Yorkie negatively regulates embryonic tracheal tube length

Yki activity typically promotes cell division and can increase cell size, so we expected that reduced Yki activity would decrease tracheal tube length and/or diameter. Surprisingly, we found that the dorsal trunks (DTs) of stage 16 yki mutant embryos had a convoluted appearance and were 16% longer than DTs of wild-type (WT) embryos (Figure 1A,B,I; p<0.005). This over-elongated phenotype closely resembles the long tracheal phenotype caused by previously characterized tracheal tube-size control mutants such as sinu [14] and cora [18], [46], and yki trachea are indeed as long as sinu or cora mutant trachea (Fig. 1D,I; Fig. 2G, J). We confirmed that loss of yki activity was responsible for this over-elongated phenotype using two approaches. First, embryos homozygous for the small chromosomal deficiency Df(2R)BSC356 that completely deletes yki have the same long trachea phenotype as the yki^B5 mutant (Fig. 1I). Second, the long trachea phenotype caused by the yki^B5 mutation is rescued by expressing a WT form of yki in tracheal cells using the tracheal-specific breathless-GAL4 (btl-GAL4) driver in yki mutant embryos (Fig. 1C,I) [47], [48]. Significantly, this result also demonstrates that yki is acting in tracheal cells to control tracheal tube length.

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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), yki^B5 mutant trachea (B) have an over-elongated phenotype similar to the SJ mutant sinu (D). Expression of UAS-yki in the trachea of yki^B5 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.

https://doi.org/10.1371/journal.pone.0101609.g001

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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 nrv2^23B (B). The SJ paracellular barrier prevents a 10 kD fluorescent dye (white) from leaking into the tracheal lumen (dashed line) in WT and yki^B5 trachea (D,F), but is defective in the SJ mutant nrv2^23B, in which the dye fills the tracheal lumen (E). Scale bar for (A–F) in (F), 10 µm. (G–Q) Double mutant combinations of yki^B5 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 sinu^nwu7 and the shortest Hippo pathway mutant, kib⁴, reveal that kib⁴ is epistatic to sinu^nwu7, 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 yki^B5 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.

https://doi.org/10.1371/journal.pone.0101609.g002

To test if Yki was not only necessary but also sufficient for controlling tube size, we expressed Yki-GFP in the trachea of WT embryos. This expression decreased tracheal length, as did overexpression of one of Yki's transcriptional co-activators, the TEAD-family member Scalloped (Sd) (Fig.1I, p<0.05 for both). Co-expression of Yki and Sd further reduced tracheal length compared to expression of Yki alone (p<0.05, Fig. 1I). Together, these results suggest that Yki has an instructive role in regulating tracheal tube length, even though Yki activity causes the opposite of what would typically be expected for a positive regulator of cell growth.

The Hippo pathway positively regulates embryonic tracheal tube length

To determine if Yki is regulated by the Hippo pathway in the embryonic trachea, we examined zygotic mutants of the core Hippo pathway components Hpo, Warts (Wts), Salvador (Sav), Mob as tumor suppressor (Mats) and Kib [41], [49], [50]. Consistent with negative regulation of Yki by the Hippo pathway observed in other tissues, mutations in sav, wts, mats and kib had short trachea (Fig. 1F–I). Trachea in homozygous hpo mutants were not short, presumably due to maternal contribution (Fig. 1E,I). However, we were unable to confirm this possibility as embryos lacking both zygotic and maternal hpo arrested development prior to tracheal morphogenesis. Notably, the length reductions caused by mutations in Hippo pathway components were comparable to the reductions caused by overexpression of Yki and Sd, suggesting that the Hippo pathway acts through Yki to control tracheal tube size.

To more directly demonstrate that the Hippo pathway regulates Yki activity, we utilized transcriptional reporters that respond to Yki activity. These reporters are transposable element insertions in the Yki target genes thread (th) and expanded (ex) that produce a nuclear-localized lacZ in response to Yki activity [35]. Quantification of anti-β-gal fluorescence revealed an approximately 2-fold difference in reporter signal between trachea heterozygous or homozygous for the reporter insertions, indicating that the reporters should have sufficient dynamic range to detect changes in Yki activity. (Fig. 3A–E, Material and Methods). We verified that the ex reporter responds to Yki activity in the trachea by determining that reporter activity is decreased by 31% in yki^B5 mutant trachea (p = 0.006, Fig. 3F).

