The Interactive Fly

Genes involved in tissue and organ development

Tracheae and spiracles

  • What are tracheae and spiracles?
  • Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems
  • Mitotic cell rounding accelerates epithelial invagination
  • Tracheal development in the Drosophila brain is constrained by glial cells
  • Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila
  • Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control
  • A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila
  • Dual origin of tissue-specific progenitor cells in Drosophila tracheal remodeling
  • The role of apoptosis in shaping the tracheal system in the Drosophila embryo
  • uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in Drosophila
  • A matrix metalloproteinase mediates airway remodeling in Drosophila
  • A Cathepsin-L is required for invasive behavior during air sac primordium development in Drosophila melanogaster
  • A cellular process that includes asymmetric cytokinesis remodels the dorsal tracheal branches in Drosophila larvae
  • Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment
  • Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth
  • Developmental compartments in the larval trachea of Drosophila
  • Microtubule-dependent apical restriction of recycling endosomes sustains adherens junctions during morphogenesis of the Drosophila tracheal system
  • Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes
  • The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity
  • Specification of leading and trailing cell features during collective migration in the Drosophila trachea
  • Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila
  • Common origin of insect trachea and endocrine organs from a segmentally repeated precursor
  • A dynamic microtubule cytoskeleton directs medial actomyosin function during tube formation
  • Compensatory branching morphogenesis of stalk cells in the Drosophila trachea
  • Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion
  • Multigenerational effects of rearing atmospheric oxygen level on the tracheal dimensions and diffusing capacities of pupal and adult Drosophila melanogaster
  • Failure to burrow and tunnel reveals roles for jim lovell in the growth and endoreplication of the Drosophila larval tracheae
  • Centrosome amplification increases single-cell branching in post-mitotic cells
    Genes expressed in tracheae and spiracles

    What are tracheae and spiracles?

    The tracheaee constitute the respiratory system of the fly. Made up of tubules that ramify throughout the body, trachea terminate in enlargements called air sacs, through which gas exchange takes place. Spiracles are the external tracheal apertures, repeated segmentally on either side of the thorax and abdomen.

    Embryonic development of tracheae and spiracles

    The embryonic tracheal system is an epithelial tubular network established from defined sets of ectodermal precursor cells. At stage 10 of embryonic development, lateral ectodermal clusters of cells on both sides of the 10 posterior (thoracic 2 and 3 and abdominal 1 through 8) parasegments assume a tracheal placode fate [Images]. These cells, about 80 in each placode invaginate at stage 11 to form tracheal pits. The subsequent formation of the tracheal tree occurs without further cell division. First the cells migrate in a distinct pattern, then they fuse with other tracheal cells from adjacent segments to form a continuous tubular network. Terminal tracheal cells send long extentions toward the target cells. (Wilk, 1996).

    Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems

    Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. Early events that trigger leg specification have been elucidated and it is shown that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. It is concluded that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. An evolutionary scenario is proposed that accommodates the available data (Franch-Marro, 2006).

    The Drosophila tracheal system has a clearly metameric origin, arising from clusters of cells, on either side of each thoracic and abdominal segment, that express the tracheal inducer genes trachealess (trh) and ventral veinless (vvl). Conversely, the leg precursors can be recognized as clusters of cells that express the Distal-less (Dll) gene, on either side of each thoracic segment; these will give rise both to the Keilin's Organs (KOs, the rudimentary legs of the larvae) and to the three pairs of imaginal discs that will give rise to the legs of the adult fly (Franch-Marro, 2006).

    To investigate whether there is a direct physical association between the leg and tracheal primordia, Drosophila embryos co-stained for the expression of trh and early markers of leg primordia were examined. Although Dll is one of the most commonly used markers for the leg primordia, it is not the earliest gene required for their specification. Instead, a couple of related and apparently redundant genes, buttonhead (btd) and Sp1, act upstream of Dll in the specification of these primordia (Estella, 2003). Examining the specification of tracheal cells with respect to btd expression, tracheal cells were observed to appear in close apposition to btd-expressing cells, from the earliest stages of their appearance (by stage 9/early stage 10). Interestingly, unlike Dll, btd is initially expressed both in the thoracic and abdominal segments, and its expression is restricted to the thoracic segments later, under the influence of the BX-C. Thus, the cells of the respiratory system in Drosophila always arise in close proximity to the cells that are fated to give rise to the legs (Franch-Marro, 2006).

    To fully endorse this conclusion it is necessary to show that the btd-expressing cells in the abdomen correspond to cryptic leg primordia. This may be a key point because, although many of the genes required for leg development are already known, it has not yet been possible to induce leg development in abdominal segments (except by transforming these segments into thoracic ones). In particular, although the Dll promoter contains BX-C binding sites that repress its expression in the abdominal segments, no ectopic appendage has been reported by misexpressing Dll in the abdomen. These observations have lead to some doubts as to whether a leg developmental program is at all compatible with abdominal segmental identity (Franch-Marro, 2006).

    Since the initial expression of btd in the abdominal segments is downregulated by the BX-C genes, it was reasoned that sustained expression of btd might overcome the repressive effect of the BX-C genes and force the induction of leg structures in the abdomen. To test this, a btd-GAL4 driver was used to drive btd expression, expecting that the perdurance of the GAL4/UAS system would ensure a more persistent expression of btd in its endogenous expression domain. No sign was ever obtained of ectopic Dll expression or KOs in the abdominal segments, but the increased expression of btd had an effect on the KOs of the thoracic segments, which had more sensory hairs than the three normally found in wild-type KOs. Thus, on its own, btd seems unable to overcome BX-C repression of leg development (Franch-Marro, 2006).

    One possibility would be that the BX-C genes could suppress appendage development in the abdomen by independently repressing both btd and Dll in this region. To assess this possibility, the same btd-GAL4 driver was used to simultaneously induce the expression of both btd and Dll. Under these circumstances, it was observed that KOs develop in otherwise normal abdominal segments; as in the previous experiment, the newly formed KOs have more than three sensory hairs. These results suggest that expression of btd and Dll in the btd-expressing abdominal primordia is sufficient to induce the development of leg structures in the abdomen, overcoming the repressive effect of the BX-C genes. Furthermore, these results demonstrate that these clusters of btd-expressing cells in the abdomen are indeed cryptic leg primordia. These results clearly show that tracheal cells are specified in close proximity to the leg primordia, in both thoracic and abdominal segments (Franch-Marro, 2006).

    Previous results have shown that the leg primordia are specified straddling the segmental stripes of wingless (wg) expression in the early embryonic ectoderm, whereas tracheal cells are specified in between these stripes. To investigate whether wg might play a role in determining the fate of these primordia, what happens when the normal pattern of wg expression is disrupted was studied. In wg mutant embryos, trh and vvl from the earliest stages of their expression are no longer restricted to separate clusters of cells; instead larger patches of expression add up to a continuous band of cells running along the anteroposterior axis of the embryo, while btd expression is suppressed in this part of the embryonic ectoderm. Conversely, ubiquitous expression of wg suppresses trh expression, while causing an expansion of btd expression along the embryo. Restricted activation or inactivation of the wg pathway by the expression of a constitutive form of armadillo or a dominant-negative form of dTCF, respectively, are also able to specifically induce or repress trh and btd expression. trh/vvl and btd seem to respond independently to wg signalling and there is no sign of cross-regulation among them, since btd expression is normal in trh vvl double mutants, and trh and vvl expression is normal in mutants for a deficiency uncovering btd and Sp1 (Franch-Marro, 2006).

    The role of wg as a repressor of the tracheal fate is further illustrated by looking at the behaviour of transformed cells: the clusters of cells that have lost btd expression and gained trh and vvl expression in wg mutant embryos begin a process of invagination that is characteristic of tracheal cells. Furthermore, these cells also express the dof (stumps) gene, a target gene of both trh and vvl in the tracheal cells. Although further development of these cells is hard to ascertain because of gross abnormalities in wg- embryos, these results indicate that they have been specified as tracheal cells. Thus, wg appears to act as a genetic switch that decides between two mutually exclusive fates in this part of the embryonic ectoderm: the tracheal fate, which is followed in the absence of wg signalling; and the leg fate, which is followed upon activation of the wg pathway. Given that there are no cell lineage restrictions setting apart the cells of the tracheal and leg primordia, these two cell populations could be considered as a single equivalence group, with the differences in their fate controlled by the activation state of the wg signalling pathway (Franch-Marro, 2006).

    A link between respiratory organs and appendages is also found in many primitively aquatic arthropods, like crustaceans, where gills typically develop as distinct dorsal branches (or lobes) of appendages called epipods. Following the current observations, which suggest a link between respiratory organs and appendages in Drosophila, whether further similarities could be found between insect tracheal cells and crustacean gills was examined. Specifically, whether homologues of the tracheal inducing genes might have a role in the development of appendage-associated gills in crustaceans was considered (Franch-Marro, 2006).

    RT-PCR was used to clone fragments of the vvl and trh homologues from Artemia franciscana and from Parhyale hawaiensis, representing two major divergent groups of crustaceans (members of the branchiopod and malacostracan crustaceans, respectively). In the case of Artemia vvl, a fragment was cloned that corresponds to the APH-1 gene and an antibody was generated for immunochemical staining in developing Artemia larvae. It was observed that Artemia Vvl is initially absent from early limb buds; it becomes weakly and uniformly expressed while the limb is developing its characteristic branching morphology, and becomes strongly upregulated in one of the epipods as its cells begin to differentiate. Uniform weak expression persists in mature limbs, but expression levels in the epipod are always significantly higher. Expression of the trh homologue from Artemia appears to be restricted to the same epipod as Vvl. Similarly, homologues of vvl and trh were cloned from Parhyale hawaiensis and their expression was studied by in situ hybridization. Both genes are specifically expressed in the epipods of developing thoracic appendages. Besides epipods, the Artemia trh and vvl homologues are also expressed in the larval salt gland, an organ with osmoregulatory functions during early larval stages of Artemia development (Franch-Marro, 2006).

    What is the significance of the two Drosophila tracheal inducer genes being specifically expressed in crustacean epipods/gills? One possibility is that the expression of these two genes was acquired independently in insect tracheae and in crustacean gills. Alternatively, tracheal systems and gills may have inherited these expression patterns from a common evolutionary precursor, perhaps a respiratory/osmoregulatory structure that was already present in the common ancestors of crustaceans and insects (Franch-Marro, 2006).

    The latter possibility is considered unlikely by conventional views, because of the structural differences between gills and tracheae (external versus internal organs, discrete segmental organs versus fused network of tubes), and the difficulty to conceive a smooth transition between these structures. Yet, analogous transformations have occurred during arthropod evolution: tracheae can be organized as large interconnected networks or as isolated entities in each segment (as in some apterygote insects), invagination of external respiratory structures is well documented among groups that have made the transition from aquatic to terrestrial environments (terrestrial crustaceans, spiders and scorpions), and conversely evagination of respiratory surfaces is common in animals that have returned to an aquatic environment (tracheal gills or blood gills in aquatic insect larvae). A very similar (but independent) evolutionary transition is, in fact, thought to have occurred in arachnids, where gills have been internalised to give rise to book lungs, and these in turn have been modified to give rise to tracheae in some groups of spiders. Thus, a relationship between insect tracheae and crustacean gills is plausible (Franch-Marro, 2006).

    A particular type of epipod/gill has also been proposed as the origin of insect wings, a hypothesis that has received support from the specific expression in a crustacean epipod of the pdm/nubbin (nub) and apterous (ap) genes - that have wing-specific functions in Drosophila. In fact, the Artemia nub and ap homologues are expressed in the same epipod as trh and vvl, raising questions as to the specific relationship of this epipod with either tracheae or wings. A resolution to this conundrum becomes apparent when one considers the different types of epipods/gills found in aquatic arthropods, and their relative positions with respect to other parts of the appendage (Franch-Marro, 2006).

    The primary branches of arthropod appendages, the endopod/leg and exopod, develop straddling the anteroposterior (AP) compartment boundary, which corresponds to a widely conserved patterning landmark in all arthropods. Different types of epipods/gills, however, differ in their position with respect to this boundary. For example, in the thoracic appendages of the crayfish, some epipods develop spanning the AP boundary [visualized by engrailed (en) expression running across the epipod], whereas others develop exclusively from anterior cells (with no en expression). Given that wing primordia comprise cells from both the anterior and posterior compartments, wings probably derived from structures that were straddling the AP boundary. Conversely, given that tracheal primordia arise exclusively from cells of the anterior compartment (anterior to en and even wg-expressing cells), it seems probable that tracheal cells evolved from a population of cells that was located in the anterior compartment. In this respect, it is interesting to note that the former type of epipods express nub, whereas the latter do not (Franch-Marro, 2006).

