Author response: A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea
2015-11-02

Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation. https://doi.org/10.7554/eLife.09373.001 eLife digest Animal cells can secrete proteins and molecules into the space around them to create a support they can attach to. This structure – known as the extracellular matrix – comes in various forms and can help to shape tissues or influence the way in which cells behave. Inside cells, filaments made of a protein called actin also provide structural support. In fruit fly larvae, “tracheal” cells create a network of tubes that will form the airways of the adult fly. Once this network is complete, these cells secrete the materials to make an extracellular matrix in the internal (apical) surface of the tubes. This matrix has a series of spiralling ridges made from a molecule called chitin. These ridges run along the tubes, spanning several cells and providing the mechanical strength needed to keep the airways open. The ridges appear to form through a co-ordinated effort between the cells, and recent studies suggest that actin filaments may be involved in this process. Here, Öztürk-Çolak et al. investigate this idea further by carrying out a detailed analysis of the relationship between the extracellular matrix and the tracheal cells as the airways develop. The experiments reveal that rings of actin filaments form on the apical side of tracheal cells before the ridges appear. These rings generate regular folds in the membrane that surrounds each tracheal cell and are required for an enzyme to accumulate in the cells. This enzyme produces chitin, leading to its deposition in stripes above the actin rings. Further experiments show that the junctions between cells play an important role in organising the pattern of the extracellular matrix. The active form of a protein called Src42A – which is known to regulate the way actin filaments are organized inside cells – accumulates at these junctions. Excessive Src42A activity in tracheal cells alters the networks of actin filaments and disrupts the formation of the matrix. Öztürk-Çolak et al. also find evidence of a “feedback” mechanism, in which the presence of chitin reduces the activity of Src42A to maintain the correct patterning of actin. These findings reveal that actin and junctions between cells play a central role in co-ordinating the formation of the extracellular matrix in fruit fly airways. The next challenge will be to understand which proteins and other molecules are involved in the process that allows the extracellular matrix to communicate with the cells. https://doi.org/10.7554/eLife.09373.002 Introduction “The anatomically internal lining of the trachea consists of a chitinous layer which presents what is usually termed a 'spiral thickening', but whose form is really that of a helicoid. (...) then we have to explain just how it is that the cells of the tracheal epithelium can cooperate unconsciously so as to form a helicoid thickening continuous from one end of the trachea to another, especially since each cell produces not merely one section of the continuous filament, but several parallel sections of unequal length.” (Thompson, 1929) The morphology of organs, and hence their proper physiology, relies to a considerable extent on the extracellular matrix (ECM) secreted by their cells. On the one hand, the ECM has a key role in cell signalling events, either acting as a storage compartment for growth factor molecules or by modifying and/or stabilising such growth factors. On the other hand, the ECM provides an environment with a variable degree of stiffness that, until recently, has been considered a physical support to ensure the arrangement of softer parts of body organs. However, increasing evidence indicates that the ECM not only provides a passive contribution to organ shape but also impinges on the cell behaviour and genetic programmes of the organ (Daley and Yamada, 2013). The ECM is emerging as a direct modulator of many aspects of cell biology, rather than as a mere physical network that supports cells (Hynes, 2014). But how does the ECM influence cell biology and which molecules are involved? There are many types of ECMs, ranging from cell walls and basement membranes to highly specialised structures such as tendons and shells. The assembly of these structures requires a complex set of diverse events involving protein secretion, as well as the modification of these proteins. In spite of the relevance of ECMs, it is not yet well understood how they are generated. In particular, a fascinating aspect of ECMs is the fact that they usually constitute a structure contributed to and shared by many cells. But how do individual cells participate in the generation of a supracellular ECM with an overall common organisation that overruns cellular borders? We have addressed these issues by studying the apical ECM (aECM) of Drosophila melanogaster