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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 [48]. Embryos were fixed [8], stained for GFP to visualize tracheal cell bodies and nuclei (G, green) and for Trachealess to visualize nuclei [51]. 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 yki^B5. 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 yki^B5 and cora⁵ are comparisons to WT. (I) Quantification of yki^B5 cell volumes and segment lengths at stage 16 normalized to WT measurements. yki^B5 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.

https://doi.org/10.1371/journal.pone.0101609.g003

Consistent with the Hippo pathway acting through Yki to regulate tracheal length, Yki reporter activity was increased by 79% (p<0.0005) in wts mutant trachea (Fig. 3F), which are shorter than WT (Fig. 1A vs. G, I). Reporter activity was not significantly changed in hpo mutant trachea, which have WT length (Fig. 1A vs. E, I, p>0.05). However, sav mutant trachea, which are slightly shorter than WT trachea, did not show a significant change in reporter activity, suggesting that reporter activity as measured in stage 16 embryos may not fully reflect the spatiotemporal regulation of Yki that controls tracheal length.

Yorkie acts separately from the SJ, basal polarity, and Src pathways

We next investigated whether yki mutations affect SJs or the organization of the lumenal matrix. In contrast to mutations in SJ components, such as nrv2 [18], that cause other SJ proteins to be mislocalized around the cell periphery (e.g. Cora in Fig. 2B) and loss of paracellular barrier function (Fig. 2E), in yki mutants the canonical SJ protein Cora is correctly localized to the apical region of the lateral cell-cell contacts (Fig. 2C) and paracellular barrier function is intact (Fig. 2F). Further, whereas SJ mutations prevent the secretion of the lumenal matrix protein Verm (Fig. 2B), Verm secretion appears normal in yki mutants (Fig. 2C). Thus, the long tracheal phenotype in yki mutants is not caused by grossly disrupted SJs or lumenal matrix formation.

To determine if SJs could act through Yki to control trachea tube length, we tested whether Yki reporter activity was down-regulated in SJ mutants. Null mutations of the SJ components nrv2, cora and sinu all cause tracheal length increases similar to those caused by mutations in yki (Fig. 1D,I; Fig. 2G,J,L), yet reporter activity was neither consistently nor strongly altered by these SJ mutants (Fig. 3F). Consistent with Yki and SJs acting in separate pathways, double mutant combinations of the yki^B5 null mutation and nrv2^23B null mutations have longer trachea than either the yki^B5 or the nrv2^23B mutations alone (Fig. 2G,M) and double mutant combination of yki^B5 and nrv2^23B had significantly more disruptions of lumenal matrix in the lateral branches (Fig. 2M, arrowhead). Interestingly, cora⁵, yki^B5 double mutant trachea are somewhat shorter than either cora⁵ or yki^B5 alone, but still clearly longer than WT trachea (Fig. 2G,J,K). Although unexpected, this result is nonetheless consistent with SJs and Yki not acting in a single linear pathway.

To further test the relationship between the SJ and Hippo pathways, was asked if the kib⁴ mutation, the Hippo pathway mutation that causes the shortest trachea, could suppress the long tracheal phenotype cause by the sinu^nwu7 mutation. kib⁴ is completely epistatic to sinu^nwu7 (Fig. 2R-T), demonstrating that the Hippo pathway can profoundly influence tracheal cell shape and that the Hippo pathway acts downstream or in parallel to the SJ pathway in regulating tracheal tube size.

We also investigated whether Yki acted in either of the apicobasal polarity pathways that we had previously showed control tracheal tube length [12], [16]. Apicobasal polarity of the tracheal cells was not obviously affected in yki mutants, as SJ junctional components, whose localization depends on cellular polarization and adherens junction formation, were correctly localized to the apicolateral cell-cell contacts in yki mutants (Fig. 2C). Further, the combination of a mutation in the basolateral polarity gene Lethal giant larvae (Lgl) and yki was as long or longer than either single mutant (Fig. 2G,I,N–O). Finally, Yki reporter activity was not substantially altered in embryos lacking the basolateral polarity components yurt or scrib (Fig. 3F). Thus, it appears that yki acts distinctly from apicobasal polarity pathways.