    In summary, it is suggested that the ancestors of arthropods had specific areas on the surface of their body that were specialized for osmoregulation and gas exchange. Homologues of trh and vvl were probably expressed in all of these cells and played a role in their specification, differentiation or function. Some of these structures were probably associated with appendages, in the form of epipods/gills or other types of respiratory surfaces. A particular type of gill, straddling the AP compartment boundary, is likely to have given rise to wings, whereas respiratory surfaces arising from anterior cells only may have given rise to the tracheal system of insects. Confirmation of this hypothetical scenario may ultimately come from the discovery of new fossils, capturing intermediate states in the transition of insects from an aquatic to a terrestrial lifestyle (Franch-Marro, 2006).

    Mitotic cell rounding accelerates epithelial invagination

    Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).

    The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).

    To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).

    After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).

    To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).

    Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).

    Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).

    To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).

    Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).

    A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).

    Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).

    Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).

    These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).

    Tracheal development in the Drosophila brain is constrained by glial cells

    The Drosophila brain is tracheated by the cerebral trachea, a branch of the first segmental trachea of the embryo. During larval stages the cerebral trachea splits into several main (primary) branches that grow around the neuropile, forming a perineuropilar tracheal plexus (PNP) at the neuropile surface. Five primary tracheal branches whose spatial relationship to brain compartments is relatively invariant can be distinguished, although the exact trajectories and branching pattern of the brain tracheae is surprisingly variable. Immuno-histochemical and electron microscopic demonstrate that all brain tracheae grow in direct contact with the glial cell processes that surround the neuropile. To investigate the effect of glia on tracheal development, embryos and larvae lacking glial cells as a result of a genetic mutation or a directed ablation, using the glial cells missing gene and a UAS-hid;rpr construct expressed in glial cells by the nrv2-Gal4 driver. In these animals, the tracheal branching pattern was highly abnormal. In particular, the number of secondary branches entering the central neuropile was increased. Wild type larvae possess only two central tracheae, typically associated with the mushroom body and the antenno-cerebral tract. In larvae lacking glial cells, six to ten tracheal branches penetrate the neuropile in a variable pattern. This finding indicates that glia-derived signals constrained tracheal growth in the Drosophila brain and restrict the number of branches entering the neuropile (Pereanu, 2006).

    Outgrowth and branching morphogenesis of the tracheal system has been investigated in great detail for the tracheal network underlying the epidermis. Three phases, all of them dependent on the FGF signaling pathway, have been distinguished. In the early embryo, shortly after gastrulation, the epidermal ectoderm of segments T2-A8 gives rise to metameric pairs of tracheal placodes. Shortly after invagination, placodes resemble simple, round cups. Soon primary tracheal branches grow out at stereotypic positions from each cup. These branches grow in length and form secondary and tertiary branches. Growth of the primary (and some of the secondary) tracheal branches is induced by the FGF homolog Bnl which is expressed in strategically positioned clusters of epidermal cells. The pattern of the Bnl expressing epidermal cells foreshadows the later pattern of primary branches; both patterns are highly invariant (Pereanu, 2006).

    The second phase of tracheal branching morphogenesis includes the formation of secondary branches which occur mainly at the growing tips of primary branches. High levels of the Bnl signal induce the expression of intrinsic activators of branching behavior, such as the ETS transcription factor Pointed, as well as inhibitory factors (e.g., sprouty) that restrict the formation of secondary branches to positions that are somewhat remote from the primary branch tip. The resulting pattern of secondary and higher order branches is considerably more variable, given that no 'hard-wired' prepattern exists. The third and terminal phase of tracheal branching describes the formation of a filigrane network of tracheoles that sprout from the tips of secondary tracheal branches. This process occurs largely after hatching of the embryo and depends on extrinsic factors, among them local oxygen levels. It is therefore a plastic, demand-based process. Again, the FGF signaling cascade plays a central role in terminal branching (Pereanu, 2006).

    The development of the ganglionic tracheal branches (GBs) that supply oxygen to the ventral nerve cord is initiated as a typical 'phase 1' process where a ventrally located cluster of epidermal cells guides the formation of a secondary branch towards the edge of the neural primordium. Subsequently, the tip of the GB follows the roots of the peripheral nerve towards the neuropile. It curves around the ventral edge of the neuropile and then turns dorsally. The formation of short tertiary tracheal branches that interconnect the corresponding ganglionic branches of the left and right side, as well as neighboring ganglionic branches of the same side, constitutes a 'phase 2' event and, accordingly, leads to a relatively variable plexus of tertiary tracheae. Aside from FGF signaling, the Slit/Robo pathway forms part of the molecular network that controls the phase 2 branching morphogenesis of the GB. Slit is expressed by midline glial cells, a subset of neuropile glia derived from the mesectoderm. GB cells express the Slit receptors Robo and Robo 2. The latter is required to attract the GB towards midline; the former inhibits crossing (Pereanu, 2006).

    Tracheation of the brain appears to be fundamentally similar to that of the epidermis and ventral nerve cord, and it is reasonable to apply to brain tracheation the three-step model. Complicating this analysis is the fact that the entire brain, including the gnathal part of the ventral nerve cord that later becomes the subesophageal ganglion, is tracheated by a single trachea, the cerebral trachea, which splits into five relatively invariant main branches (called 'primary' branches in this paper) that form the perineuropilar plexus around the brain neuropile. Strictly speaking, the cerebral trachea represents a primary branch of the first tracheal primordium, and the main tracheae [basomedial cerebral trachea (BMT), basolateral cerebral trachea (BLT), basocentral cerebral trachea (BCT), centromedial cerebral trachea (CMT), and centroposterior cerebral trachea (CPT)] would therefore constitute secondary branches. In line with such interpretation, the trajectories and branching patterns of the brain tracheae is much more variable than that of typical primary branches in the trunk. In contrast, with respect to their diameter and expansion, brain tracheae resemble primary tracheal branches of the trunk. More analysis is required to evaluate brain tracheation in comparison to the segmentally organized tracheation of the ventral nerve cord. Of particular importance will be comparative studies that reconstruct tracheation patterns in more primitive insects. Thus, it is likely that the lack of segmental tracheal primordia in the gnathal segments and, possibly, even in the preoral head segments of Drosophila, is derived from a primitive condition in which such segmental primordia were present. The main ('primary') brain tracheae which in Drosophila arise as secondary branches of a single tracheal primordium could in the primitive condition have been formed as primary branches of one or more segmental tracheal primordia (Pereanu, 2006).

    From the existing descriptions of tracheation of the ventral nerve cord it seems likely that throughout development, the GBs and their branches are in contact with glial cell precursors that surround the peripheral nerve roots and the neuropile. A direct contact between tracheae and glia does certainly occur for all tracheae of the brain, as shown in this study. Furthermore, experimental ablation of glia reveals inhibitory interactions between glial and tracheal cells. The molecular nature of these interactions remains to be established. It is tempting to invoke FGF signaling as part of the mechanism, given its pervasive involvement in other aspects of tracheal development, and the fact that the FGF receptor heartless (htl) is expressed and required for glial development. Loss of Htl prevents the formation of glial processes enwrapping the neuropile, and application of FGF soaked beads in grasshopper embryos provokes the outgrowth of such processes. Thus a scenario exists where two different FGF receptors, Htl and Btl, are expressed by adjacent tissues, namely glia and trachea, respectively. Competition for a common source of the FGF signal (from brain neurons?) or other, more complex interactions could guide the formation of glia and tracheae, and form the basis of the inhibitory action of the former on the latter (Pereanu, 2006).

    The comparison of vascular patterning in the vertebrate brain and tracheal patterning in Drosophila reveals a number of similarities. In both systems, epithelial vessels penetrate the neural primordium from the outside, follow radially organized glial elements, and branch out to form a vascular/tracheal plexus, the subependymal plexus in vertebrates and the perineuropilar plexus in Drosophila. Furthermore, vascular growth in vertebrates and tracheal growth in flies is guided by specialized tip cells. Similar to the tips of extending axons, vascular/tracheal tip cells have a growth cone whose filopodia explore cues of the microenvironment, which primarily consists of glia and the extracellular matrix produced by these cells (Pereanu, 2006).

    Numerous signaling mechanisms guiding vascular/tracheal tip cells are shared between vertebrates and Drosophila, among them FGF signaling which plays a preeminent role. In addition to FGF signaling, other receptor tyrosine kinases and their ligands were identified as mediators of vascular patterning in the vertebrate brain. During their radial growth into the neural primordium, vertebrate capillary tip cells, expressing VEGF and PDGF receptors, follow a gradient of VEGF. A corresponding role of the Drosophila VEGFR/PDGFR homolog, PVR, cannot be ascertained, at least during the embryonic and larval phase. Drosophila PVR is expressed and required for hemocyte differentiation and migration, but has not been found in the tracheal system (Pereanu, 2006).

    Other signal-receptor systems, among them members of the semaphorin and neuropilin families of proteins, as well as adhesion molecules such as N-cadherin and integrin, modify the response of developing capillaries to FGF and VEGF. Many of these signaling mechanisms require the close interaction between capillaries and radial glia/astrocytes. Astrocyte-derived cues are also responsible for the structural changes in capillary endothelia that underlie the formation of the blood-brain barrier (BBB), which consists of specialized tight junctions, as well as a number of enzymatic transport systems which regulate molecular movements across the endothelial cell membrane. In addition, factors derived from endothelial cells, including leukemia inhibitory factor (LIF), modulate astrocytic differentiation, demonstrating that endothelium and astrocytes are engaged in a two-way inductive process. In Drosophila, cadherins (e.g. E-cadherin) are essential for tracheal morphogenesis. A more specific role of these and other adhesion systems in the tracheation of the nervous system awaits to be studied (Pereanu, 2006).

    In conclusion, as in so many other instances where comparisons between organogenetic processes in Drosophila and vertebrates were conducted, one is confronted with a surprising degree of similarity, both in structural morphogenetic mechanisms and their molecular control. Given that there is little support for true homologies [tracheae are highly derived structures that only appear in insects, and even glia as a tissue might not have been present in the bilaterian ancestor. it is reasonable to assume that the constraints that acted during the independent evolution of a tracheated insect nervous system and a vascularized vertebrate nervous system were similar. In both scenarios, epithelial tubes, branching in a dichotomous way, penetrate the neural primordium at an early stage. In branching out and permeating the volume of the growing nervous system, tracheae/blood vessels had to adopt to the special microenvironment presented by the neural primordium, a microenvironment that is composed of conserved elements like axonal growth cones, a specific extracellular matrix, signaling systems controlling midline crossing, and others. Some of these elements are most likely homologous, i.e., existed in the bilaterian ancestor; one only need to look at the conserved role of the Slit/Robo system, which regulates midline crossing of axons in animals ranging from flies to humans. Novel elements that, later in evolution and possibly multiple times, were added to this conserved microenvironment presented by the neural primordium, made use of the elements already present for their own development, and thereby convergently evolved a large number of similar characteristics (Pereanu, 2006).

    Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila

    The development of air-filled respiratory organs is crucial for survival at birth. A combination of live imaging and genetic analysis was used to dissect respiratory organ maturation in the embryonic Drosophila trachea. Tracheal tube maturation was found to entail three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, gas-filling-deficient mutants were identified showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 was shown to be required for protein secretion, luminal matrix assembly, and diametric tube expansion. sar1 encodes a small GTPase that regulates COPII vesicle budding from the endoplasmic reticulum (ER) to the Golgi apparatus. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation (Tsarouhas, 2007).

    Branched tubular organs are essential for oxygen and nutrient transport. Such organs include the blood circulatory system, the lung and kidney in mammals, and the tracheal respiratory system in insects. The optimal flow of transported fluids depends on the uniform length and diameter of the constituting tubes in the network. Alterations in the distinct tube shapes and sizes cause pronounced defects in animal physiology and lead to serious pathological conditions. For example, tube overgrowth and cyst formation in the collecting duct are intimately linked to the pathology of Autosomal Dominant Polycystic Kidney Disease. Conversely, stenoses, the abnormal narrowing of blood vessels and other tubular organs, are associated with ischemias and organ obstructions (Tsarouhas, 2007 and references therein).

    While the early steps of differentiation, lumen formation, and branch patterning begin to be elucidated in several tubular organs, only scarce glimpses into the cellular events of lumen expansion and tubular organ maturation are available. De novo lumen formation can be induced in three-dimensional cultures of MDCK cells. Recent studies in this system revealed that PTEN activation, apical cell membrane polarization, and Cdc42 activation are key events in lumen formation in vitro (Martin-Belmonte, 2007). In zebrafish embryos and cultured human endothelial cells, capillary vessels form through the coalescence and growth of intracellular pinocytic vesicles (Kamei, 2006). These tubular vacuoles then fuse with the plasma membranes to form a continuous extracellular lumen. Salivary gland extension in Drosophila requires the transcriptional upregulation of the apical membrane determinant Crumbs (Crb), but the cellular mechanism leading to gland expansion remains unclear (Tsarouhas, 2007 and references therein).

