Tracheal Length Changes are Correlated with Yki Activity, but Not with Tracheal Cell Size.

<p>(A–F) Quantification of Yki reporter activity in WT and tracheal mutant backgrounds. Embryos homozygous for <i>DIAP-lacZ</i> reporter (A, green) or <i>ex-lacZ</i> (C, red) have approximately twice the signal (E) of nuclear β-gal staining in tracheal cells compared to embryos heterozygous for these reporters (B,D). (F) <i>yki</i> mutant embryos show strongly reduced reporter activity, while <i>wts</i> mutant embryos have dramatically increased reporter activity. Mutations in SJ and basolateral polarity pathways do not consistently or strongly affect reporter activity. Measurements for <i>yki</i>, <i>hpo</i>, <i>nrv2</i>, <i>cora</i>, and <i>Src42</i> used ex-lacZ, while <i>sav</i>, <i>wts</i>, <i>sinu</i>, <i>scrib</i>, and <i>yrt</i> used DIAP-lacZ. Normalized values are shown as the fold-change of the signal in trachea homozygous for a mutation compared to heterozygous control trachea on the same slide. Scale bar for (A–D) in (D), 10 µm. (G–I) Tracheal segment length is not correlated with cell volume. Cell volumes were measured in embryos expressing a cytoplasmic and nuclear GFP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101609#pone.0101609-Shiga1" target="_blank">[48]</a>. Embryos were fixed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101609#pone.0101609-Samakovlis1" target="_blank">[8]</a>, stained for GFP to visualize tracheal cell bodies and nuclei (G, green) and for Trachealess to visualize nuclei <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101609#pone.0101609-Wilk1" target="_blank">[51]</a>. Cell numbers, volumes, and segment lengths were analyzed using Volocity software (materials and methods). (H) Quantification of average tracheal cell volume (in light and dark blue) and metamere length (in light and dark orange) for segments 2, 5, and 8 at stages 14 (light blue and light orange) and 16 (dark blue and dark orange) for both WT and <i>yki^B5</i>. Average cell volume and metamere length were also determined for <i>cora⁵</i> at stage 16. Notably, cell volumes consistently decrease (but are not statistically significant) between stages 14 and 16, whereas segment length increases. Statistics shown for <i>yki^B5</i> and <i>cora⁵</i> are comparisons to WT. (I) Quantification of <i>yki^B5</i> cell volumes and segment lengths at stage 16 normalized to WT measurements. <i>yki^B5</i> mutant trachea are longer (orange) than WT, but cell volumes (blue) are not statistically significant different than WT. Legend in (H) also applies to (I). *, p<0.05; **, p<0.005; ***, p<0.0005; n.s., not statistically significant; p<i>-</i>values determined using Student's <i>t</i>-test. Error bars show s.e.m.