We also tested if Yki acted in the pathway containing Src42, which controls both the orientation and amount of growth of tracheal cells and acts distinctly from SJs and apicobasal polarity pathways [25]. Double mutant combinations of yki^B5 and Src42^26-1, a putative null allele, have the same short trachea phenotype of Src42^26-1 mutants, indicating that Src42 is largely epistatic to yki (Fig. 2G,P-Q). In contrast to SJ mutations, which do not modify the Src42 length phenotype [25], the yki^B5 mutation slightly lengthens the Src42 mutant trachea (p<0.05), suggesting that Yki may act in parallel to the Src42 pathway. Consistent with this possibility, Yki reporter activity was reduced by only 10% in Src42 mutant trachea. While it is possible that the ability of yki mutations to affect the Src42 mutant phenotype results from maternal Src42 contributions, the above results suggest that Yki activity represents a distinct input into mechanisms that control tracheal tube size from the known pathways involving SJs, apicobasal polarity and Src42.

Yorkie mutants increase tube length without increasing cell volume or number

As Yki plays a critical role in controlling cell proliferation in many tissues and can also control cell size [30], [31], [34], we investigated the possibility that loss of Yki alters tracheal cell volume or cell number. To allow comparison with normal development, we first characterized trachea cell volume in WT embryos, which had not been previously done. Tracheal cell bodies and nuclei were visualized by staining for a tracheal-GFP and for the transcription factor Trachealess (Trh) [51] (Fig. 3G and materials and methods). Cell volumes and number measurements revealed several unexpected findings. First, tracheal cell volume steadily increases along the length of the dorsal trunk. Surprisingly, at stage 16, anterior cells (DT2) have 44% less volume than posterior cells (DT8; 32 µ³±3 vs. 57 µ³±2, respectively, p<0.0005; Fig. 3H). This anterior-to-posterior differential is not observed in epidermal cells that gave rise to the tracheal cells approximately six hours earlier, but does account for anterior tracheal cells having a more squamous (flatter) profile than the more cuboidal posterior cells (epidermal cell volume for segments 2, 5 and 8, respectively: 95 µ³±12, 92 µ³±11, 103 µ³±8). Second, cell volume is not linearly proportional to cell length (Fig. 3H). For example, the posterior segment DT8 is only 22% longer than the anterior segment DT2, yet DT8 cells have a 60% larger volume than DT2 cells, and there are 31% more cells in DT8 than DT2 (Fig. 3H, 4J). Third, despite the length of tracheal segments increasing over the 4-hour period from stage 14–16, there is a consistent, but not statistically significant, decrease in tracheal volume over this time (Fig. 3H). Thus, while tracheal cell volume is dynamically regulated during development, tube length increases are not driven by increases in cell volume.

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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 th^J5C8 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 th^6B 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 yki^B5 mutants is sufficient to rescue tracheal length to WT. (I) Expression of th in WT trachea shortens trachea, similar to the GOF th^6B allele and Ice mutants. Yellow lines indicate dorsal trunk measurements. Lumens visualized by 2A12 staining. (J) Quantification of tracheal cell numbers in w¹¹¹⁸, cora⁵, yki^B5, th^J5C8, 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.

https://doi.org/10.1371/journal.pone.0101609.g004

Having established a baseline of WT development, we determined tracheal cell number and volume in yki mutants and, for comparison, the SJ mutant cor. Consistent with the almost complete absence of cell death or division during most of tracheal morphogenesis [9], [10], cell number was not significantly altered in yki mutants (Fig. 4J). Tracheal cell volume in yki mutants was also not statistically different than in WT (Fig. 3I). Thus, the increased tracheal length in yki mutants is not driven by increased tracheal cell volume. Surprisingly, in the SJ mutant cor, cell volumes were significantly increased compared to WT (Fig. 3H; p<0.05 for DT2 and DT5, p<0.005 for DT8), providing further evidence that Yki does not act in the same pathway as SJ components. In combination, these results suggest that while cell volume is dynamically regulated in the tracheal system, cell volume changes do not drive tube length increases.

