as Drosha and Pasha indicated above, suggesting a role of the canonical miRNA pathway in the ovary development. Furthermore, genome-wide screening and genetic studies identify a set of Drosha-controlled miRNAs including miR-8, miR-14, miR-33, miR-184, miR-317 and let-7-C that function in this gonadogenesis. Taken together, this study provides the first ever demonstration that miRNA-mediated regulation is involved in the Drosophila larval ovary morphogenesis.

Single-cell transcriptome sequencing reveals that Wolbachia induces gene expression changes in Drosophila ovary cells to favor its own maternal transmission.

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia. Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia, revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCEWolbachia, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain wMel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia. Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host.

Targeting Endogenous Loci That Function in Drosophila Germline Stem Cells.

CRISPR-Cas9 genome editing technology can be used to manipulate the genome of Drosophila melanogaster. The ability to delete genes, make specific mutations, add tags, or make other genetic manipulations is useful for studying germline stem cell biology. In this chapter, we will describe a method to use CRISPR-Cas9 genome editing technology to make knock-out and knock-in flies. We will cover everything from guideRNA (gRNA) and donor plasmid design and cloning to screening for positive edits.

Ultrastructural Analysis of Cell-Cell Interactions in Drosophila Ovary.

The Drosophila ovary is an exceptional model for studying cell-cell interactions in vivo. Cells communicate with each other in a highly coordinated manner. Accurate spatiotemporal regulation of cell-cell interaction is critical for the development of eggs. Ultrastructural analysis using electron microscopy (EM) permits the visualization of both cells and subcellular signaling structures with high resolution. Here we describe a method for the processing of intact fly ovaries by scanning electron microscopy (SEM).

The Osa-Containing SWI/SNF Chromatin-Remodeling Complex Is Required in the Germline Differentiation Niche for Germline Stem Cell Progeny Differentiation.

The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. Here, we report that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, we show that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation.

Ovaries absent links dLsd1 to HP1a for local H3K4 demethylation required for heterochromatic gene silencing.

Heterochromatin Protein 1 (HP1) is a conserved chromosomal protein in eukaryotic cells that has a major role in directing heterochromatin formation, a process that requires co-transcriptional gene silencing mediated by small RNAs and their associated argonaute proteins. Heterochromatin formation requires erasing the active epigenetic mark, such as H3K4me2, but the molecular link between HP1 and H3K4 demethylation remains unclear. In a fertility screen in female Drosophila, we identified ovaries absent (ova), which functions in the stem cell niche, downstream of Piwi, to support germline stem cell differentiation. Moreover, ova acts as a suppressor of position effect variegation, and is required for silencing telomeric transposons in the germline. Biochemically, Ova acts to link the H3K4 demethylase dLsd1 to HP1a for local histone modifications. Therefore, our study provides a molecular connection between HP1a and local H3K4 demethylation during HP1a-mediated gene silencing that is required for ovary development, transposon silencing, and heterochromatin formation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy.

The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.