Apontic regulates somatic stem cell numbers in Drosophila testes

BACKGROUND: Microenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells. RESULTS: This study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. We determined that the transcriptional regulator Apontic (Apt) acts in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. We found Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population. CONCLUSIONS: Genetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, we determined JAK/STAT signaling promotes apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. We propose that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation

Identification of Novel Regulators of the JAK/STAT Signaling Pathway that Control Border Cell Migration in the Drosophila Ovary

The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway is an essential regulator of cell migration both in mammals and fruit flies. Cell migration is required for normal embryonic development and immune response but can also lead to detrimental outcomes, such as tumor metastasis. A cluster of cells termed “border cells” in the Drosophila ovary provides an excellent example of a collective cell migration, in which two different cell types coordinate their movements. Border cells arise within the follicular epithelium and are required to invade the neighboring cells and migrate to the oocyte to contribute to a fertilizable egg. Multiple components of the STAT signaling pathway are required during border cell specification and migration; however, the functions and identities of other potential regulators of the pathway during these processes are not yet known. To find new components of the pathway that govern cell invasiveness, we knocked down 48 predicted STAT modulators using RNAi expression in follicle cells, and assayed defective cell movement. We have shown that seven of these regulators are involved in either border cell specification or migration. Examination of the epistatic relationship between candidate genes and Stat92E reveals that the products of two genes, Protein tyrosine phosphatase 61F (Ptp61F) and brahma (brm), interact with Stat92E during both border cell specification and migration

Additional file 5: Figure S5. of Apontic regulates somatic stem cell numbers in Drosophila testes

Apt limits the GSC population at the hub interface and E-cadherin expression. A-B) Single optical sections of testes stained with antibodies that recognize Vasa (magenta), Tj (white), and Fas3 (white, to label the hub). The hub is outlined in blue. Scale bars = 10 μm. Testes from homozygous apt KG05830 males exhibit a significant increase in GSCs (magenta) contacting the hub (B), relative to wild type (A). C) Number of GSCs at the hub interface for the indicated genotypes. D-E) Images of testes stained with antibodies specific for E-cadherin (magenta and insets), Vasa (white), and DAPI (blue). Scale bars = 20 μm. An increase or mislocalization of E-cadherin expression is observed in the cells surrounding the hub, including the Vasa + GSCs (arrows) in a testis from an apt KG05830 homozygous male (E), compared to a w 1118 testis (D, arrows), where it is barely detected outside the hub. Images were taken under the same conditions. F-G) Single optical sections of testes stained for Apt (magenta and insets), Tj (white), and DAPI (blue). Arrows indicate GSCs; arrowheads show CySCs. Scale bars = 10 μm. F) apt KG05830 /+ heterozygotes show no significant reduction of Apt protein in CySCs and a mild reduction in GSCs. G) In homozygous mutant males, Apt expression is reduced in CySCs (first tier of Apt+/Tj + cells proximal to the hub: arrowheads) but is not detected in the germline (arrows, the presence of a cell is indicated by DAPI). H) Quantification of the relative expression levels of Apt protein in the stem cell populations adjacent to the hub for the indicated genotypes. "n" is the number of testes examined with the number of cells in parentheses. Statistical significance was tested via two-tailed t-tests, where *p < 0.05, ***p < 0.005, ****p < 0.0001, and n.s. = not significant. Experimental genotypes were tested against Canton S, unless indicated by a bar. (TIF 14942 kb

Additional file 4: Figure S4. of Apontic regulates somatic stem cell numbers in Drosophila testes