Yki acts through thread/DIAP to control tracheal tube length

Since Yki does not appear to control tracheal size by regulating cell growth, we examined embryos mutant for the known Yki transcriptional targets: thread (th, which encodes the protein DIAP1- Drosophila Inhibitor of Apoptosis), expanded (ex), and bantam (ban). Tracheal length was not substantially changed in ban or ex mutants (Fig. 4E,F,K). However, embryos homozygous for the reduction-of-function th^J5C8 mutation, which results from a transposable element insertion in the th 5′ UTR [52], [53], had dramatically longer trachea, comparable to length increases in yki^B5 mutants (p<0.005; Fig. 4A,K). We attempted to confirm this result using additional loss-of-function alleles of th: th⁴, which substitutes a tyrosine for an invariant histidine in the BIR2 domain, and th⁷, which is a small deletion the removes part of the BIR2 domain [54]. Unfortunately, most homozygous th⁴ embryos had severe morphogenesis defects and it was not possible to meaningfully determine tracheal length of this genotype (data not shown). On the other hand, th⁷ mutants had normal embryonic morphology, but also had WT tracheal length, presumably because of a combination of maternal contribution and th⁷ being a partial loss-of-function mutation.

To further investigate the role of DIAP in tracheal size control, we also examined embryos homozygous for three gain-of-function mutations in the th locus, each of which has a different spectrum of interactions with the pro-apoptotic proteins Grim, Reaper and Hid, and with the caspases Dronc and Ice [54]–[56]. We examined th^SL, which is a valine to methionine substitution in the BIR1 domain [54], th^21-2s which is a semi-lethal allele resulting from a proline to serine change also in the BIR1 domain [55] and th^6B which substitutes tyrosine for an invariant cysteine in the RING domain [54]. Tracheal length was normal in homozygous th^SL and th^21-2s st. 16 embryos. However embryos homozygous for th^6B had shortened trachea that were as short as the strongest SWH mutants (Fig. 4C,I; [57]). Since the th^6B can act as a dominant negative mutation with regard to Hid induced-killing in the eye [54], this result supports the model that DIAP mediates the effects of Yki activity and implicates Hid as a potential regulator of tracheal length. Notably, th^SL mutation does not act as dominant negative with regard to Hid [54]. The significance of the semi-viable th^21-2s mutation not causing a tracheal phenotype is unclear since the mutation only causes partial lethality and was scored by Goyal et al. as a weak suppressor of Hid in the eye. Although the genetics of DIAP1 mutations is complex [56], and further complicated by maternal contribution in the embryo, these results clearly implicate DIAP as a downstream effector of the Yki in tracheal size control.

To more directly assess the role of DIAP in tracheal size control, we expressed WT th in the trachea of otherwise WT embryos. Consistent with th mediating the effects of yki on tracheal size, th expression resulted in shortened tracheal tubes that were approximately the same length as Hippo pathway mutants (Fig. 4I,K). To confirm the overall relationship of the Hippo pathway and th, we examined kib⁴, th^J5C8 double mutants (a short kib mutant in combination with a long th mutant), however the mid-body tracheal segments were grossly defective in these embryos and we were unable to determine tracheal length (data not shown). Critically, expressing th in the trachea of yki^B5 mutants restored tracheal length to WT (Fig. 4H,K), strongly supporting the model that th/DIAP1 is the primary mediator of Yki function in tube size control.

A non-apoptotic function of the caspase Ice mediates tracheal tube size control

How does the th/DIAP1 control tube size? A critical function of DIAP1 is to regulate cell death by binding and inhibiting the pro-apoptotic caspases Dronc and Ice [58], [59]. We tested if caspases control tracheal size by characterizing the phenotypes of mutations in Dronc and Ice. Tracheal length was not altered by the Dronc⁵¹ null mutation, however, the loss-of-function Ice¹⁷ and Ice^Δ1 mutations decreased tracheal length as much as the th^6B and wts^338/1489 mutations (Fig. 4C–D,K). Given that Dronc is thought to be required to activate Ice (reviewed in [56]), it is somewhat surprising that Ice but not Dronc mutations affect tracheal length. However, it is likely that a role for Dronc in tracheal size control is obscured by the significant maternal contribution of Dronc [56]. To confirm a role for Ice and its proteolytic activity in size control, we used the tracheal specific driver btl-Gal4 to express baculovirus p35, a well-characterized caspase inhibitor that covalently attaches to and inhibits Ice. p35 expression decreased tracheal length similar to Ice and th^6B mutations. Because p35 blocks proteolytic activity of Ice without necessarily eliminating Ice protein, this result suggests the proteolytic activity of Ice is required for tracheal size control, even though Baer et al. did not observe significant staining of an antiserum that binds cleaved caspases in the dorsal trunks of late stage embryos [10]. Strikingly, expressing p35 in yki^B5 mutant trachea restored tracheal length to WT, strongly supporting the model that Yki acts through DIAP1 and subsequently Ice to regulate tube size (Fig. 4K).

