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.

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.

Summary Serotonin (5-HT) is a neuromodulator involved in regulating mood, appetite, memory, learning, pain, and establishment of left-right (LR) asymmetry in embryonic development. To explore the role of 5-HT in a variety of physiological contexts, we have created two forms of “caged” 5-HT, BHQ-O-5HT and BHQ-N-5HT. When exposed to 365- or 740-nm light, BHQ-O-5HT releases 5-HT through 1- or 2-photon excitation, respectively. BHQ-O-5HT mediated changes in neural activity in cultured primary sensory neurons from mouse and the trigeminal ganglion and optic tectum of intact zebrafish larvae in the form of high amplitude spiking in response to light. In Xenopus laevis embryos, 5-HT released from BHQ-O-5HT upon exposure to light increased the occurrence of LR patterning defects. Maximal rates of LR defects were observed when 5-HT was released at stage 5 compared to stage 8. These experiments show the potential for BHQ-caged serotonins in studying 5-HT-regulated physiological processes.

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.

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, sperm cells, using mating experiments. We show that repeated mating reduced the sperm pool and increased the percentage of GScs in M-and S-phase of the cell cycle. the increase in dividing GScs depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNAi) constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males. consistent with a role for the G-proteins in regulating GSc division frequency, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to increase the numbers of dividing GSCs in response to mating. Metazoan tissues undergo homeostasis wherein stem cells divide and their daughter cells proliferate and differentiate to replace lost cells. The human hematopoietic stem cells, for example, renew a remarkable number of about one trillion blood cells per day 1,2. Stem cells have to maintain a baseline mitotic activity for the production of daughter cells that account for the daily turnover of differentiated cells. However, whether stem cells can modulate their mitotic activity in response to demands that challenge the system is not fully explored. In some instances, stem cells respond to physiological cues; for example, murine hematopoietic stem cells divide more frequently during pregnancy due to increased oestrogen levels 3. In Drosophila melanogaster, intestinal stem cells initiate extra cell divisions upon ablation of differentiated gut cells, and GSCs modulate their mitotic activity in response to environmental conditions, such as nutrient availability and temperature 4-7. Drosophila is an excellent model for identifying the molecules and mechanisms that regulate and fine-tune tissue homeostasis. A plethora of genetic tools are available for manipulating and monitoring dividing adult stem cells. The small size of the fly, the short generation cycle, and the fairly low costs covering their maintenance allow for high throughput screens. Here, we subjected several thousand male and several million virgin female flies to mating experiments, a task challenging to perform with vertebrate model organisms. We discovered that repeated mating caused a reproducible and significant increase in GSC division frequency in Drosophila wild-type (wt) males. Our analysis revealed that this response to mating was dependent on the activity of G-proteins. Impairing G-protein activity from the germline cells eliminated the ability of the GSCs to in...