trachea, the insect respiratory system. Once the different branches of the tracheal system have been established to cover the overall embryonic body, tracheal cells begin to secrete the components of a chitin-rich aECM that lines up the lumen of the tracheal tubes and can be visualised by the incorporation of chitin-binding probes (Moussian et al., 2005). A distinctive feature of this aECM are taenidial folds, a series of cuticle ridges that compose a helical structure running perpendicular to the tube length along the entire lumen (Wigglesworth, 1990). Taenidia are believed to confer mechanical strength to the tubes and have been compared to a coiled spring within a rubber tube (Thompson, 1929) or to the corrugated hose of a vacuum cleaner (Manning and Krasnow, 1993). From the very first descriptions, it was noticed that taenidia are unaffected by the presence of cell boundaries (Thompson, 1929), thereby indicating that they are a supracellular structure and suggesting a substantial degree of intercellular coordination. More recently, it has been reported that taenidial organisation correlates with that of the apical F-actin bundles in underlying cells—the formation of these bundles preceding the appearance of taenidia (Matusek et al., 2006; Kondo et al., 2007). However, the relationship between these bundles and taenidia is still poorly understood. In addition, physical modelling has recently revealed that the interaction of the apical cellular membrane and the aECM determines the stability of biological tubes (Dong et al., 2014), thus generating more questions about how this interaction occurs. Here, we report that there is a dynamic relationship between sub-apical F-actin and taenidial folds during tracheal lumen formation. We show that cell-cell junctions participate in organising F-actin bundles and the taenidial fold supracellular aECM and that this chitinous aECM contributes to regulating F-actin organisation in a two-way regulatory mechanism. Results and discussion Time course of actin ring and taenidial fold formation In order to obtain a detailed framework of taenidial fold formation during embryonic development, we began by performing a detailed analysis of the timing of taenidial formation. We focused on the main branch of the trachea, the dorsal trunk (DT), where taenidia are more conspicuous. It is worth mentioning that, prior to taenidial fold formation, a transient chitin filament is formed inside the tracheal lumen. This filament has been postulated to regulate tube length and diameter expansion (Tonning et al., 2005; Moussian et al., 2006a; Luschnig and Uv, 2014). As this filament is a transient structure, its appearance in and disappearance from the lumen of the DT is a useful landmark to precisely stage embryos. Taenidia began to be detectable by late stage 16 when the chitin filament was still present in the tracheal lumen (Figure 1A). Optical section analysis showed that taenidia develop at the more external luminal sections, while the chitin filament lies in a central position inside the lumen (Figure 1A). From early stage 17, a stage when the luminal chitin fibre is already absent (Moussian et al., 2006b), taenidia became increasingly more prominent (Figure 1E). As mentioned above, taenidial folds were organised as spiral rings that span many distinct cells (Figure 1L). Figure 1 with 1 supplement see all Download asset Open asset Dynamics of taenidial fold and actin ring formation. (A–F) Dorsal Trunk detail of wild-type embryos stained with fluostain to label the chitin structures. Maximum projections of confocal Z sections showing the dynamics of intraluminal chitin filament and taenidial folds during late stages of embryonic development. Chitin structures are schematically represented under each image. Chitin filament: at late stage 16, intraluminal chitin filament is thick and dense (A); as the embryo develops, it becomes less and less dense (B, C) until it turns into a thin chitin fibre that runs in zigzags along the tube diameter (D); in the last steps of embryogenesis, the intraluminal chitin filament is completely cleared from the lumen (E, F). Taenidial folds: at late stage 16, taenidial folds are newly formed and thin (A); as the taenidial folds become thicker, it is apparent that the taenidial folds at fusion points are not formed yet (B); later, the taenidial folds at fusion points are also formed which generates a continuous taenidial structure along the tube (C); in the final steps, as the intraluminal chitin filament is cleared from the lumen, the taenidial folds reach their the most mature form (D–F). Scale bars = 10 μm. (G–J) wild-type embryonic (G–H) and 3rd instar larval (I–J) trachea stained with fluostain (red) and phalloidin (green) showing taenidial folds and F-actin bundles together (G, H, I, J) or separately (G’, G’’, H’, H’’ and I’, I’’). F-actin organisation in structures perpendicular to the main tube axis occurs at stage 16 