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. 2014 Jul 18;9(7):e101609.
doi: 10.1371/journal.pone.0101609. eCollection 2014.

Non-canonical Roles for Yorkie and Drosophila Inhibitor of Apoptosis 1 in Epithelial Tube Size Control

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Free PMC article

Non-canonical Roles for Yorkie and Drosophila Inhibitor of Apoptosis 1 in Epithelial Tube Size Control

Renée M Robbins et al. PLoS One. .
Free PMC article

Abstract

Precise control of epithelial tube size is critical for organ function, yet the molecular mechanisms remain poorly understood. Here, we examine the roles of cell growth and a highly conserved organ growth regulatory pathway in controlling the dimensions of the Drosophila tracheal (airway) system, a well-characterized system for investigating epithelial tube morphogenesis. We find that tracheal tube-size is regulated in unexpected ways by the transcription factor Yorkie (Yki, homolog of mammalian YAP and TAZ) and the Salvador/Warts/Hippo (SWH) kinase pathway. Yki activity typically promotes cell division, inhibits apoptosis, and can promote cell growth. However, reducing Yki activity in developing embryos increases rather than decreases the length of the major tracheal tubes, the dorsal trunks (DTs). Similarly, reduction of Hippo pathway activity, which antagonizes Yki, shortens tracheal DTs. yki mutations do not alter DT cell volume or cell number, indicating that Yki and the Hippo pathway regulate cell shape and apical surface area, but not volume. Yki does not appear to act through known tracheal pathways of apical extracellular matrix, septate junctions (SJs), basolateral or tubular polarity. Instead, the Hippo pathway and Yki appear to act downstream or in parallel to SJs because a double mutant combination of an upstream Hippo pathway activator, kibra, and the SJ component sinu have the short tracheal phenotype of a kibra mutant. We demonstrate that the critical target of Yki in tube size control is Drosophila Inhibitor of Apoptosis 1 (DIAP1), which in turn antagonizes the Drosophila effector caspase, Ice. Strikingly, there is no change in tracheal cell number in DIAP1 or Ice mutants, thus epithelial tube size regulation defines new non-apoptotic roles for Yki, DIAP1 and Ice.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Yorkie and the Hippo Pathway Regulate Tracheal Tube Size.
(A–D) Maximum projections of confocal sections showing tracheal lengths. Compared to stage 16 WT embryos (A), ykiB5 mutant trachea (B) have an over-elongated phenotype similar to the SJ mutant sinu (D). Expression of UAS-yki in the trachea of ykiB5 mutants rescues tracheal length defects (C). Yellow lines indicate dorsal trunk measurements. Tracheal lumens visualized by anti-2A12 marker staining. (E–H) Although embryos trans-heterozygous for hpo mutations have WT tracheal length (E), mutations in Hippo pathway components sav, wts and kib shorten tracheal length (F–H, respectively). (I) Quantification of tracheal length shows that decreased Yki activity lengthens trachea, while increased Yki activity or decreased Hpo pathway activity reduces tracheal length. Tracheal length was measured in three dimensions in confocal stacks, normalized to embryo length and then normalized to WT (w1118, red line). Error bars show normalized s.e.m.; *: p<0.05; **: p<0.005; ***: p<0.0005. p-values determined using Student's t-test. Scale bar for (A–G) in (H), 50 µm.
Figure 2
Figure 2. Yki Acts Separately from the Verm/aECM, SJ, Basolateral Polarity, and Src Pathways.