    The epithelial cells of the Drosophila tracheal network form tubes of different sizes and cellular architecture, and they provide a genetically amenable system for the investigation of branched tubular organ morphogenesis. Tracheal development begins during the second half of embryogenesis when 20 metameric placodes invaginate from the epidermis. Through a series of stereotypic branching and fusion events, the tracheal epithelial cells generate a tubular network extending branches to all embryonic tissues. In contrast to the wealth of knowledge about tracheal patterning and branching, the later events of morphogenesis and tube maturation into functional airways have yet to be elucidated. As the nascent, liquid-filled tracheal network develops, the epithelial cells deposit an apical chitinous matrix into the lumen. The assembly of this intraluminal polysaccharide cable coordinates uniform tube growth. Two luminal, putative chitin deacetylases, Vermiform (Verm) and Serpentine (Serp), are selectively required for termination of branch elongation. The analysis of verm and serp mutants indicates that modifications in the rigidity of the matrix are sensed by the surrounding epithelium to restrict tube length. What drives the diametric expansion of the emerging narrow branches to their final size? How are the matrix- and liquid-filled tracheal tubes cleared at the end of embryogenesis (Tsarouhas, 2007)?

    This study used live imaging of secreted GFP-tagged proteins to identify the cellular mechanisms transforming the tracheal tubes into a functional respiratory organ. The precise sequence and cellular dynamics were characterized of a secretory and an endocytic pulse that precede the rapid liquid clearance and gas filling of the network. Analysis of mutants with defects in gas filling reveals three distinct but functionally connected steps of airway maturation. Sar1-mediated luminal deposition of secreted proteins is tightly coupled with the expansion of the intraluminal matrix and tube diameter. Subsequently, a Rab5-dependent endocytotic wave frees the lumen of solid material within 30 min. The precise coordination of secretory and endocytotic activities first direct tube diameter growth and then ensure lumen clearance to generate functional airways (Tsarouhas, 2007).

    Two strong, hypomorphic sar1 alleles were identified in screens for mutants with tracheal tube defects. In wild-type embryos, the bulk of luminal markers 2A12, Verm, and Gasp has been deposited inside the DT lumen by stage 15. However, in zygotic sar1P1 mutants (hereafter referred to as sar1), luminal secretion of 2A12, Verm, and Gasp was incomplete. The tracheal cells outlined by GFP-CAAX partially retained those markers in the cytoplasm. sar1 zygotic mutant embryos normally deposited the early luminal marker Piopio by stage 13. Luminal chitin was also detected in sar1 mutants at stage 15. However, the luminal cable was narrow, more dense, and distorted compared to wild-type. To test if the sar1 secretory phenotype in the trachea is cell autonomous, Sar1 was reexpressed specifically in the trachea of sar1 mutants by using btl-GAL4. Such embryos showed largely restored secretion of 2A12, Verm, and Gasp. Thus, it is concluded that tracheal sar1 is required for the efficient secretion of luminal markers, which are predicted to associate with the growing intraluminal chitin matrix (Tsarouhas, 2007).

    sar1 mRNA has been reported to be abundantly maternally contributed. At later stages, zygotic expression of sar1 mRNA is initiated in multiple epithelial tissues. To monitor Sar1 zygotic expression in the trachea, a Sar1-GFP protein trap line was used. Embryos carrying only paternally derived Sar1-GFP show a strong zygotic expression of GFP in the trachea. An anti-Sar1 antibody was used to analyze Sar1 expression in the trachea of wild-type, zygotic sar1P1, and sar1EP3575Δ28 null mutant embryos were generated. Both zygotic mutants showed a clear reduction, but not complete elimination, of Sar1 expression in the trachea. To test the effects of a more complete inactivation of Sar1, transgenic flies were generated expressing a dominant-negative sar1T38N form in the trachea. In btl > sar1T38N-expressing embryos, early defects were observed in tracheal branching and epithelial integrity as well as a complete block in Verm secretion. In contrast to btl > sar1T38N-expressing embryos, zygotic sar1P1 mutant embryos show normal early tracheogenesis with no defects in branching morphogenesis and epithelial integrity (Tsarouhas, 2007).

    In summary, tracheal expression of Sar1 is markedly reduced in zygotic sar1 mutant embryos. While maternally supplied Sar1 is sufficient to support early tracheal development, zygotic Sar1 is required for efficient luminal secretion (Tsarouhas, 2007).

    Given the conserved role of Sar1 in vesicle budding from the ER, its subcellular localization in the trachea was determined by using anti-Sar1 antibodies. Sar1 localizes predominantly to the ER (marked by the PDI-GFP trap). Continuous COPII-mediated transport from the ER is required to maintain the Golgi apparatus and ER structure. To test if zygotic loss of Sar1 compromises the integrity of the ER and Golgi in tracheal cells, sar1 mutant embryos were stained with antibodies against KDEL (marking the ER lumen) and gp120 (highlighting Golgi structures). In sar1 mutant embryos, a strongly disrupted ER structure and loss of Golgi staining was observed in dorsal trunk (DT) cells at stage 14. Additionally, TEM of stage-16 wild-type and sar1 mutant embryos showed a grossly bloated rough ER structure in DT tracheal cells. Consistent with its functions in yeast and vertebrates, Drosophila Sar1 localizes to the ER and is not only required for efficient luminal protein secretion, but also for the integrity of the early secretory apparatus (Tsarouhas, 2007).

    To analyze tracheal maturation defects in sar1 mutant embryos, sar1 strains were generated and imaged that carry either btl > ANF-GFP btl-mRFP-moe or btl > Gasp-GFP. In sar1 mutants, luminal deposition of both ANF-GFP and Gasp-GFP is reduced. Like endogenous Gasp in the mutants, Gasp-GFP was clearly retained in the cytoplasm of sar1 embryos. ANF-GFP was also retained in the tracheal cells of sar1 mutants, but to a lesser extent. Strikingly, sar1 mutants failed to fully expand the luminal diameter of the DT outlined by the apical RFP-moe localization. This defect was quantified by measuring diametric growth rates in metamere 6 for wild-type and sar1 mutant embryos. While early lumen expansion commences in parallel in both genotypes, the later diametric growth of sar1 mutants falls significantly behind compared to wild-type embryos. The DT lumen in sar1 mutants reaches only an average of 70% of the wild-type diameter at early stage 16. Identical diametric growth defects were detected in fixed sar1 mutant embryos expressing btl > GFP-CAAX by analysis of confocal yz sections or TEM. Reexpression of sar1 in the trachea of sar1 mutant embryos not only rescued secretion, but also the lumen diameter phenotype at stage 16. In contrast to the diametric growth defects, DT tube elongation in sar1 embryos was indistinguishable from that in wild-type. This demonstrates distinct genetic requirements for tube diameter and length growth. It also reveals that the sar1 DT luminal volume reaches less than half of the wild-type volume. Prolonged live imaging showed that sar1 mutants are also completely deficient in luminal protein and liquid clearance. Up to 80% of the rescued embryos also completed luminal liquid clearance, suggesting that efficient tracheal secretion and the integrity of the secretory apparatus are prerequisites for later tube maturation steps. Taken together, the above-described results show that tracheal Sar1 is selectively required for tube diameter expansion. Additionally, subsequent luminal protein and liquid clearance fail to occur in sar1 mutants (Tsarouhas, 2007).

    Do the tracheal defects of sar1 reflect a general requirement for the COPII complex in luminal secretion and diameter expansion? To test this, lethal P element insertion alleles were examined disrupting two additional COPII coat subunits, sec13 and sec23. Mutant sec13 and sec23 embryos were stained for luminal Gasp and for Crb and α-Spectrin to highlight tracheal cells. At stage 15, embryos of both mutants show a clear cellular retention of Gasp. Furthermore, stage-16 sec13 and sec23 embryos show significantly narrower DT tubes when compared to wild-type. The average diameter of the DT branches in metamere 6 was 4.8 μm and 4.4 μm in fixed sec13 and sec23 embryos, respectively, compared to 6.3 μm in wild-type. Therefore, sec13 and sec23 mutants phenocopy sar1. The phenotypic analysis of three independent mutations disrupting ER-to-Golgi transport thus provides a strong correlation between deficits in luminal protein secretion and tube diameter expansion (Tsarouhas, 2007).

    The live-imaging approach defines the developmental dynamics of functional tracheal maturation. At the organ level, three sequential and rapid developmental transitions were identified: (1) the secretion burst, followed by massive luminal protein deposition and tube diameter expansion, (2) the clearance of solid luminal material, and (3) the replacement of luminal liquid by gas. Live imaging of each event additionally revealed insights into the startlingly dynamic activities of the tracheal cells. ANF-GFP-containing structures and apical GFP-FYVE-positive endosomes rapidly traffic in tracheal cells during the secretion burst and protein clearance. The direct live comparison between wild-type and mutant embryos further highlights the dynamic nature of epithelial activity during each pulse (Tsarouhas, 2007).

    This study identified several mutations that selectively disrupt distinct cellular functions and concurrently interrupt the maturation process at specific steps. This clearly demonstrates the significance of phenotypic transitions in epithelial organ maturation and establishes that secretion is required for luminal diameter expansion and endocytosis for solid luminal material clearance (Tsarouhas, 2007).

    The sudden initiation of an apical secretory burst tightly precedes diametric tube expansion. The completion of both events depends on components of the COPII complex, further suggesting that the massive luminal secretion is functionally linked to diametric growth. How does apical secretion provide a driving force in tube diameter expansion? In mammalian lung development, the distending internal pressure of the luminal liquid on the epithelium expands the lung volume and stimulates growth. Cl channels in the epithelium actively transport Cl ions into luminal liquid. The resulting osmotic differential then forces water to enter the lung lumen, driving its expansion (Olver, 2004). By analogy, the tracheal apical exocytic burst may insert protein regulators such as ion channels into the apical cell membrane or add additional membrane to the growing luminal surface. Since the ER is a crucial cellular compartment for intracellular traffic and lipogenesis, its disruption in sar1 mutants may disrupt the efficient transport of so far unknown specific regulators or essential apical membrane addition required for diametric expansion. Alternatively, secreted chitin-binding proteins (ChB) may direct an increase of intraluminal pressure and tube dilation. Overexpression of the chitin-binding proteins Serp-GFP or Gasp-GFP was insufficient to alter the diametric growth rate of the tubes, suggesting that lumen diameter expansion is insensitive to increased amounts of any of the known luminal proteins. In sar1 mutants, the secretion of at least two chitin-binding proteins, Gasp and Verm, is reduced. Chitin, however, is deposited in seemingly normal quantities, but assembles into an aberrantly narrow and dense chitinous cable. This phenotype suggests that the correct ratio between chitin and multiple interacting proteins may be required for the correct assembly of the luminal cable. Interestingly, sar1, sec13, and sec23 mutant embryos form a severely defective and weak epidermal cuticle (Abrams, 2005). The luminal deposition of ChB proteins during the tracheal secretory burst may orchestrate the construction and swelling of a functional matrix, which, in turn, induces lumen diameter dilation. While this later hypothesis is favored, it cannot be excluded that other mechanisms, either separately or in combination with the dilating luminal cable, drive luminal expansion (Tsarouhas, 2007).

    During tube expansion, massive amounts of luminal material, including the chitinous cable, fill the tracheal tubes. This study found that Dynamin, Clathrin, and the tracheal function of Rab5 are required to rapidly remove luminal contents, indicating that endocytosis is required for this process. Several lines of evidence argue that the tracheal epithelium activates Rab5-dependent endocytosis to directly internalize luminal material. First, the tracheal cells of rab5 mutants show defects in multiple endocytic compartments. These phenotypes of rab5 mutants become apparent during the developmental period matching the interval of luminal material clearance in wild-type embryos. Second, tracheal cells internalize two luminal markers, the endogenously encoded Gasp and the dextran reporter, exactly prior and during luminal protein clearance. The number of intracellular dextran puncta reaches its peak during the clearance process and ceases shortly thereafter. Lastly, intracellular puncta of both Gasp and dextran colocalize with defined endocytic markers inside tracheal cells. The colocalization of Gasp and dextran with GFP-Rab7 and of Gasp with GFP-LAMP1 suggests that the luminal material may be degraded inside tracheal cells. Taken together, these data show that the tracheal epithelium activates a massive wave of endocytosis to clear the tubes (Tsarouhas, 2007).

    Endocytic routes are defined by the nature of the internalized cargoes and the engaged endocytic compartments. What may be the features of the endocytic mechanisms mediating the clearance of luminal material? The phenotype of chc mutants and the presence of intracellular Gasp in CCVs indicate that luminal clearance at least partly relies on Clathrin-mediated endocytosis (CME). In addition to CME, Dynamin and Rab5 have also been implicated in other routes of endocytosis, suggesting that multiple endocytic mechanisms may be operational in tracheal maturation. The nature of the endocytosed luminal material provides an additional perspective. While cognate uptake receptors may exist for specific cargos such as Gasp, Verm, and Serp, the heterologous ANF-GFP, degraded chitin, and the fluid-phase marker dextran may be cleared by either fluid-phase internalization or multifunctional scavenger receptors. Interestingly, Rab5 can regulate fluid-phase internalization in cultured cells by stimulating macro-pinocytosis and the activation of Rabankyrin-5. The defective tracheal internalization of dextran in rab5 mutants provides further loss-of-function evidence for Rab5 function in fluid-phase endocytosis in vivo. The above-described arguments lead to the speculation that additional Rab5-regulated endocytic mechanisms most likely cooperate with CME in the clearance of solid luminal material (Tsarouhas, 2007).

