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

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

Simulating the three dimensional model results in collective migration.

<p>A simulation showing six border cells (green), two polar cells (red), the epithelium (transparent green), and the surface of the oocyte (black, right) at three time points during the migration. Fifteen nurse cells are situated inside the egg chamber, but are not plotted so as to maintain clarity of this three dimensional structure. Polar cells are surrounded by border cells, making them hard to distinguish. (A) At 2 minutes, cells are beginning to invade between nurse cells. (B) At 2.4 hours, the cluster is about halfway to its destination. (C) At 5.6 hours, the border cell cluster has reached the edge of the oocyte. See also Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122799#pone.0122799.s003" target="_blank">S3 Movie</a>.</p

Composite non-dimensional parameter values used for simulation.

<p>The parameter <i>α</i> is the time scale (an hour) and <i>D</i> is the diameter of an IMC (7<i>μ</i>m). We can consider the <i>α</i> and <i>D</i> scaling the damping viscosity coefficient, <i>μ</i>, then the parameters in the table represent a ratio of force exerted on the IMC by movement through the heterogeneous medium and the force exerted by the adhesion, repulsion, migration and stochastic forces.</p

Simulations with four, six, and eight border cells at the same time point (<i>t</i> = 1.8 hours) during migration.

<p>The cluster with four border cells (A) has moved significantly less distance than the cluster with six (B) or eight (C) border cells.</p

Polar cell positions along main axis of migration.

<p>(A) The distance of the polar cells from the anterior of the egg chamber versus time. (B) The relative positions of the two polar cells to one another, along the axis that runs from anterior to posterior through the egg chamber. Each line corresponds to one of the polar cells. As the cluster moves forward, we observe that the polar cells are changing position with respect to one another along this axis, including a complete switch at 0.8 hours. This simulation modeled six border cells.</p

Border cell migration in <i>Drosophila melanogaster</i> egg development.

<p>(a) At the beginning of stage eight, the polar cells (yellow) and border cells (green) lie in the follicular epithelium of the developing egg chamber. In stage nine, these cells coalesce to form a cluster that detaches from the epithelium. The cluster then translocates between large nurse cells through the egg chamber to reach the developing oocyte (gray) by stage ten. The border cell cluster migrates about 150<i>μ</i>m over approximately 4–6 hours. (b) Still images from a time-lapse movie of wild-type border cell migration. The motile cells are marked in green by expression of Slbo-life-Act-GFP. The oocyte, which autofluoresces, is indicated by the dashed line. The nuclei of all cells, including the large, polyploid nurse cells, are seen in blue. In the image at time 0, border cells have already clustered (arrow) and begun moving towards the oocyte. In this example, the border cells reached the oocyte border by 3.5h (arrow on right-most panel). (c) Still images at a higher magnification from a time-lapse movie of a different egg chamber. Images differ by 30 minute intervals. The border cells are marked by a membrane-tethered GFP, and show wild-type behavior. Cells can be observed to change relative positions with respect to the front of the cluster as they move toward the right (arrow and arrowhead indicate the same cell over time). See also Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122799#pone.0122799.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122799#pone.0122799.s002" target="_blank">S2</a> Movies.</p

The total time and relative time taken for computational clusters of 4, 6 and 8 border cells to complete migration.

<p>The total time and relative time taken for computational clusters of 4, 6 and 8 border cells to complete migration.</p

The Phenotype of <i>M⁻ Z⁻ tre1</i> Mutant Embryos

<div><p>Anterior is left in all figures.</p> <p>(A–F) Embryos are stained with anti-Vasa (brown) to mark germ cells. (A–D) Lateral views. (E–F) Top views. (A), (C), and (E) are wild-type embryos. (B), (D), and (F) are <i>tre1</i> mutant embryos. Wild-type germ cells migrate out of the PMG at stage 10 (A) and migrate toward mesoderm at stage 11 (C) and finally to the gonad at stage 13 (E), but in <i>tre1</i> mutant embryos, germ cells fail to leave the PMG ([B] shows stage 10 and [D] shows stage 11) and are mostly found “clumped” together in the middle of the gut at stage 13 (F).</p> <p>(G–J) High magnification view of wild-type (G and H) and <i>tre1</i> mutant (I and J) embryos stained with anti-Neurotactin (red) to mark cell membranes of midgut epithelium and germ cell-specific anti-Vasa (green). Wild-type germ cells are migrating out of the PMG at early stage 10 (G) and are outside of the PMG and thus at a different optical plane than PMG at late stage 10 (H). <i>tre1</i> germ cells, in contrast, do not migrate out of the PMG at stage 9/10 (I) and are still left inside the PMG and thus at the same optical level as the PMG cells at late stage 10 (J). Punctate appearance of anti-Vasa staining in <i>tre</i> mutant germ cells is likely due to heat fixation protocol used as it can also be observed in wild-type germ cells (data not shown).</p></div