prior to taenidial fold appearance (G). When taenidia become apparent (H), they are positioned over the actin bundles and this co-localisation continues throughout larval stages (I, J). Scale bars = 10 μm. (K) TEM detail of wild-type DT taenidia, the open triangle points to actin filament cross-sections (diameter around 7 nm). Scale bar 250 nm. (L) Detail of DT showing apical cell borders (labelled by anti-DE-Cad in green) and how taenidia (labelled by fluostain in red) span continuously beyond cell-cell borders from one cell to the other. https://doi.org/10.7554/eLife.09373.003 Given the close correlation between taenidia and the rings of actin bundles (Matusek et al., 2006), we next analysed the developmental time course of these two structures in the same embryo. For this purpose, we used fluostain and phalloidin to visualise chitin and F-actin, respectively (Moussian et al., 2005; Araújo et al., 2005). At early stage 16, when taenidia were not yet detectable, we distinguished some actin rings in the cells of the DT (Figure 1G). It is noteworthy that the first actin rings to appear in the trachea were those corresponding to the fusion cells, which are not related to taenidia but instead to the fusion between the lumen of adjacent segments of the DT (Lee and Kolodziej, 2002). Shortly after, actin rings in other cells were detected throughout the length of the DT, but these were much weaker than those present in the fusion cells (Figure 1G’’). At this stage, remnants of the chitin filament were still detectable but taenidial folds were not (Figure 1G’). As the chitin filament faded away and taenidia became more visible, the actin rings became more defined and prominent (Figure 1H; for a more detailed time course of actin ring and taenidial fold formation see Figure 1—figure supplement 1). To further study the functional link between actin rings and taenidial folds, we addressed whether these two structures are generated in frame. Maximum projection from confocal planes obtained from embryos stained with phalloidin and fluostain supported this notion (Figure 1H), similarly to what has been shown upon F-actin labelling in fluorescent and DIC images at larval stages (Figure 1I, J and [Matusek et al., 2006]). In embryos, it is more difficult to reach a clear conclusion from confocal projection images, as the two structures are in different planes. However, transmission electron microscopy (TEM) images of the embryonic tracheal DT show the presence of electron dense structures in the cytoplasm underneath the taenidia (Figure 1K, open arrow). These densities are likely to be cross-sections of actin filaments and not vesicles or microtubules because of their size. Whereas microtubules appear as circular structures of about 25 nm in diameter and vesicles are generally 5–20 times larger, actin filaments are only about 7 nm in diameter (Grazi, 1997). Taken together these observations, lead us to conclude that actin filaments and taenidia are 'in-frame'. Impairment of apical actin rings similarly reshapes taenidia Alteration of the tracheal apical F-bundles by mutants in genes encoding actin polymerisation proteins also cause defects in taenidial arrangement (Matusek et al., 2006). Such is the case for mutants for tarsal-less (tal), also known as polished rice (pri), a gene transcribed into a polycistronic mRNA that contains short ORFs encoding 11 or 32 amino acid-long peptides (Kondo et al., 2007; Galindo et al., 2007). Interestingly, tal/pri is essential for the formation of actin bundles that prefigure two chitin structures, namely denticles in the embryonic cuticle and taenidia in the trachea (Kondo et al., 2007). To further address the contribution of tracheal actin bundles to the arrangement of taenidia, we examined these two structures in the same mutant trachea to assess whether they are strictly correlated. In most tal/pri mutant embryos, the tracheal F-actin bundles formed but they were misoriented and did not follow the ring distribution found in the wild-type (Figure 2B,D); in some extreme cases, the bundles were completely disorganised. In all the cases studied, we found taenidia to be organised along the same pattern as the F-actin bundles, either running parallel to the tube axis when actin bundles were oriented in this way (Figure 2C), completely disorganised, or completely misshapen in twisted tracheal tubes when F-actin fibres were aligned in a twisted manner. Figure 2 Download asset Open asset Taenidial folds and F-actin rings in tal/pri, DAAM, and Btk29A mutants. Wild-type (A, B), tal/pri (C-D), DAAM (E-F), and Btk29A (G-H) mutant embryos stained with fluostain (A, C, E, G) to label taenidial folds and phalloidin (B, D, F, H) to label F-actin bundles. Both taenidial folds and F-actin bundles run perpendicular to the tube axis in wild-type embryos (A, B) while in most of the pri mutants they are parallel to the tube axis (C, D). In DAAM (E-F) and Btk29A (G-H) mutant