    How is liquid cleared from the lumen? While very little is known about this fascinating process, some developmental and mechanistic arguments suggest that this last maturation step is mechanistically distinct. First, the interval of luminal liquid clearance is clearly distinct from the period of endocytic clearance of solids. Second, the dynamic internalization of dextran and the abundance of GFP-marked endocytic structures decline before liquid clearance. Finally, assessment of liquid clearance further suggests that it requires a distinct cellular mechanism (Tsarouhas, 2007).

    Viewing the entire process of airway maturation in conjunction, some general conclusions may be drawn. First, the three epithelial pulses are highly defined by their sequence and exact timing, suggesting that they may be triggered by intrinsic or external cues. Second, the analysis of mutants that selectively reduce the amplitude of the secretory or endocyic pulses demonstrates the requirement for each epithelial transition in the completion of the entire maturation process. These pulses are induced in the background of basal secretory and endocytic activities that operate throughout development. Third, specific cellular activities exactly precede each morphological transition. Finally, the separate transitions are interdependent in a sequential manner. Efficient secretion is a prerequisite for the endocytic wave. Similarly, protein endocytosis is a condition for luminal liquid clearance. This suggests a hierarchical coupling of the initiation of each pulse to completion of the previous one in a strict developmental sequence (Tsarouhas, 2007).

    This study provides a striking example of how pulses of epithelial activity drive distinct developmental events and mold the nascent tracheal lumen into an air delivery tube. These findings are likely to be relevant beyond the scope of tracheal development. The uniform growth of salivary gland tubes in flies and the excretory canal and amphid channel lumen in worms also require the assembly of a luminal matrix for uniform tube growth (Abrams, 2006; Perens, 2005). Luminal material is also transiently present during early developmental stages in the distal nephric ducts of lamprey. Thus, the coordinated, timely deposition and removal of transient luminal matrices may represent a general mechanism in tubulogenesis (Tsarouhas, 2007).

    Rab9 and retromer regulate retrograde trafficking of luminal protein required for epithelial tube length control

    Apical extracellular matrix filling the lumen controls the morphology and geometry of epithelial tubes during development, yet the regulation of luminal protein composition and its role in tube morphogenesis are not well understood. This study shows that an endosomal-retrieval machinery consisting of Rab9, retromer and actin nucleator WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) regulates selective recycling of the luminal protein Serpentine in the Drosophila trachea. Secreted Serpentine is endocytosed and sorted into the late endosome. Vps35, WASH and actin filaments differentially localize at the Rab9-enriched subdomains of the endosomal membrane, where Serpentine containing vesicles bud off. In Rab9, Vps35 and WASH mutants, Serpentine was secreted normally into the tracheal lumen, but the luminal quantities were depleted at later stages, resulting in excessively elongated tubes. In contrast, secretion of many luminal proteins was unaffected, suggesting that retrograde trafficking of a specific class of luminal proteins is a pivotal rate-limiting mechanism for continuous tube length regulation (Dong, 2013).

    Maturation of the tracheal tube involves diameter expansion triggered by a cell-intrinsic programme of luminal material secretion and apical membrane growth, and the tube’s longitudinal growth is negatively regulated by the presumed conversion of chitin to chitosan through deacetylation by chitin deacetylase. Each step involves temporally regulated apical secretion, but the regulatory mechanism underlying this selective cargo secretion has not been understood. This study identified Serp as a novel cargo for the Rab9-mediated retrograde recycling pathway. Using time-lapse imaging, it was demonstrated that Serp in the endocytic compartment is sorted out by the budding of actin- and WASH-enriched portions of the LEs. The similarity of the Rab9, Vps35, WASH and Serp tracheal tube-length phenotypes, and the colocalization of those in the Serp-containing endosomal-budding sites in S2 cells, suggest that Serp is one of the major cargoes of the retrograde trafficking mediated by Rab9, Vps35 and WASH. It was also found that association of WASH and actin filaments with retrograde cargo is Rab9 and Vps35 independent. Modular organization of machineries for cargo retrieval (retromer) and membrane remodelling (WASH) may allow flexibility in endosomal sorting systems. This work connects retrograde trafficking to luminal retention of the key rate-limiting protein required to restrict the tracheal tube length to the proper level and has identified a new regulatory process for tracheal tube size (Dong, 2013).

    Previous works in mammalian cells revealed that recruitment of the cargo recognition subcomplex of retromer (Vps35, Vps29 and Vps26) to the endosomal membrane requires Rab5 and Rab7. Rab7 has been shown to bind directly to Vps35/29/26 complex, but no evidence is available for association of Rab5 or Rab9 with those components. This analysis of the Drosophila counterparts have shown that conversion of early to LE is accompanied by gradual reduction of relative abundance of GFP-Rab5 to RFP-Rab9 signals. Throughout the process, Vps35-mRFP is present as distinct puncta associated with endosomes. The following order of Rab9-Vps35 assembly is proposed. In early endosome, Rab9 recruits Vps35 to the endosomal membrane through physical binding. This process requires Rab5. After initial recruitment of Vps35, endosomal maturation proceeds with exchange of Rab5 with Rab7, which promotes retromer assembly and cargo concentration, followed by membrane scission induced by WASH and F-actin. Together, these results indicate that Rab5, Rab9 and Rab7 acts on retromer assembly at different stages of endosomal maturation and tubular membrane formation, where Serp and other cargos are retrieved for retrograde pathway (Dong, 2013).

    In contrast to the tracheal diameter expansion, which is triggered by a cell autonomous burst of exocytosis, axial elongation is a slow and continuous process spanning stage 14–16. Therefore, any mechanism that restricts elongation is expected to be active throughout the elongation process. Previous studies showed that the two chitin deacetylases Serp and Verm act additively in tube length control and the overexpression of either one causes the same tube length defects, indicating that the amount of luminal chitin deacetylase must be precisely maintained. Furthermore, the expression of Serp mRNA in the trachea declines at stage 16, when luminal Serp protein is still abundant, implying that Serp synthesized in the previous stage must be effectively retained and reused. This study has shown that Serp is endocytosed and associates with GFP-Rab9. The requirement for Rab9 and Vps35 for the luminal retention of Serp suggests that the endocytosed Serp is sorted from LEs to the TGN for secretion into the lumen, and this recycling pathway actively maintains the steady-state level of Serp to optimize the tube elongation process. Moreover, the retrieval transport through the TGN might help promote the modification of the endocytosed Serp, to recover its activity or to bind to an adaptor for its subsequent polarized secretion (Dong, 2013).

    In rab9, vps35 and wash mutants, Serp was mislocalized at the apical cortex of tracheal cells. This mislocalization may have been due to the trapping of Serp in endosomes marked with Rab7 or Rab11 which are apically localized in a Rab9-independent manner. Consistent with this idea, a recent report showed that the suppression of Vps35 expression in NLT cells and neurons causes the accumulation of its cargo protein β-secretase (BACE1) in endosomes. The second possibility is that the loss of Rab9 diverts the trafficking route of Serp from the endosomes to the cell surface, as observed in Rab9-deficient HeLa cells. In support of this idea, an enhancement of Serp accumulation at the surface of the epidermis was observed in rab9 mutants. The third possibility is that a defect in endocytosis arrested Serp at the apical membrane, because Vps35 has been shown to be involved in endocytosis in S2 cells. Further analysis will be required to understand the diverse influences of retrograde trafficking on endosomal dynamics (Dong, 2013).

    The apical localization of Crb depends on the retromer complex, but it was unaffected in rab9 mutants. One possible explanation for this finding is that the alternative recycling pathway of Crb, which involves Rab11, compensates for the defect in retrograde trafficking in rab9 mutants. Another possibility is that Crb-retromer and Serp-retromer complexes use different trafficking pathways distinguished by sorting nexins, which organize endosomal membranes into distinct morphological and functional regions for transport to diverse destinations. Further study of the different sorting nexins should uncover the role of Rab9 in retromer cargo specificity (Dong, 2013).

    The formin Dia and motor protein Myosin V (Didum) are required for the secretion of a number of markers, including 2A12 antigen, Pio and artificial ANF-GFP, into the tracheal lumen. On the other hand, the secretion of Serp and Verm and the cell-surface localization of Crb are normal in Dia mutants, indicating that a Dia-independent secretory pathway regulates these protein localizations in the trachea8. The spectrum of secreted protein localizations affected in the rab9 and vps35 mutants was nearly complementary to that of Dia, suggesting that the Dia-dependent and retrograde trafficking-dependent mechanisms are the two major apical secretory pathways in the trachea. Verm localization was not affected in the rab9 and vps35 mutants, suggesting that this putative chitin deacetylase behaves differently from Serp. The difference in cargo specificity for each pathway allows cells versatility in controlling the localization of different proteins according to distinct schedules, so that the secretory burst that triggers tube diameter expansion and the continuous recycling of chitin deacetylases in axial elongation are controlled separately. The results provide a molecular basis for the roles of distinct trafficking pathways in controlling tubule growth and geometry (Dong, 2013).

    A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila

    The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. This study shows that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp is expressed by the fat body, and the secreted Serp is taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process is defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. Fat body-derived Serp reaches the tracheal lumen after establishment of epithelial barrier function and is retained in the lumen in a chitin synthase-dependent manner. These results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development (Dong, 2014).

    Dual origin of tissue-specific progenitor cells in Drosophila tracheal remodeling

    During Drosophila metamorphosis, most larval cells die. Pupal and adult tissues form from imaginal cells, tissue-specific progenitors allocated in embryogenesis that remain quiescent during embryonic and larval life. Clonal analysis and fate mapping of single, identified cells show that tracheal system remodeling at metamorphosis involves a classical imaginal cell population and a population of differentiated, functional larval tracheal cells that reenter the cell cycle and regain developmental potency. In late larvae, both populations are activated and proliferate, spread over and replace old branches, and diversify into various stalk and coiled tracheolar cells under control of fibroblast growth factor signaling. Thus, Drosophila pupal/adult tissue progenitors can arise both by early allocation of multipotent cells and late return of differentiated cells to a multipotent state, even within a single tissue (Weaver, 2008).

    Drosophila larval tissues are composed of differentiated larval cells and imaginal cells. Imaginal cells are pupal and adult tissue progenitors that reside in clusters embedded in or attached to larval tissue. They remain quiescent during embryogenesis and part or all of larval life, then proliferate and differentiate into pupal and adult tissues at metamorphosis. By contrast, larval cells cease dividing and differentiate early in development; however, they typically enlarge and become polyploid during larval life. At metamorphosis, most larval cells die. Although some larval neurons and muscles are retained in adult tissues, no differentiated cells are known to reenter the cell cycle and generate new cells and tissues. This study shows that tracheal (respiratory) system remodeling at metamorphosis is carried out by a classical imaginal cell population and another progenitor population that, like facultative stem cells in mammals, arises from differentiated cells (Weaver, 2008).

    During embryogenesis, the tracheal system develops from segmentally repeated groups of ~80 cells that express Trachealess transcription factor and invaginate, forming sacs attached to epidermis by a stalk of spiracular branch (SB) cells. Branches bud from the sacs and cells diversify primarily under control of Branchless FGF (fibroblast growth factor), which activates Breathless FGFR (FGF Receptor) on tracheal cells. At metamorphosis, posterior tracheal segments Tr6 to Tr10 are lost; new branches form in Tr4 and Tr5 to supply posterior tissues and in Tr2 to supply flight muscle. Although most branches in Tr1 to Tr5 are retained, most of their cells are replaced by imaginal cells (Weaver, 2008)..

    Previous work indicated that imaginal tracheal cells (tracheoblasts) compose the SB. Unlike other tracheal cells, SB cells express imaginal marker escargot (esg), remain small and quiescent during embryonic and early larval life, and do not form gas transport tubes. Bromodeoxyuridine (BrdU) incorporation studies showed that SB cells enter S phase at the beginning of the third larval period (L3) and divide 12 to 16 hours later. Proliferation continues for 24 hours, generating an expanding cluster of tracheoblasts at the SB-transverse connective (TC) junction. SB cells in the embryo and early larva express trachealess (trh) but, unlike most other tracheal cells, do not express the Trachealess target gene breathless (btl): The tracheal program is apparently arrested at this step. When activated in L3, they turn down esg and turn on btl as they proliferate and leave the SB (Weaver, 2008).

    A 'molecular timer' strain was developed that highlights the burst of btl expression in activated tracheoblasts, which allowed distinguishing them from the larval tracheal cells they migrate over and replace. SB tracheoblasts followed stereotyped paths. In Tr4, they migrated along the TC onto the visceral branch; later, some differentiated into coiled tracheolar (CT) cells. Tracheoblasts respected specific boundaries, never spreading into neighboring tracheal segments or populating the dorsal trunk (DT). However, tracheoblasts were observed on the other side of the DT, along the dorsal branch (DB) of Tr4 and other anterior DBs (Weaver, 2008).