(A–F) The localization of lumenal Verm (green) and the SJ marker Cora (red) is similar in the trachea of WT (A) and yki mutant (C) embryos (arrow). In contrast, Verm secretion is dramatically reduced (lack of green staining) and Cora is mislocalized to the lateral membranes (arrowhead) in the SJ mutant nrv223B (B). The SJ paracellular barrier prevents a 10 kD fluorescent dye (white) from leaking into the tracheal lumen (dashed line) in WT and ykiB5 trachea (D,F), but is defective in the SJ mutant nrv223B, in which the dye fills the tracheal lumen (E). Scale bar for (A–F) in (F), 10 µm. (G–Q) Double mutant combinations of ykiB5 with representative mutants in known tracheal pathways all show additive effects of the mutations, suggesting that yki acts in a distinct pathway. (R–T) Double mutant combination of the long SJ mutant sinunwu7 and the shortest Hippo pathway mutant, kib4, reveal that kib4 is epistatic to sinunwu7, suggesting that the Hippo pathway acts downstream or in parallel to the SJ pathway. (H–Q,S–T) Maximum projections of confocal stacks. Yellow lines indicate dorsal trunk measurements. In addition to having increased length compared to either single mutant, the yki, nrv2 double mutant had much more severe lumenal secretion and morphogenesis defects in the lateral tracheal branches than either single mutant (arrows vs. arrowhead in L and M). (G,R) Quantification of tracheal lengths in single and double mutant embryos. The red line in (G) marks the length of ykiB5 mutant trachea for comparison to double mutant combinations. Tracheal length was measured in three dimensions in confocal stacks, normalized to embryo length and then normalized to WT (w1118). Error bars are normalized s.e.m.; *: p<0.05; **: p<0.005; ***: p<0.0005. p-values determined using Student's t-test. Scale bar for (G–P) in (P), 50 µm.
Figure 3
Figure 3. Tracheal Length Changes are Correlated with Yki Activity, but Not with Tracheal Cell Size.
(A–F) Quantification of Yki reporter activity in WT and tracheal mutant backgrounds. Embryos homozygous for DIAP-lacZ reporter (A, green) or ex-lacZ (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) yki mutant embryos show strongly reduced reporter activity, while wts mutant embryos have dramatically increased reporter activity. Mutations in SJ and basolateral polarity pathways do not consistently or strongly affect reporter activity. Measurements for yki, hpo, nrv2, cora, and Src42 used ex-lacZ, while sav, wts, sinu, scrib, and yrt 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 . Embryos were fixed , stained for GFP to visualize tracheal cell bodies and nuclei (G, green) and for Trachealess to visualize nuclei . 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 ykiB5. Average cell volume and metamere length were also determined for cora5 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 ykiB5 and cora5 are comparisons to WT. (I) Quantification of ykiB5 cell volumes and segment lengths at stage 16 normalized to WT measurements. ykiB5 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-values determined using Student's t-test. Error bars show s.e.m.
Figure 4
Figure 4. Non-apoptotic functions of th/DIAP1 and Ice Mediate Yki Activity in Tracheal Size Control.
(A–C) th/DIAP1 regulates tracheal tube length. The loss-of-function allele thJ5C8 causes an over-elongated trachea (A) that closely resembles the yki phenotype (G). The hypomorphic allele th7 does not alter length (B) but the gain-of-function allele th6B shortens trachea (C). (D) A loss-of-function allele of Ice, an effector caspase normally inhibited by DIAP1, shortens the trachea, similar to the GOF th allele in (C). (E–F) Mutations in other Yki targets, ex and bantam, do not cause dramatic changes in tracheal length. (G–H) Expression of th in the trachea of ykiB5 mutants is sufficient to rescue tracheal length to WT. (I) Expression of th in WT trachea shortens trachea, similar to the GOF th6B allele and Ice mutants. Yellow lines indicate dorsal trunk measurements. Lumens visualized by 2A12 staining. (J) Quantification of tracheal cell numbers in w1118, cora5, ykiB5, thJ5C8, and Ice17 mutants. (K) Quantification of tracheal lengths for th, Ice, ex and ban mutants, and th and p35 expression experiments. Tracheal length was measured in three dimensions in confocal stacks, normalized to embryo length and then normalized to WT (w1118, red line). Error bars show normalized s.e.m.; *: p<0.05; **: p<0.005; ***: p<0.0005. p-values determined using Student's t-test. Scale bar for (A–H) in (H), 50 µm. (L) Model of genetic pathways regulating tracheal tube length.

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