embryos, taenidial folds and actin bundles are hardly detected and when so they appear mis-oriented, running both parallel and perpendicular to the tube axis. In all panels anterior is to the left and scale bars =10 μm. https://doi.org/10.7554/eLife.09373.005 Similarly to tal/pri, mutants in the gene coding for the DAAM formin also disrupt tracheal actin rings and taenidia; this is also the case for the mutants in the gene coding for the Btk29A non-receptor tyrosine kinase, which interacts genetically with DAMM (Matusek et al., 2006). In both mutants, we found heterogeneous patterns of F-actin bundling in the same trachea, with stretches of perpendicular bundles followed by stretches of parallel bundles (Figure 2F, H). In support of the close relationship between F-actin bundles and taenidia, we found the latter to reproduce the stretches of parallel and perpendicular orientation of the former (Figure 2E,G). Of note, actin bundles were harder to observe in mutants for DAAM as they were much thinner than in the wild-type (Figure 2F). We extended our analysis to other genes such as singed (sn) and forked (f), which code for a fascin and an actin-binding protein, respectively, both expressed in tracheal cells (Okenve-Ramos and Llimargas, 2014) and also required for other cuticle structures such as adult bristles (Overton, 1967). However, neither appear to be involved in the tracheal actin rings as these structures formed normally in sn and f mutants and also in sn;f double mutants (data not shown). All together, these results indicate that tracheal actin rings are not only required for proper organisation of taenidia in larvae (Matusek et al., 2006) but also appear to play an instructive role in determining their initial formation during embryonic stages. Tracheal actin rings relate to the spatial distribution of the chitin synthase How could tracheal actin rings instruct the positioning of taenidia? EM images have identified the accumulation of electron-dense material at the base of the taenidia that is thought to correspond to the chitin synthesis complex (Uv and Moussian, 2010) and that appears to be located at the top of actin fibres (Moussian et al., 2006b). Thus, the tracheal actin rings may instruct taenidial fold shaping by dictating the sites of chitin production. In the Drosophila trachea, this production is carried out by the membrane-inserted chitin synthase encoded by the krotzkopf verkehrt (kkv) gene (Ostrowski et al., 2002). The Kkv chitin synthase enzyme catalyses the linkage between N-acetyl-glucosamines supplied in the cytoplasm and extrudes the polymers across the plasma membrane (for a review see [Moussian et al., 2006b]). Therefore, we analysed Kkv distribution in tracheal cells. For this purpose, we made use of a recently generated transgenic strain carrying a kkv-GFP-tagged construct under the control of the UAS promoter sequences (Moussian et al., 2015). When driven by the trachea-specific btlGal4 driver, it rescued the tracheal phenotype of the mutants results and [Moussian et al., thereby indicating that the protein is functional and showed a highly accumulation in tracheal cells, which it difficult to clear spatial However, we did perpendicular to the tube length that the actin rings detected by phalloidin and the chitin rings detected by fluostain (Figure and and Figure supplement 1). We have the of times these with the fluostain rings and that they are more within each ring 25 in to = Figure supplement 1E). Interestingly, we also found that these are when they with the fluostain rings than when they are present (Figure supplement These findings support the notion that actin which taenidial fold in the distribution of the Kkv chitin Interestingly, we did not observe the same upon in a tal/pri mutant = Figure we found that Kkv are more aligned with the actin In tal/pri mutants, actin organisation is and taenidia are not the idea of a functional role for Kkv Figure with 1 supplement see all Download asset Open asset Kkv with F-actin rings during tracheal Detail of stage 16 wild-type embryos a in tracheal cells and stained with fluostain to reveal the chitin Kkv is detected in rings that the F-actin rings in DT detail of stage 16 wild-type embryos a in tracheal cells and stained with fluostain and phalloidin to reveal the chitin filament and the F-actin rings Detail of a stage embryo in tracheal cells, showing the of Kkv in rings throughout the length of the Detail of stage tal/pri embryos a and stained with fluostain to reveal the chitin in tal/pri mutants, Kkv is detected in a pattern throughout stages and not in lines or bundles, in to wild-type embryos. In all panels anterior is to the left and scale bars = 10 μm. Chitin deposition and actin bundling contribute to proper taenidial fold organisation We next examined the contribution of chitin deposition to the organisation of As when studying the contribution of tracheal actin rings to this we mutants that do