    If DB tracheoblasts arise from SB tracheoblasts, they would have to move across the DT to reach the DB. However, tracheoblasts were never seen crossing the DT. To exclude this possibility, the fate of SB tracheoblasts was mapped using SB-specific flipase (FLP) recombinase to permanently label SB cells and their descendants. This labeled all tracheoblasts migrating out of the SB, but not DB tracheoblasts. Hence, DB tracheoblasts arise independently (Weaver, 2008).

    To identify the source of DB tracheoblasts, early L3 larval DBs, which comprise five to seven cells were scrutinized, but no additional cells or cells were found with the distinctive small size and nuclear morphology of SB tracheoblasts. The positions and number of tracheoblast clones in a clonal analysis of larval DBs suggested that DB tracheoblasts arise from ~4 to 5 progenitors along the DB (Weaver, 2008).

    Whether differentiated stalk cells (DB3 to DB7) might be the source of DB tracheoblasts was considered. To test this, a heat-inducible FLP transgene was used to permanently label and trace the fate of individual tracheal cells identified in live L2 larvae. This demonstrated that larval DB stalk cells are the source. Individual stalk cells displayed a range of proliferative capacities, giving rise to 2 to 22 tracheoblasts, although occasionally a labeled stalk cell failed to proliferate or degenerated, the standard fate of DB1 and DB2 cells (Weaver, 2008).

    DB stalk cells have a complex morphology unlike typical progenitor or stem cells: They are tubular, with autocellular junctions, some (DB3 cells) forming Y-shaped tubes. Yet these cells become proliferating, migrating DB tracheoblasts while maintaining contacts with neighboring tracheal cells (Weaver, 2008).

    Phosphohistone H3 staining showed that DB stalk cells in anterior segments begin dividing 14 to 16 hours after the second molt. Even before they divide, they have smaller nuclei than other larval tracheal cells, including DB stalk cells in posterior segments, which are otherwise indistinguishable but do not give rise to tracheoblasts. BrdU labeling of newly molted third-instar larvae showed that anterior DB stalk cells do not incorporate the label, implying that they do not endoreplicate and presumably remain diploid, unlike posterior DB cells and other differentiated larval cells, most of which endoreplicate and become polyploid (Weaver, 2008).

    Although most DB tracheoblasts form multicellular stalks of pupal DBs, in Tr2 they form more elaborate structures. After proliferating and spreading along the DB, they aggregate, form secondary branches, and differentiate into multicellular stalks (MS), unicellular stalks (US), and Blistered (DSRF)-expressing CT cells with coiled intracellular lumens that unfurl on flight muscle. Fate mapping showed that single DB stalk cells in Tr2 routinely formed mixed clones containing MS, US, and CT cells. Thus, DB stalk cells in Tr2 transform into multipotent tracheoblasts that can proliferate and acquire different fates (Weaver, 2008).

    Bnl/Btl signaling controls cell fate selection in the embryo. To test for function in pupal tracheoblasts, btl minus clones were generated. These rarely formed CT cells. Likewise, conditional expression of dominant-negative Btl in the L3 tracheal system reduced or eliminated secondary branches and CT cells. Constitutively active receptor induced ectopic secondary branches and CT cells throughout the tracheal system, including all anterior DBs. Thus, DB tracheoblasts in anterior segments can acquire new tracheal fates, and FGF signaling also plays a critical role in reselecting cell fates when DB stalk cells are reactivated (Weaver, 2008).

    Anterior DB stalk cells are the first differentiated cells in Drosophila shown to reenter the cell cycle and regain developmental potency. They regain the same abilities to proliferate, spread, and differentiate into various tracheal cell types as SB tracheoblasts, classical imaginal cells that remain quiescent (blocked in tracheal outgrowth and cell diversification) during embryonic and most of larval life. This suggests that both types of tracheal progenitors arrive at a similar state, one by early developmental arrest (SB tracheoblasts), the other by late return to an earlier state (anterior DB stalk cells). The only known features that distinguish these cells from tracheal cells that lack progenitor potential (including posterior DB stalk cells that are otherwise indistinguishable from anterior DB stalk cells) are their small nuclear size and lack of endoreplication. These features may be part of a program that maintains, or allows cells to regain, the proliferative and diversification potential of early tracheal cells. This program is operative in imaginal tracheal cells and can apparently be implemented in other tracheal cells, independent of their differentiation program (Weaver, 2008).

    The blurring of the distinction between imaginal and differentiated larval cells in Drosophila parallels a current debate about adult stem cells in mammals. Some mammalian tissues have dedicated stem cells maintained in a primitive state. However, other tissues may rely on facultative stem cells, differentiated cells that reenter the cell cycle to replenish lost cells. The current results show that progenitors with each of these features are present in a single Drosophila tissue and that both play crucial roles. This provides a tractable system for dissection of the arrest of a tissue-specific developmental program and reversal to an earlier, more plastic state, important steps in tissue engineering, repair, and cancer (Weaver, 2008).

    The role of apoptosis in shaping the tracheal system in the Drosophila embryo

    The tubular network of the tracheal system in the Drosophila embryo is created from a set of epithelial placodes by cell migration, rearrangements, fusions and shape changes. A designated number of cells is initially allocated to each branch of the system. The final cell number in the dorsal branches is not only determined by early patterning events and subsequent cell rearrangements but also by elimination of cells from the developing branch. Extruded cells die and are engulfed by macrophages. These results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues (Baer, 2010).

    In live studies of the tracheal system using embryos expressing GFP under the control of the breathless (btl) promoter GFP-expressing motile cells were observed that were not attached to the tracheal system. The btl gene, which encodes an FGF receptor homolog, is mainly expressed in the tracheal system, but also in glial cells and a few other cell type. However, it had not previously been described as being expressed in individual cells that were dispersed in the embryo. Since it was not clear what the individual cells outside the tracheal system were, other tracheal markers were used to determine whether they indeed derived from the tracheal system, or rather represented an as yet undiscovered cell type in which the btl promoter is active. When the tracheal system was marked with lacZ expressed under the control of the promoter of another tracheal gene, trachealess (trh-lacZ), it was observed that lacZ was also expressed not only in the tubular tracheal epithelia but also in single cells detached from the tracheal system (Baer, 2010).

    In the live observations using GFP it was noticed that these cells appeared to be moving around in the embryo, preferentially along the dorsal trunk of the trachea. Previously hemocytes had been described to move along the tracheal system. Thus it was asked whether these cells might be a subpopulation of hemocytes that shared expression of some genes with the tracheal system. To assess their cell type, the cells were analyzed in embryos in which markers for the tracheal system and for hemocytes were simultaneously visualized. Hemocytes were visualized by using a croquemort-GAL4 transgene to control the expression of GFP. Croquemort is expressed in a subpopulation of hemocytes, the macrophages. For the tracheal system trh-lacZ was used (Baer, 2010).

    It was found that the individual lacZ-expressing cells that are detached from the tracheal system also expressed the crq-controlled GFP. In addition, there were also many cells that showed only crq-GFP staining and no lacZ. There are two possible explanations for these findings. Either a subset of macrophages can activate transcription of the btl and trh promoters, or the doubly-stained objects are not single cells. Consistent with the latter explanation, it was noticed that the two fluorescent markers were often localized in non-overlapping parts of the cells, suggesting that lacZ-expressing cells or cell fragments might have been engulfed by macrophages. If this were the case, then the doubly-stained objects should contain two nuclei. Indeed, in triple stainings with the nuclear marker TOTO-3 two TOTO-3 signals were observed in the majority of the doubly-stained objects, indicating the presence of two nuclei. It is concluded therefore that the objects are hemocytes that have engulfed tracheal cells that have left the tracheal epithelium (Baer, 2010).

    To test directly whether these single cells originate from the developing tracheal tree, more detailed live observations were performed. In previous experiments it was noticed that the disconnected cells can be preferentially found near the branches that undergo cellular rearrangements, for example, at the bases of dorsal branches. The areas around these sites were chosen for further studies. Live imaging of dorsal branches of the embryos expressing α-cat-GFP or sqh-GFP revealed that during the outgrowth of the dorsal branches individual tracheal cells detach from the tracheal system at, or near the site where the dorsal branches emerge from the dorsal trunk. The numbers of cells leaving the system from the base of branches and from the interbranch-regions of the dorsal trunk were compared in 12 videos of wildtype embryos. The 'base of the branch' is described as those cells that bordered at least on one side on a cell that was part of the dorsal branch. 42 branches were evaluated and cells were found leaving from the base in 20 cases, whereas in 40 interbranch-regions seven cells leaving the system were found. Thus, cells are almost threefold less likely to be extruded in the large interbranch area than from the small area at the base of the branch (Baer, 2010).

    Since the detached cells appeared to be engulfed by macrophages and one function of macrophages is to clear up apoptotic cells, whether the trh-lacZ positive cells seen inside the hemocytes displayed markers for apoptosis was tested. It was found that some but not all of the trh-lacZ cells that were detached from the tracheal system gave a positive signal in a TUNEL reaction. Since the TUNEL assay only detects cells in a brief phase of apoptosis it was not surprising that only a subset of the detached cells were labelled. Thus this result is consistent with the notion that the engulfed cells are dying or dead cells. A further hallmark of apoptosis is the activation of caspase 3, which can be detected with an antibody that specifically recognizes the activated form. Trh-lacZ-expressing cells that were detached from the tracheal system also gave a positive signal when stained with antibodies against activated caspase3 (Baer, 2010).

    Next, it was asked whether the detached cells undergo apoptosis because they have lost contact with the tracheal epithelium, or, conversely, whether they die within the epithelium and are subsequently extruded. This question was addressed by two approaches: tracing caspase activity in vivo and blocking apoptosis. To examine when the apoptotic program is induced in tracheal cells leaving the system an in vivo fluorescent sensor of caspase activity, the 'Apoliner' transgene (Bardet, 2008), was used. The Apoliner consists of two fluorophores, a membrane-anchored RFP and a GFP with nuclear localisation signal (NLS), which are linked by a caspase sensitive fragment of the DIAP1 protein. Upon caspase activation within the cell, the sensor is cleaved, resulting in translocation of GFP into the nucleus, whereas RFP stays at the membrane. The Apoliner was expressed using btl-GAL4 and the development of dorsal branches was followed in vivo. Nuclear GFP can be observed in individual cells in the tracheal system. Shortly after they begin to accumulate nuclear GFP these cells detach from the system. Thus apoptosis precedes cell removal from the tissue (Baer, 2010).

    If death is a prerequisite for expulsion, then suppressing apoptosis should reduce the number of detached cells. To test this, the tracheal system was examined in embryos homozygous for a deficiency Df(3L)H99, which removes the three pro-apoptotic genes, grim, reaper and hid, and in which apoptosis therefore cannot occur. Since homozygous Df(3L)H99 embryos show strong overall developmental defects, the analysis was restricted to the dorsal branches in segments that do not show gross morphological abnormalities, i.e. segments 3-7. Whereas wild type stage 14/15 embryos show approximately 2-4 detached cells in segments 3-7 on each side, no detached cells were observed in embryos that were homozygous for Df(3L)H99. To exclude the possibility that this was due to a non-autonomous effect of the mutant phenotype of surrounding tissues in Df(3L)H99 mutant embryos, an alternative way of blocking apoptosis only in the tracheal system was used. The baculoviral inhibitor of apoptosis p35 was expressed in tracheal cells using the btl-GAL4 driver line. To confirm that apoptosis was blocked efficiently, the Apoliner was co-expressed together with p35. In these embryos no Apoliner signal was detected, indicating the absence of caspase activity, and no cells were seen to leave the tracheal system. This shows that the completion of the apoptotic program is necessary for detachment of the tracheal cells (Baer, 2010).

    The fact that the detached cells were frequently found near the base of the dorsal branches of the tracheal system raises the question whether their leaving the epithelium might be associated with a specific developmental programme, such as the morphogenesis of the dorsal branches. Since each dorsal branch arises from six cells, but the final branch typically consists of only five cells, it has been suggested that the sixth cell migrates back to the dorsal trunk. The results point to an alternative possibility suggesting that apoptosis might be a mechanism that contributes to the determination of cell number in the dorsal branches. If the assumption that cell death is involved in determining the number of cells is correct, then dorsal branches should contain more cells if apoptosis cannot occur. To test this, cell numbers were counted in the dorsal branches of Df(3L)H99 mutant embryos and in embryos expressing p35 in the tracheal system. The embryos were stained with an antibody against Trachealess to mark specifically the nuclei of tracheal cells. The nuclei in dorsal branches were then counted under the microscope by focusing through the entire depth of the dorsal branch. Branches with an unclear course were not included in the analysis (Baer, 2010).

