Cordula Schulz

Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment.

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments [1-3]. However, how microenvironments are shaped is poorly understood. In testes of Drosophila melanogaster, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells [4]. We show that germ cells signal via the Egf class ligand Spitz (Spi) and provide evidence that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppressed the germ cell enclosure defects of the conditional spi allele. We propose that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. Our novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage.

The Drosophila cyst stem cell lineage Partners behind the scenes

In all animals, germline cells differentiate in intimate contact with somatic cells and interactions between germline and soma are particularly important for germline development and function. In the male gonad of Drosophila melanogaster, the developing germline cells are enclosed by somatic cyst cells. The cyst cells are derived from cyst stem cells (CySCs) of somatic origin and codifferentiate with the germline cells. The fast generation cycle and the genetic tractability of Drosophila has made the Drosophila testis an excellent model for studying both the roles of somatic cells in guiding germline development and the interdependence of two separate stem cell lineages. This review focuses on our current understanding of CySC specification, CySC self-renewing divisions, cyst cell differentiation, and soma-germline interactions. Many of the mechanisms guiding these processes in Drosophila testes are similarly essential for the development and function of tissues in other organisms, most importantly for gametogenesis in mammals.

Control of Germline Stem Cell Division Frequency – A Novel, Developmentally Regulated Role for Epidermal Growth Factor Signaling

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model.

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.

Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-962288, had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation.

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. We propose that different CRLs enable the responses of somatic support cells to Upd and EGF.

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.

G-Protein signaling increases germline stem cell activity in Drosophila males in response to multiple rounds of mating

Adult stem cells divide to renew the stem cell pool and replenish specialized cells that are lost due to death or usage. However, little is known about the mechanisms regulating how stem cells adjust to a demand for specialized cells. A failure of the stem cells to respond to this demand can have serious consequences, such as tissue loss, or prolonged recovery post injury. Here, we challenge the male germline stem cells (GSCs) of Drosophila melanogaster for the production of specialized cells using mating experiments. We show that repeated mating reduced the sperm pool and accelerated germline stem cell (GSC) divisions. The increase in GSC divisions depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNA-i) constructs against G-proteins or a dominant negative G-protein eliminated the increase in GSC divisions in mated males. Consistent with a role for the G-proteins in the regulation of GSC divisions, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to accelerate their GSC divisions in response to mating. Our data show that GSCs are receptive to GPCR stimulus, potentially through a network of interactions among multiple signaling pathways.Though adult tissues are maintained by homeostasis, little is known about how their precursor cells adjust to a demand for specialized cells to account for changes during development or in the environment. In the male gonad, the ability to respond to a demand for increased gamete production, commonly referred to as reproductive plasticity, is essential for the fitness of an individual and the species. Here, we show that a demand for sperm, caused by repeated male mating, increased germline stem cell (GSC) division frequency and the production of gametes. The increase in GSC divisions depended on activity of four classical G-protein coupled receptors and downstream signaling molecules within the germline cells. Thus, GSCs are reliant on the GPCR stimulus. Among the signaling molecules, Serotonin appeared sufficient to accelerate GSC divisions in non-mated males, making the highly conserved Serotonin receptors key players in the mechanism regulating tissue replenishment.