Somatic reduction of apt heightens STAT expression in the CySCs. A-B) Testes stained with antibodies recognizing STAT (magenta), Tj (white, somatic cells), and Fas3 (white, hub: blue outline) and counterstained with DAPI (blue, nuclei). Arrowheads indicate CySCs (first tier of Tj + cells around the hub). Scale bars = 20 μm. Insets display STAT expression, alone. A) Control testis shows wild - type STAT expression: most detectable STAT is found in the GSCs around the hub (labeled arrows), but it decreases in gonialblasts and CySCs (arrowheads). A Tj + cell distal from the hub shows undetectable levels of nSTAT (unlabeled arrow). B) More STAT is detectable when apt is reduced in somatic cells via Tj-Gal4. Tj + cells several cell diameters away from the hub displayed high levels of nSTAT (asterisks). C) Nuclear STAT (nSTAT) levels were quantified in CySCs and normalized to DAPI intensity. Tj staining was utilized to outline nuclei of CySCs for measurement (see Methods). Tj-Gal4;aptRNAi was normalized to the Tj-Gal4 or aptRNAi-alone controls to obtain a relative expression level. Somatic reduction of apt significantly increases nSTAT levels in CySCs. Two-tailed t-tests were used to test for significance, as indicated. “n” provides the total number of testes examined for each genotype, while the number of individual cells analyzed is given in parentheses. (TIF 8047 kb

Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target

A Mathematical Model of Collective Cell Migration in a Three-Dimensional, Heterogeneous Environment

<div><p>Cell migration is essential in animal development, homeostasis, and disease progression, but many questions remain unanswered about how this process is controlled. While many kinds of individual cell movements have been characterized, less effort has been directed towards understanding how clusters of cells migrate collectively through heterogeneous, cellular environments. To explore this, we have focused on the migration of the border cells during Drosophila egg development. In this case, a cluster of different cell types coalesce and traverse as a group between large cells, called nurse cells, in the center of the egg chamber. We have developed a new model for this collective cell migration based on the forces of adhesion, repulsion, migration and stochastic fluctuation to generate the movement of discrete cells. We implement the model using Identical Math Cells, or IMCs. IMCs can each represent one biological cell of the system, or can be aggregated using increased adhesion forces to model the dynamics of larger biological cells. The domain of interest is filled with IMCs, each assigned specific biophysical properties to mimic a diversity of cell types. Using this system, we have successfully simulated the migration of the border cell cluster through an environment filled with larger cells, which represent nurse cells. Interestingly, our simulations suggest that the forces utilized in this model are sufficient to produce behaviors of the cluster that are observed <i>in vivo</i>, such as rotation. Our framework was developed to capture a heterogeneous cell population, and our implementation strategy allows for diverse, but precise, initial position specification over a three- dimensional domain. Therefore, we believe that this model will be useful for not only examining aspects of <i>Drosophila</i> oogenesis, but also for modeling other two or three-dimensional systems that have multiple cell types and where investigating the forces between cells is of interest.</p></div

Effects of cell packing on chemoattractant distribution within a tissue

Diffusible signals provide critical information to cells in biological systems, often in a concentration-dependent manner. In animal development, such signals can determine different cell fates or guide motile cells to their proper locations. It is well-known that migrating cells respond to graded chemoattractant cues by moving toward areas of higher concentrations. However, it is not clear how cell-dense animal tissues impact the distribution of chemoattractants in three dimensions. We leverage the simple architecture of the Drosophila egg chamber to explore this idea. In this context, sixteen large germline cells are packed together, enveloped by a somatic epithelium. A small set of epithelial cells, the border cells, form a motile cell cluster and respond to guidance signals by moving across the egg chamber during oogenesis. We created a geometrically-realistic model of the egg chamber and determined the distribution of the chemoattractants through that domain using a reaction-diffusion system. We used this information to determine reasonable biophysical parameters of chemoattractant that would facilitate gradient formation in the appropriate developmental time, and to explore the effects of different secretion locations in the egg chamber. Our model revealed several interesting features: The chemoattractant is more concentrated and the gradient sets up more quickly in a cell-packed space, and cell packing creates dips in the concentration and changes in gradient along the migratory path. We simulated migration with our calculated chemoattractant gradient and compared it to that with a constant gradient. We found that with our calculated gradient, migration was slower initially than in the constant gradient, which could be due to the exponential nature of the gradient or other variation in signal due to the heterogeneous domain. Given the many situations in which cell migration occurs in complex spatio-temporal environments, including development, immune response, and cancer metastasis, we believe modeling chemoattractant distribution in heterogeneous domains is widely relevant