    All tested genotypes showed variability in cell number in the dorsal branches, but there were clear differences between the mutant and the wild type embryos. 76.3% of the 93 dorsal branches evaluated in 10 wild type embryos consisted of 5 cells, 8.6% consisted of six cells and 15.1% consisted of 3 or 4 cells. This shows that branches with six cells are possible, but 5 cells are preferred. By contrast, embryos with blocked apoptosis had 51.6% (btl-Gal4, UAS-p35 embryos) or 56.6% (Df(3L)H99 mutant embryos) dorsal branches with six cells. These results indicate that removal of the cells by apoptosis indeed contributes to the determination of the final cell number in the dorsal branches (Baer, 2010).

    It is unclear how the emigrating cell is determined, and why it is not determined in every one of the branches. The fact that this is so shows that, in contrast to many other situations in which apoptosis eliminates unwanted cells during development, cell death in this case is not the result of a hard-wired developmental programme. Instead it is more likely to be a response to unfavourable conditions in the cell's environment. Changes in the microenvironment have been shown to induce cell death in mammalian endothelial cells, with signals from the extracellular matrix influencing the balance between cell survival and apoptosis. Adhesion via integrins can protect cells from FAS mediated apoptosis, and integrins may act as mechano-transducers in this context. Thus it is possible that weakening of cell-cell junctions resulting from tissue remodelling in the tracheal system could trigger the apoptotic pathway. Junction rearrangement also affects cell death in the wing disc, where an imbalance in the junctional forces within the epithelium was found to be rebalanced by the elimination of cells. In this system, the initial irregularity arose through cell proliferation, but it is imaginable that in the tracheal system cell intercalation might affect local junctional forces, and cell extrusion can be triggered to obtain a geometrically optimal structure (Baer, 2010).

    The fact that only half of the dorsal branches in the mutant embryos had six cells shows that apoptosis cannot be the only mechanism for the removal of supernumerary cells. There must be other ways of losing cells, perhaps by re-integration into the dorsal trunk, as previously suggested. Such cases were indeed observed. More stringently, even embryos in which a larger number of branches have six cells, namely btl-Gal4, UAS-p35 embryos, can develop normally and hatch. It is of course possible that subtle defects, for example a reduced efficiency in oxygen delivery, would not manifest themselves in easily measurable phenotypes in a laboratory setting (Baer, 2010).

    In summary, the results are consistent with a scenario in which cells that find themselves in a sub-optimal epithelial context either move away, or die and leave the epithelium. If the apoptotic pathway is blocked, they may be forced to use the option of moving elsewhere, or induce neighbouring cells to rearrange further, or they remain in place, and the system is able to tolerate a sub-optimal structure (Baer, 2010).

    uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in Drosophila

    The tracheal system of Drosophila has proven to be an excellent model system for studying the development of branched tubular organs. Mechanisms regulating the patterning and initial maturation of the tracheal system have been largely worked out, yet important questions remain regarding how the mature tubes inflate with air at the end of embryogenesis, and how the tracheal system grows in response to the oxygen needs of a developing larva that increases nearly 1000-fold in volume over a four day period. This study describes the cloning and characterization of uninflatable (uif), a gene that encodes a large transmembrane protein containing carbohydrate binding and cell signaling motifs in its extracellular domain. Uif is highly conserved in insect species, but does not appear to have a true ortholog in vertebrate species. uif is expressed zygotically beginning in stage 5 embryos, and Uif protein localizes to the apical plasma membrane in all ectodermally derived epithelia, most notably in the tracheal system. uif mutant animals show defects in tracheal inflation at the end of embryogenesis, and die primarily as larvae. Tracheal tubes in mutant larvae are often crushed or twisted, although tracheal patterning and maturation appear normal during embryogenesis. uif mutant larvae also show defects in tracheal growth and molting of their tracheal cuticle (Zhang, 2009).

    This study has described the initial characterization of a novel gene, uninflatable, which encodes a single-pass type I transmembrane protein with several different carbohydrate binding motifs and epidermal growth factor repeats in its extracellular domain. This gene is highly conserved in insect species, but does not appear to have a true ortholog in mammals or other vertebrate organisms. Uninflatable is expressed on the apical surface of ectodermally-derived epithelial cells including the epidermis, trachea, salivary gland and fore- and hindgut, although it is most highly expressed in tracheal epithelia starting as the tracheal placode invaginates and persisting throughout its development. Three mutant alleles of uif were isolated, and the predominant defects associated with loss of uif were found to be defective air inflation at the end of embryogenesis, and tracheal growth and tracheal molting defects in larvae (Zhang, 2009).

    The most obvious defects observed in uif mutant late embryos and newly hatched first instar larvae were incomplete inflation of the trachea coupled with tracheal tubes that often appeared pinched or stretched. Live imaging of individual uif mutant embryos and larvae revealed that these tracheal inflation defects result from the inability to completely inflate the trachea at the end of embryogenesis rather than a defect in the maintenance of an inflated tracheal system after hatching. Interestingly, tracheae in uif mutant embryos are normally patterned and appear to mature in a manner indistinguishable to tracheae in wild type animals. Thus this tracheal inflation defect likely results from a requirement for uif function late during embryogenesis (Zhang, 2009).

    Several genes have been characterized whose defects include incomplete tracheal inflation. Included in these genes are those that function at the end of embryogenesis to clear the trachea of solid luminal material. This process requires clathrin-mediated endocytosis and includes the proteins clathrin heavy chain, the GTPase dynamin (encoded by shibire), and a clathrin binding transmembrane protein encoded by wurst. Notably, mutations in all of these genes also result in elongated tracheal tubes suggesting a defect in tracheal tube size control. Gas filling defects were also observed for mutations in serp and verm, two putative matrix chitin deacytlases that function to regulate tracheal tube length. Tracheal maturation appears normal in uif mutant embryos, including the secretion of chitin into the tracheal lumen and the subsequent uptake of luminal solids near the end of embryogenesis. In addition, uif mutant tracheae do not show diameter or length defects during embryogenesis, suggesting that the luminal chitin cylinder forms normally and that Serp and Verm are correctly functioning during the maturation process. Taken together these results suggest that the air filling defects observed in uif mutant embryos occurs through a mechanism independent of that linked to endocytosis of solid luminal material (Zhang, 2009).

    A second class of proteins that likely function in proper air inflation is the epithelial sodium channels (ENaCs). In mammals, ENaC proteins are required to help remove liquid from embryonic lung prior to birth. There are 16 ENaC genes (also referred to as pickpocket or ppk genes) in the Drosophila genome, of which 9 are expressed in the embryonic tracheal system. It is thought that the influx of sodium through these channels drives water from the lumen into the epithelial cells, and that degassing of this liquid inflates the trachea. Although there are no loss of function mutations in ENaCs that result in air filling defects in Drosophila, RNA interference mediated knockdown of ppk4 and ppk11 results in partially liquid-filled tracheae in larvae. In addition, inhibiting these channels using amiloride also results in fluid filled tracheae in larvae. Although both of these examples affected air filling after a molt, it seems likely that ENaC proteins may also contribute to air filling at the end of embryogenesis. At this point it is not possible to exclude the possibility that uif may regulate the expression or activity of ENaC encoded proteins in tracheal epithelia (Zhang, 2009).

    A third possibility, and one that is favored, is that uif is playing primarily a structural role in embryonic tracheal maturation. Two pieces of evidence support this notion. First, in early stage 17 mutant embryos the tracheae are of normal length and diameter and have a stereotypic appearance that is indistinguishable from that of wild type animals, whereas in newly hatched mutant larvae the tubes are often crushed or twisted. Second, while dissecting tracheae from wild type and uif mutant third instar larvae it was clear that the mutant tracheae were more brittle. Wild type tracheae have an elastic property that makes them difficult to break, whereas we had to be very careful dissecting uif tracheae in order to get a section that included more than one metameric unit. Together these observations suggested a model in which the mechanical properties of uif mutant tracheae are compromised, thereby allowing the tracheal cuticle to fail when the embryo initiates violent muscular contractions prior to hatching. As noted in the live imaging, tracheal inflation initiates well after the embryo begins these dramatic muscular contractions. It is predicted that a combination of crushed tubes and small cracks in the tracheal cuticle prevent complete inflation (Zhang, 2009).

    uif mutant larvae that survive to second or third instar show striking defects in tracheal growth and tracheal molting. The tracheae in these uif mutant larvae have short dorsal trunks that are well out of proportion to the body length of the animal. It is difficult to accurately assess the diameter of the dorsal trunk in these animals, however, because the mutant animals fail to completely shed their tracheal cuticle, and often only the first instar lumen is inflated. This tracheal molting defect was nearly completely penetrant in all third instar uif2B7 and uif1A15 mutant larvae examined, but the severity seemed to vary along the anterior-posterior axis, with some middle and anterior sections showing no molting defects, whereas the sections near the posterior spiracles showed at least two and sometimes three tracheal cuticles. A previous study carried out morphometric analyses of larval tracheal growth and observed that tracheae lengthen in a continuous fashion, whereas the diameter increases in a stepwise manner coincident with the molt, suggesting that tube length is controlled independently from tube diameter. The current results indicate that uif plays a role in regulating the growth of the tracheae along their length (Zhang, 2009).

    How might uif regulate larval tracheal tube length? One possibility is that Uif might regulate the interface between the tracheal epithelium and the cuticle, possibly by providing lubrication through its hyalin domains. Loss of uif would therefore result in a situation where the epidermis is too tightly bound to the overlying cuticle to allow for growth along the long axis of the tracheae. This type of mechanism could also account for the tracheal molting defects and thereby couple these phenotypes. An earlier study identified mutations in the Matrix Metalloproteinase encoded by Mmp1; similar defects were found in tracheal elongation. It was speculated that the Mmp1 tracheal defects might be caused by the inability of the tracheal epithelial cells to loosen their attachment to the cuticle. Since uif2B7 mutant larvae have no full length Uif protein, it is unlikely that Uif is a primary target of MMP1 in tracheal cells. In addition, this study observed no genetic interaction between uif and Mmp1 by second-site noncomplementation (Zhang, 2009).

    An alternative hypothesis is that Uif might serve as a receptor or co-receptor for a systemic signal that couples tracheal growth with larval growth. The extracellular domain of Uif contains multiple EGF domains and a laminin G domain, and the cytoplasmic domain is remarkably conserved in all insect species. A combination of structure/function analysis and genetic and biochemical approaches to identify Uif interacting proteins should help to shed light on this functions of uif. Understanding how Uif couples tracheal growth to the growth of the larva may serve as an important paradigm for similar couplings of organ growth to organismal growth in other species (Zhang, 2009).

    Finally, hypoxia may account for all the other larval phenotypes associated with loss of uif, including early larval lethality, slow growth, developmental arrest and failure to pupariate. Most uif mutant larvae die as first instars, and those that die early almost invariably have the most severe tracheal inflation defects. uif mutant larvae that survive to second or third instar grow much slower than their heterozygous siblings. btl>uifRNAi larvae that have only lost uif function in their tracheae show an identical slow growth phenotype. This study consistently observed uif mutant larvae wandering away from food, suggesting that they were experiencing hypoxia. Not surprisingly these larvae accumulate less fat and have a transparent appearance. Examination of the tracheae in these mutant animals revealed that the inflated portion of the trachea was often just through the first instar tracheal lumen, and therefore these animals were likely oxygen starved as well. Consistent with this notion, additional tracheal branching was observed in these mutant animals suggesting that the hypoxia induced factor pathway had been engaged. Thus, hypoxia could explain the growth defects observed in uif mutant larvae. These growth defects may have then resulted in the observed developmental delays. For example, mutant third instar larvae may not have reached a critical weight threshold needed for pupariation, and thus would not have been able to pupariate even if they experienced the metamorphic pulse of ecdysone. Interestingly, many uif mutant animals even failed to advance to third instar, but this did not reflect a defect in epidermal molting or ecdysis, as no evidence was found for an extra set of head skeleton or epidermal cuticle. Rather the animals just arrested as first or second instars. It is possible that hypoxia-induced growth defects contributed to this phenotype as well, since larvae have to attain a critical size to be competent for molting, just as they do for pupariation (Zhang, 2009).

    A matrix metalloproteinase mediates airway remodeling in Drosophila

    Organ size typically increases dramatically during juvenile growth. This growth presents a fundamental tension, as organs need resiliency to resist stresses while still maintaining plasticity to accommodate growth. The extracellular matrix (ECM) is central to providing resiliency, but how ECM is remodeled to accommodate growth is poorly understood. This study investigated remodeling of Drosophila respiratory tubes (tracheae) that elongate continually during larval growth, despite being lined with a rigid cuticular ECM. Cuticle is initially deposited with a characteristic pattern of repeating ridges and valleys known as taenidia. For tubes to elongate, this study found that the extracellular protease Mmp1 is required for expansion of ECM between the taenidial ridges during each intermolt period. Mmp1 protein localizes in periodically spaced puncta that are in register with the taenidial spacing. Mmp1 also degrades old cuticle at molts, promotes apical membrane expansion in larval tracheae, and promotes tube elongation in embryonic tracheae. Whereas work in other developmental systems has demonstrated that MMPs are required for axial elongation occurring in localized growth zones, this study demonstrates that MMPs can also mediate interstitial matrix remodeling during growth of an organ system (Glasheen, 2010).