To confirm that changes in overall tracheal cell number were not influencing tracheal system length, we counted the tracheal cells in yki, th and Ice mutants (Fig. 4J). Tracheal cell numbers in these mutants were within the WT range and not correlated with tracheal length (Fig. 4J and [8], [9]). Thus, control of epithelial tube length by Yki-DIAP1-Ice is a non-apoptotic function of this evolutionarily conserved cell death pathway.

Fly reagents

Fly stocks and alleles were obtained from Bloomington Drosophila Stock Center [70] or were generously provided by A. Bergmann (Ice¹⁷), R. Carthew (yw hsFLP; FRT42D, FRT42B ovo^D1/MS(2)M1, Sp/CyO), R. Fehon (yki^B5;UAS-FLAG-yki), A. Ghabriel (wts^{miracle-gro338}, wts^{miracle-gro1483}, wts^{miracle-gro1489}), G. Halder (yki^B5, ex^e1), I. Hariharan (wts^MGH1, sav¹), B. Hay (Ice^Δ1), R. Holmgren (UAS-p35), J. Kennison (th⁷), K. Moberg (th^J5C8, ex⁶⁹⁷), H. Steller (th^21-2s), H. Stocker (kib⁴), and Kristin White (th^6B, th^SL). Fly stocks were maintained at room temperature and were balanced with dfd-GMR-YFP marked balancer chromosomes [71], which also allowed genotyping of embryos. Imaging performed at stage 16 unless otherwise noted. Germline mosaic clones were attempted using the hpo^KS240 allele and published approaches [72]–[74].

Immunohistochemistry

The following antibodies were used: mouse (ms) anti-2A12 1∶2, rabbit (rb) anti-Verm 1∶300 (S. Luschnig), rat anti-Trachealess 1∶50 (B. Shilo [51]), guinea pig (gp) anti-Cora 1∶10000 (R. Fehon), rb anti-β-galactosidase 1∶75 (Capel), ms anti-GFP (Molecular Probes, A11120), and rb anti-GFP (Molecular Probes, A11122). Alexa Fluor 488, 546, and 647 were used for secondary antibodies (Molecular Probes). The lumenal antibody 2A12, embryo fixation, staining, and staging procedures were performed as previously described [8]. All embryos were imaged with a Leica SP2 or SP5 confocal microscope. Control heterozygous embryos were imaged on the same slide as the experimental homozygous embryos using the same laser settings such that relative signal levels could be directly compared. For Yki reporter activity experiments, lasers were set such that in a control embryo, the maximum signal for pixels in tracheal nuclei did not saturate the detector.

Measurements

Confocal image analysis was performed using the Perkin Elmer Volocity software program. Normalized tracheal lengths were calculated by measuring the length of the dorsal trunk from DB2 to DB10 and dividing by embryo body length. For Yki activity experiments, signal levels of 20–30 nuclei in DT7 and DT8 tracheal cells (3–5 sections/segment) per embryo were measured and averaged. Tracheal metamere volumes were determined from confocal sections of fixed embryos that had expressed a cytoplasmic and nuclear GFP in the trachea. GFP was visualized with a fluorescent secondary antibody. Trachea were automatically detected as “objects” with a fluorescence intensity above a threshold value. To fill missing volumes in the cell bodies due to non-uniform expression of GFP, automatic functions of the Volocity software were used to “dilate”, “close” and “fill” spaces within the confocal stack. This resulted in a contiguous filled object for the cell bodies of the trachea. A representative image of processed trachea is shown in Fig. 3G. Tracheal metameres were manually segmented and the volume of segmented object was calculated. For statistical analyses, p-values were calculated using Student's t-test with two-tailed unpaired variance.

Dye exclusion assay

Paracellular barrier function of tracheal SJs was assayed as previously described by injecting a 10 kD Texas Red-conjugated dextran (Molecular Probes) into the body cavities of stage 16 embryos [75]. Injected embryos were imaged on a Leica TCS SP2 confocal microscope within 20 min of injection.