not completely chitin as these mutants tracheal development, thus analysis of the of Thus, we to an response gene et al., et al., that the Drosophila of the factor protein gene and whose mutants have been reported to have misshapen trachea completely of taenidia et al., 2006). mutant embryos were compared to the wild-type (Figure B), a phenotype with weaker embryonic by the deposition or organisation of chitin (Ostrowski et al., 2002). with this mutants showed a cuticle with denticles (Figure B), their phenotype is weaker than that of the chitin synthase mutants (Ostrowski et al., 2002). This that, while chitin deposition is some still in the cuticle of mutant embryos. In support of this we detected levels of fluostain in the trachea of mutants compared to the wild-type (Figure D). Thus, we to find similarly less taenidia, which was the However, the most feature of taenidia was their as they were not organised in folds perpendicular to the tube axis but instead parallel to it (Figure Given the close correlation between taenidia and actin we examined actin arrangement in mutants and found that it was In most mutants examined = we did not observe tracheal actin rings (Figure 1 and However, in the mutant embryos in which we detected apical actin bundles = these were oriented in parallel to the tube length (data not the chitin structures (Figure Thus, as is the case for the other mutant examined so in embryos the of a proper arrangement of taenidial folds correlates with either the or pattern of actin rings. Figure Download asset Open asset Taenidial folds, F-actin bundles, and chitin levels in mutant embryos. of wild-type and embryos visualised under In the mutant the cuticle and the chitin structures at the are when compared to wild-type C) and mutant embryos stained with fluostain to label chitin structures. in the same the images are into in which different levels of fluorescent are with different The code is shown on the of in the control DT and are in the mutant DT there are and indicating that the fluorescent of fluostain is in the mutant Scale bars = 10 μm. Wild-type (E, G) and mutant embryos H) stained with fluostain (E, to label taenidial folds and phalloidin (G, H) to label F-actin bundles. Both the taenidial folds and F-actin bundles run perpendicular to the tube axis in wild-type embryos (E, G) while in most of the mutants F-actin bundles to form and taenidial folds run parallel to the tube axis The images are of confocal Scale bars = 10 μm. 1 Download asset This be in because does support may still the for Download as Download as Download as images of a wild-type embryo carrying construct visualised from a the accumulation of F-actin at the apical surface leading to the polymerisation of F-actin bundles in the form of rings at the end of the 2 Download asset This be in because does support may still the for Download as Download as Download as images of a mutant embryo carrying construct visualised from a that the accumulation of F-actin at the apical surface occurs but F-actin to form bundles in the form of rings at the end of the at the fusion analysis by TEM the close between actin and chitin in both tal/pri and mutants. In wild-type embryos, each is formed by a plasma membrane and the taenidia have a regular shape (Figure D). of plasma membrane in tal/pri and mutant tracheal cells is (Figure C, E, F). At the end of embryogenesis, the of these taenidia is very in it is highly variable in tal/pri and mutants nm = pri = and nm at the of the taenidia the plasma see and for This is in with the that proper F-actin ring organisation and chitin deposition are for taenidial morphogenesis. Figure Download asset Open asset Taenidial structure in tal/pri and embryos. of sections of the dorsal trunk in tal/pri and embryos. In wild-type stage 16 embryos, regular of the apical plasma membrane of dorsal trunk cells the extracellular taenidial cuticle that consists of the and the In tal/pri and mutant stage 16 embryos, of the apical plasma membrane are with extended The taenidial cuticle these and the is In wild-type stage embryos, prior to the taenidial cuticle is by folds of In in tal/pri and mutant stage embryos the of the taenidial folds is highly Scale bar nm to all The chitinous aECM the organisation of the tracheal actin rings The of an of a in a gene required for proper chitin arrangement on actin bundling was To assess whether the of on actin organisation was a of chitin deposition in the tracheal cuticle rather than the of a direct and yet role of in F-actin we examined tracheal actin organisation in mutants for a gene required for chitin only (Moussian et al., 2005). mutants also actin rings (Figure thereby indicating a feedback role of proper tracheal cuticle in F-actin organisation. In addition, F-actin bundles formed normally and in mutants (Figure and This indicates that a proper cuticle is not required for the of the F-actin rings but instead for their This that proper