    This study found that larval tracheal tubes elongate their apical matrix at discrete sites along the long axis of the tube. Immediately after a molt in wild-type animals, the new apical matrix (cuticle) is constructed with taenidial ridges at a fixed interval of ~ 0.8 µm. During intermolt tube elongation, this matrix expands the taenidial interval to ~ 1.6 µm. At molting, this fully expanded matrix is discarded and replaced with a new matrix with a taenidial interval again at 0.8 µm, which will again expand about two-fold. This precise remodeling of the taenidia is accomplished by the extracellular protease Mmp1, which is localized in discrete apical puncta, each associated with an individual taenidium. In Mmp1 mutants, the taenidial ridges do not expand, the taenidial interval remains fixed, and larvae cannot elongate their tracheae as their bodies elongate, causing stretched tubes that eventually break. In normal tube elongation, ECM expansion is coupled with cellular apical membrane expansion, and Mmp1 is required for the coordination of ECM and cellular expansion; Mmp1 is able to promote both aspects of tube expansion when overepressed in embryos. Mmp1 is also required for the other important tracheal cuticle remodeling event in larvae: degrading cuticle into pieces that can be discarded at each molt. Thus, Mmp1 tracheal tubes cannot elongate because they cannot remodel apical extracellular matrix either to expand it or to discard it (Glasheen, 2010).

    On first inspection, it is difficult to account for the progressively deteriorating phenotype of Mmp1 mutants. Although the tracheal system appears morphologically normal at hatching in Mmp1 null mutants, within several hours, they develop taut stretched tracheal systems. As has been previously reported, this phenotype worsens throughout larval life: by second instar, many animals have broken dorsal trunks and some death occurs; by third instar, nearly all animals have tracheal breaks and all eventually die (Page-McCaw, 2003). This presents an apparent paradox as early third instar mutants, with shortened tracheal systems, have normal spacing of their taenidia. This paradox is resolved, however, when one considers that in each instar taenidial expansion is a requirement for tube elongation. Thus, at the start of second instar, although the taenidia are correctly spaced, they comprise a tube that is already shorter than wild-type and is virtually unexpandable, and thus, tubes frequently break as the animal grows. By third instar, although the taenidia are again deposited with normal spacing, the collective failures of elongation in the previous instars make the entire tracheal system very short and highly abnormal. Taken alone, the taenidial expansion data might suggest that the Mmp1 tubes fail to elongate at all. If Mmp1 mutant tubes were unable to achieve any elongation, then the tracheal system of third instar larvae would be ~ 1/8 the length of wild-type controls; it was observed that an Mmp1 tracheal segment is about 1/4 the length of wild-type. Thus, despite the lack of taenidial expansion, there appears to be some kind of other elongation at work in these mutant tubes. Possibilities include an aberrant brute-force stretching of unremodeled cuticle, or a burst of tube elongation during molts when the tube releases cuticle (Glasheen, 2010).

    In electron micrographs of late Mmp1 mutants, the cuticle appears to separate from the epithelial layer in late Mmp1 samples, but not in wild-type; the explanation of artefactual sample fracturing is unlikely, as cell membranes appeared intact in TEMs from both genotypes. Two models were envisioned to explain this separation. One possibility is that matrix components are still secreted by the mutant cells, but they require matrix remodeling to become incorporated into cuticle, and so they accumulate between the cells and the unremodeled cuticle, creating a gap. They would have to be electron-transparent to be consistent with the images, and there are reports of electron-transparent cuticle layers in insects; indeed, the taenidial cores of the TEMs can appear electron-transparent. Alternatively, it is possible that Mmp1 is required for processing adhesion molecules that hold the cuticle to the cell layer, so that adhesion is lost in the absence of the Mmp1. The first model is favored since it accounts for the fate of the matrix components that should have been deposited in the cuticle in the absence of remodeling (Glasheen, 2010).

    An important question arising from this study is how MMP localization and activation is controlled to produce uniform tube elongation. One simple model is that the cells can sense tension and respond by secreting or activating Mmp1. However, this model is not supported by the observation that Tubby (Tb) mutants, with slack in the tracheal tubes, still increase the intertaenidial distance during the intermolt period. Thus, the hypothesis is favored that tube elongation is under developmental regulation. The Tb mutant phenotype indicates that such a tube elongation program is independent of body elongation. One possible mechanism is that the developmental program could trigger Mmp1 secretion from cytoplasmic vesicles to the apical ECM; how MMP secretion is controlled is an open question. The protease appears to be localized in extracellular puncta precisely coordinated with the taenidia. This localization pattern may be ultimately directed by the actin cytoskeleton, which appears to pattern the taenidia during new cuticle secretion. Consistent with the possibility of actin localizing Mmp1, the actin cytoskeleton generally regulates apical secretion in the embryonic tracheal system; and in cultured neurons, it has been observed that MMP-containing vesicles traffic along microfilaments. Mmp1 mutants that lack a hemopexin domain, or dominant-negative mutants that interfere specifically with the Mmp1 hemopexin domain, are still able to elongate their tubes, indicating that the hemopexin domain is not required for secretion, localization, or activation of Mmp1 in these discrete puncta (Glasheen, 2010).

    The interstitial remodeling of tracheal cuticle stands in contrast to other developmental mechanisms of ECM deposition and remodeling that occur during axial growth of rigid or load-bearing structures. During vertebrate long-bone growth, elongation takes place at the growth plates near the ends of the bones, concentrating ECM remodeling and deposition distally. In plants, axial elongation takes place at the meristem regions, which are located distally like growth plates, again confining ECM deposition to distal regions. These cases of spatially restricted remodeling occur in contexts where maintaining the integrity of a rigid ECM presents a structural hurdle to simultaneous remodeling. Interestingly, MMPs appear to be required for both these kinds of growth. Mouse MMP13 mutants cannot remodel the cartilage at the growth plate to make bone, and MMP9 is also required for normal long-bone growth. In Arabidopsis, the MMP mutant At2-mmp1 cannot extend shoots, and in the Loblolly pine, MMP expression is correlated with embryonic root (radicle) protrusion, which is hindered by an MMP inhibitor. In Drosophila larval tracheae, the cuticle is not fully sclerotized and so retains some plasticity, which might be expected as the cuticle does not provide rigidity along the axis but instead protects circumferentially from crushing forces. This different set of structural requirements probably explains why in tracheae deposition of matrix is not confined to distal regions or even to one region per segment, but rather is dispersed across the length of the tissue. The requirement for tube rigidity in the circumferential axis, but not along the body axis, also addresses why tracheal elongation can occur continually, modifying an existing cuticle. In contrast, tube circumferential expansion cannot occur continually but is limited to the molt, when the cuticle is completely replaced. The importance of MMPs in axial growth is underscored by the common requirement for MMPs during all these cases, despite the different structural contexts for matrix remodeling in bone elongation, plant growth, and tracheal elongation (Glasheen, 2010).

    In Mmp1 mutants, where the cuticle remains unelongated, it is striking that elongation of the underlying cells and their apical membranes is also impaired. Although there is some excess of apical membrane in Mmp1 mutants, the excess is much less than expected had the cells underlying the cuticle expanded their membranes to the wild-type extent. These results suggest that either Mmp1 activity or cuticle elongation is required for cells and apical membranes to elongate normally (Glasheen, 2010).

    How are Mmp1 activity and/or cuticle remodeling coordinated with membrane expansion? Although it is possible that matrix remodeling and membrane expansion represent distinct activities of Mmp1, the simplicity of having a direct causal relationship between matrix remodeling and membrane expansion is appealing. One example of a combined model is that extracellular matrix elongation, mediated by Mmp1, places cells under tension, and cells respond by elongating apical membrane. Another model is that Mmp1 proteolytic activity, which remodels cuticle, may simultaneously generate an inductive signal (or inactivate a negative regulator) that causes the underlying cells to elongate, thus coordinating the elongation of both the rigid cuticle and the underlying cells. Production of such a signal would be analogous to mammalian MMPs cleaving laminin-5 or collagen IV to unmask cryptic signaling sites that promote cell migration; this model would also be consistent with recent findings that release of a collagen IV domain by MT2-MMP is required for branching morphogenesis of the submandibular gland in mice. Consistent with Mmp1 activity generating a cuticle-derived signal, misexpression of Mmp1 in embryonic tracheae causes tube elongation only when cuticle is present (Glasheen, 2010).

    These results show that extracellular matrix remodeling is a critical aspect of tube elongation in larval tracheae. Mmp1 mutants cannot remodel cuticle and cannot properly elongate their tubes or degrade unnecessary matrix material to be discarded at molts. These remodeling events are regulated separately from the initial deposition and patterning of cuticle, as Mmp1 mutants are able to secrete normally patterned cuticles. Additionally, Mmp1 appears to regulate cell elongation, perhaps directly by processing a signaling molecule, or indirectly by regulating the ECM. Hence, a matrix metalloproteinase appears to act as a critical coregulator of matrix and cellular growth. Finally, it is significant that tracheal remodeling but not initial tracheal morphogenesis requires a matrix metalloproteinase. This analysis of this remodeling phenotype reinforces the notion that matrix metalloproteinases are specialists for remodeling existing tissues, rather than forming tissues, likely because of the need to alter existing ECM that limits plasticity (Glasheen, 2010).

    A Cathepsin-L is required for invasive behavior during air sac primordium development in Drosophila melanogaster

    The Drosophila Air Sac Primordium (ASP) has emerged as an important structure where cellular, genetic and molecular events responsible for invasive behavior and branching morphogenesis can be studied. This report presents data which demonstrate that a Cathepsin-L encoded by the gene Cysteine proteinase-1 (CP1) in Drosophila is necessary for invasive behavior during ASP development. CP1 is expressed in ASP and knockdown of CP1 results in suppression of migratory and invasive behavior observed during ASP development. It was further shown that CP1 possibly regulates invasive behavior by promoting degradation of Basement Membrane. These data provide clues to the possible role of Cathepsin L in human lung development and tumor invasion, especially, given the similarities between human lung and Drosophila ASP development (Dong, 2015).

    Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment

    To characterize the cellular mechanisms of subcellular tube formation, this study refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using these methods, it was found that subcellular tracheal tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. Correlative light/TEM procedures were developed to identify labeled cells in TEM-fixed larval samples. Using this technique, it was found that the vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, these methods should be applicable to analyzing the many cell biological problems which can be addressed using Drosophila larvae (Nikolova, 2015).

    A cellular process that includes asymmetric cytokinesis remodels the dorsal tracheal branches in Drosophila larvae

    Tubular networks are central to the structure and function of many organs, such as the vertebrate lungs or the Drosophila tracheal system. Their component epithelial cells are able to proliferate and to undergo complex morphogenetic movements, while maintaining their barrier function. Little is known about the details of the mitotic process in tubular epithelia. This study presents a comprehensive model of cellular remodeling and proliferation in the dorsal branches of third-instar Drosophila larvae. Through a combination of immunostaining and novel live imaging techniques, this study identified the key steps in the transition from a unicellular to a multicellular tube. Junctional remodeling precedes mitosis and, as the cells divide, new junctions are formed through several variations of what is refered to as 'asymmetric cytokinesis'. Depending on the spacing of cells along the dorsal branch, mitosis can occur either before or after the transition to a multicellular tube. In both instances, cell separation is accomplished through asymmetric cytokinesis, a process that is initiated by the ingression of the cytokinetic ring. Unequal cell compartments are a possible but rare outcome of completing mitosis through this mechanism. The Dpp signaling pathway was found to be required but not sufficient for cell division in the dorsal branches (Denes, 2015).

    Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth

    Seamless tubes form intracellularly without cell-cell or autocellular junctions (see Labarsky, 2003). Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. This study has found that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes (Schottenfeld-Roames, 2013).

    Three tube types -- multicellular, autocellular and seamless -- are found in the Drosophila trachea. Most tracheal cells contribute to multicellular tubes or make themselves into unicellular tubes by wrapping around a lumenal space and forming autocellular adherens junctions, but two specialized tracheal cell types, fusion cells and terminal cells, make 'seamless' tubes. How seamless tubes are made and how they are shaped are largely unknown. One hypothesis holds that seamless tubes are built by 'cell hollowing', in which vesicles traffic to the centre of the cell and fuse to form an internal tube of apical membrane, whereas an alternative model proposes that apical membrane is extended internally from the site of intercellular adhesion. In both models, transport of apical membrane would probably play a key role. As terminal cells make seamless tubes continuously during larval life, they serve as an especially sensitive model system in which to dissect the genetic program (Schottenfeld-Roames, 2013).

