The importance of a halotyrosine dehalogenase for Drosophila fertility.

The ability of iodotyrosine deiodinase to salvage iodide from iodotyrosine has long been recognized as critical for iodide homeostasis and proper thyroid function in vertebrates. The significance of its additional ability to dehalogenate bromo- and chlorotyrosine is less apparent, and none of these functions could have been anticipated in invertebrates until recently. Drosophila, as most arthropods, contains a deiodinase homolog encoded by CG6279, now named condet (cdt), with a similar catalytic specificity. However, its physiological role cannot be equivalent because Drosophila lacks a thyroid and its associated hormones, and no requirement for iodide or halotyrosines has been reported for this species. We have now applied CRISPR/Cas9 technology to generate Drosophila strains in which the cdt gene has been either deleted or mutated to identify its biological function. As previously shown in larvae, expression of cdt is primarily limited to the fat body, and we now report that loss of cdt function does not enhance sensitivity of the larvae to the toxic effects of iodotyrosine. In adult flies by contrast, expression is known to occur in testes and is detected at very high levels in this tissue. The importance of cdt is most evident in the decrease in fertility observed when either males or females carry a deletion or mutation of cdt Therefore, dehalogenation of a halotyrosine appears essential for efficient reproduction in Drosophila and likely contributes to a new pathway for controlling viability in arthropods.

Polycomb Group Gene E(z) Is Required for Spermatogonial Dedifferentiation in Drosophila Adult Testis.

Dedifferentiation is an important process to replenish lost stem cells during aging or regeneration after injury to maintain tissue homeostasis. Here, we report that Enhancer of Zeste [E(z)], a component of the Polycomb repression complex 2 (PRC2), is required to maintain a stable pool of germline stem cells (GSCs) within the niche microenvironment. During aging, germ cells with reduced E(z) activity cannot meet that requirement, but the defect arises from neither increased GSC death nor premature differentiation. Instead, we found evidence that the decrease of GSCs upon the inactivation of E(z) in the germline could be attributed to defective dedifferentiation. During recovery from genetically manipulated GSC depletion, E(z) knockdown germ cells also fail to replenish lost GSCs. Taken together, our data suggest that E(z) acts intrinsically in germ cells to activate dedifferentiation and thus replenish lost GSCs during both aging and tissue regeneration.

A non-cell autonomous role of E(z) to prevent germ cells from turning on a somatic cell marker.

In many metazoans, germ cells are separated from somatic lineages early in development and maintain their identity throughout life. Here, we show that a Polycomb group (PcG) component, Enhancer of Zeste [E(z)], a histone transferase that generates trimethylation at lysine 27 of histone H3, maintains germline identity in Drosophila adult testes. We find excessive early-stage somatic gonadal cells in E(z) mutant testes, which originate from both overproliferative cyst stem cells and germ cells turning on an early-stage somatic cell marker. Using complementary lineage-tracing experiments in E(z) mutant testes, a portion of excessive early-stage somatic gonadal cells are found to originate from early-stage germ cells, including germline stem cells. Moreover, knocking down E(z) specifically in somatic cells caused this change, which suggests a non-cell autonomous role of E(z) to antagonize somatic identity in germ cells.

MicroRNAs downregulate Bag of marbles to ensure proper terminal differentiation in the Drosophila male germline.

In many adult stem cell lineages, the continuous production of functional differentiated cells depends on the maintenance of progenitor cells in an undifferentiated and proliferative state, as well as the subsequent commitment to proper terminal differentiation. In the Drosophila male germline stem cell (GSC) lineage, a key differentiation factor, Bag of marbles (Bam), is required for the transition from proliferative spermatogonia to differentiating spermatocytes. We show that bam mRNA, but not Bam, is present in spermatocytes, suggesting that bam is regulated post-transcriptionally. Consistent with this, repression of Bam accumulation is achieved by microRNAs via the bam 3'UTR. When the bam 3'UTR was substituted with the 3'UTR of a constitutively expressed α-Tubulin, Bam became stabilized in spermatocytes. Moreover, such a persistent expression of Bam in spermatocytes was recapitulated by specifically mutating the putative miR-275/miR-306 recognition site at the bam 3'UTR. In addition, overexpression of miR-275 or miR-306 in spermatogonial cells resulted in a delay of the proliferation-to-differentiation transition and resembled the bam loss-of-function phenotype, suggesting that these microRNAs are sufficient to downregulate Bam. Finally, the failure of Bam downregulation in spermatocytes affected spermatid terminal differentiation and resulted in increased male sterility. Our results demonstrate that microRNAs control the stem cell differentiation pathway through regulating Bam, the downregulation of which is crucial for proper spermatid terminal differentiation.

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage.

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here, we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription.

The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling.

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.

Epigenetic regulation of germ cell differentiation.

Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization. The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic circuitries as well as epigenetic mechanisms. Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. The past decade has witnessed an ever-increasing understanding of epigenetic regulation in many different cell types/tissues during embryonic development and adult homeostasis. In this review, we focus on recent discoveries of epigenetic regulation of germ cell differentiation in various metazoan model organisms, including worms, flies, and mammals.

Auxilin is essential for Delta signaling.

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin, an endocytic factor first identified in vertebrate cells. Here, we show in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. We find that like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. We discuss these observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling.

Identification of genes that interact with Drosophila liquid facets.

We have performed mutagenesis screens of the Drosophila X chromosome and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets gene encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter protein. In Drosophila, Liquid facets is a core component of the Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain, Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.

Endocytosis, endosome trafficking, and the regulation of Drosophila development.

Endocytosis and endosome trafficking regulate cell signaling in unexpected ways. Here we review the contribution that Drosophila research has made to this exciting field. In addition to attenuating signaling, endocytosis shapes morphogen gradients, activates ligands, and regulates spatially receptor activation within a single cell. Moreover, some receptors signal from within endosomes, and the ability of a specific type of endosome to form controls the ability of cells to signal. Experiments in Drosophila reveal that through regulation of a variety of cell signaling pathways, endocytosis controls cell patterning and cell fate.