    Tracheal cells are initially organized into epithelial sacs with their apical surface facing the sac lumen. During tubulogenesis, γ-tubulin becomes localized to the lumenal membrane of each tracheal cell, generating microtubule networks oriented with minus ends towards the apical membrane. Terminal and fusion cells are first selected as tip cells that undergo a partial epithelial-to-mesenchymal transition and initiate branching morphogenesis: they lose all but one or two cell-cell contacts and become migratory. Branchless-FGF signalling induces a subpopulation of tip cells to differentiate as terminal cells. During larval life, terminal cells ramify on tissues spread across several hundred micrometres, with branching patterns that reflect local hypoxia. A single seamless tube forms within each branched extension of the terminal cell (Schottenfeld-Roames, 2013).

    How trafficking contributes to seamless tube morphogenesis is unknown. Despite clues that vesicle transport plays a role in the genesis of seamless tubes, the tube morphogenesis genes remain elusive. This study characterized the cytoskeletal polarity of larval terminal cells, shows that a minus-end-directed microtubule motor complex is required for seamless tube growth, and characterizes mutations in whacked (wkd) that uncouple seamless tube growth from the normal spatial cues. Sequence analysis indicates that wkd encodes a RabGAP, and it was shown that Rab35 is the essential target of Wkd, and that together, Wkd and Rab35 can polarize the growth of seamless tubes (Schottenfeld-Roames, 2013).

    Apical-basal polarity and cytoskeletal organization was examined in mature larval terminal cells. The lumenal membrane was decorated by puncta of Crumbs, a definitive apical membrane marker. Actin filaments were found enriched in three distinct subcellular domains: surrounding seamless tubes, decorating filopodia and outlining short stretches of basolateral membrane. The microtubule cytoskeleton also seemed polarized, with γ-tubulin lining the seamless tubes and enriched at tube tips. These data are consistent with tracheal studies in the embryo. EB1::GFP analyses of growing (plus-end) microtubules demonstrated that some are oriented towards the soma and others towards branch tips. Stable acetylated microtubules ran parallel to the tubes and extended beyond the lumen at branch tips where they may template tube growth. Consistent with such a role, microtubule-tract-associated fragments of apical membrane were observed distal to the blind ends of the seamless tubes. Filopodia extended past the stable microtubules as expected. These data indicate that mature terminal cells maintain the polarity and organization described for embryonic terminal cells. On the basis of γ-tubulin localization, it is inferred that a subset of microtubules is nucleated at the apical membrane, and that apically targeted transport along such microtubules would require minus-end motor proteins. Indeed, homozygous mutant Lissencephaly-1. As γ-tubulin lines the entire apical membrane, growth through minus-end-directed transport might be expected to occur all along the length of seamless tubes, and indeed, a pulse of CD8::GFP (transmembrane protein tagged with GFP) synthesis uniformly labelled the apical membrane as it first became detectable (Schottenfeld-Roames, 2013).

    The cytoplasmic dynein motor complex drives minus-end-directed transport of intracellular vesicles in many cell types; to test for its requirement in seamless tube formation, terminal cells were examined mutant for any of four dynein motor complex genes: Dynein heavy chain 64C (Dhc64C), Dynein light intermediate chain (dlic), Dynactin p150 (Glued) and Lis-1. Mutant terminal cells showed a cell autonomous requirement for these genes. Mutant terminal cells had thin cytoplasmic branches that lacked air-filling, and antibody staining revealed that seamless tubes did not extend into these branches although acetylated microtubules often did. It was also noted that formation of filopodia at branch tips is disrupted in dynein motor complex mutants, which may account for the decreased number of branches in mutant terminal cells. Ectopic seamless tubes that were not air-filled were detected near the nucleus, as described below. Interestingly, discontinuous apical membrane fragments (similar to those in Lis-1 embryos) were found in terminal branches lacking seamless tubes, and were associated with microtubule tracts. Whereas γ-tubulin was enriched on truncated tubes and on these presumptive seamless tube intermediates, diffuse γ-tubulin staining was detected throughout the mutant cells, indicating that assembly of apical membrane is required to establish or maintain γ-tubulin localization. Likewise, Crumbs seemed reduced and aberrantly localized. Reduced levels of acetylated microtubule staining in these cells may reflect loss of apical γ-tubulin. Importantly, these data show that stable microtubules extend through cellular projections that lack seamless tubes. Thus, without minus-end-directed transport, stable microtubules are insufficient to promote seamless tube formation, but stable cellular projections are formed and maintained in the absence of seamless tubes (Schottenfeld-Roames, 2013).

    In contrast to these defects in seamless tube generation, mutations in wkd confer overly exuberant tube growth. Examination of wkd terminal cell tips revealed a 'U-turn'; phenotype in which seamless tubes executed a series of 180 degree turns - the possibility is entertained that branch retraction, similar to that observed in talin mutants, could contribute to the U-turn defect (Schottenfeld-Roames, 2013).

    Homozygous wkd animals survived until pharate adult stages, and, other than the seamless tube defects, had normal tracheal tubes at the third larval instar. Mosaic analysis revealed a terminal cell autonomous requirement for wkd. Mutant clones in multicellular tubes, and in unicellular tubes that lumenize by making autocellular adherens junctions, were of normal morphology. Strikingly, fusion cells, which also form seamless tubes, were unaffected by loss of wkd (Schottenfeld-Roames, 2013).

    To determine the molecular nature of wkd, a positional cloning approach was taken. Mapping techniques defined a candidate gene interval of ~ 75 kilobases (kb). Focused was placed on CG5344 as it encodes a protein containing a TBC (Tre2/Bub2/Cdc16) domain characteristic of Rab GTPase-activating proteins, and hence was likely to participate in vesicular trafficking, a process that could lie at the heart of seamless tube formation. A single nucleotide change was identified that resulted in mis-sense (PC24) and non-sense (220) mutations in the CG5344 coding sequence. Pan-tracheal knockdown of wkd by RNA-mediated interference (RNAi) caused terminal cell-specific U-turn defects (other defects characteristic of the ethyl methanesulfonate (EMS)-induced alleles of wkd were detected at a low frequency). A genomic rescue construct for CG5344 rescued wkd mutants, confirming gene identity. On the basis of these results, it is concluded that wkd is CG5344 and that it probably regulates vesicular trafficking during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

    To determine the Rab target(s) of Wkd regulation, whether tracheal expression of constitutively active 'GTP-locked'; Rab isoforms (henceforth, RabCA) might phenocopy wkd was investigated. RabCA for 31 of the 33 Drosophila Rabs were tested individually in the tracheal system. Rab35CA alone conferred terminal-cell-specific U-turns defects (Schottenfeld-Roames, 2013).

    To evaluate Wkd overexpression, UAS-wkd was expressed in wild-type animals in a pan-tracheal pattern. Excess Wkd caused formation of ectopic seamless tubes surrounding the terminal cell nucleus. At higher levels of expression, small spheres of apical membrane were found adjacent to the nucleus and less abundantly at more distal sites. Consistent with Wkd regulation of vesicle trafficking by modulation of Rab35, expression of a dominant-negative Rab35 (henceforth, Rab35DN) caused formation of ectopic proximal tubules (Schottenfeld-Roames, 2013).

    Attempts were made to determine whether Rab35 was the essential target of Wkd GAP activity. Wkd primary structure is equally conserved in three human RabGAPs. All three act as Rab35GAPs, although each has been proposed to have additional targets. To further determine if Wkd acts as a Rab35GAP, whether Rab35DN could suppress wkd mutants was examined; tracheal-specific expression of Rab35DN strongly suppressed the 'U-turn'; defects of wkd-null animals and, surprisingly, also rescued the lethality of wkd. Since mutant Rab35 isoforms phenocopy wkd gain and loss of function, Rab35DN bypasses the requirement for wkd and human Wkd orthologues are Rab35GAPs, it is concluded that the critical function of Wkd is as a GAP for Drosophila Rab35 (Schottenfeld-Roames, 2013).

    In other systems Rab35 is implicated in polarized membrane addition to plasma membrane compartments - for example, immune synapse, cytokinetic furrow and so on - or, in actin regulation. A role for actin in fusion cell seamless tube formation has been proposed, so whether Wkd and Rab35 act by modulation of the terminal cell actin cytoskeleton was examined. As the actin-bundling protein Fascin (Drosophila singed) was recently identified biochemically as a Rab35 effector, a role of singed in terminal cell tubes was examined, but found no evidence was found for one. Furthermore, overexpression of Wkd, or of Rab35DN, did not significantly alter the terminal cell actin cytoskeleton, leading to the conclusion that actin regulation is not a primary function of Wkd/Rab35 during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

    The alternative model -- that Rab35 acts in polarized membrane addition -- was found to be attractive, because extra Rab35-GTP activity promoted seamless tube growth at branch tips whereas depletion of Rab35-GTP promoted tube growth at the cell soma. To test this model, advantage was taken of the information that expression of an activated Breathless-FGFR (lambdaBtl in terminal cells induces robust growth of ectopic seamless tubes surrounding the nucleus; whether growth of the ectopic tubes could be redirected from the soma to the branch tips was investigated by eliminating wkd. The activated FGFR phenotype was not altered in wkd heterozygotes, but in wkd mutant animals (or wkd-RNAi animals) the site of ectopic seamless tube growth was strikingly different. In some cells, extra tubes were found throughout the cell - in the soma and at branch tip - whereas in others extra tubes were present only at the branch tip. Thus, the position of seamless tube growth is dependent on Wkd activity, although Wkd itself is not essential for tube formation. These data provide evidence against branch retraction (as occurs in talin mutants) as the mechanism for generating a U-turn phenotype, because branch retraction would not redirect ectopic tube growth (Schottenfeld-Roames, 2013).

    To better understand how Wkd and Rab35 determined the site of seamless tube growth, their subcellular distribution was examined. Pan-tracheal expression of mKate2-tagged Wkd (Wkd::mKate2) rescued wkd-null animals. The steady-state subcellular localization of Wkd::mKate2 was restricted to the lumenal membrane with higher accumulation at the growing tips of seamless tubes. At lower levels, cytoplasmic puncta of Wkd::mKate2 were noted that could reflect vesicular localization, as well as labeling of filopodia. It was found that YFP::Rab35 was distributed in a diffuse pattern throughout the terminal cell cytoplasm with some apical enrichment, and notable localization to filopodia. Substantial co-localization of Wkd::mKate2with YFP::Rab35 was found at the apical membrane, in cytoplasmic puncta, and in filopodia. Among endosomal Rabs, Rab35 seemed uniquely abundant within filopodia, and showed the greatest overlap with Wkd at the apical membrane. Substantial overlap was noted between Wkd/Rab35 and acetylated microtubules, including at positions distal to the blind end of seamless tubes. The enrichment of Wkd along seamless tubes indicates that Rab35 functions in an apical membrane trafficking event, leading to the speculation that recycling endosomes at filopodia might be targeted to the growing seamless tube by minus-end motor transport (Schottenfeld-Roames, 2013).

    In a similar vein, it is speculated that vesicles might be transported from the soma towards branch tips in a process regulated by Wkd and Rab35. Disruption of such transport might explain why overexpression of Wkd leads to ectopic seamless tube growth in the soma. Whether Wkd::mKate2 localization was compromised in dynein motor complex mutants was examined. As these cells have branches that lack apical membrane/seamless tubes, disruption in the localization pattern of Wkd was anticipated, but it was wondered whether co-localization with acetylated tubulin would be intact, indicative of a microtubule association independent of dynein motor transport. It was found that Wkd::mKate2 is broadly distributed throughout the cytoplasm of dynein motor complex mutants, and does not show enrichment on acetylated microtubule tracts; indeed, substantial basal enrichment was detected of Wkd::mKate2. If Wkd/Rab35-dependent trafficking of apical vesicles was dynein motor complex dependent, ectopic seamless tubes would be expected in the soma of dynein motor complex mutants, similar to those seen with Wkd overexpression or expression of Rab35DN. In fact, such ectopic tubes were consistently found in the dynein motor complex mutants, consistent with dynein-dependent trafficking of Rab35 vesicles. It cannot be ruled out that these defects are due to dynein-dependent processes unrelated to Wkd and Rab35; however, whether the ectopic tubes could be redirected distally by expression of Rab35CA, or elimination of Wkd, was examined. The motor complex ectopic tube phenotype could not be altered, indicating that the phenotype does not arise as an indirect consequence of altered Wkd localization or Rab35 activity (Schottenfeld-Roames, 2013).

    The roles of RabGAP proteins have started to become clear only in recent years. Historically, it has been difficult to determine which Rab proteins are substrates of specific RabGAPs. Tests of in vitro GAP activity produced conflicting results, and in some cases did not seem indicative of in vivo function. Indeed, the specificity of Carabin (also known as Wkd orthologue TBC1D10C) has been controversial: it was first shown to act as a RasGAP, whereas later studies indicate a Rab35-specific GAP activity. The in vivo genetic data for wkd, together with recent studies characterizing the function of all three human Wkd-like TBC protein, make a compelling case that this family of proteins acts as GAPs for Rab35. Furthermore, this study establishes a role for classical vesicle trafficking proteins in seamless tube growth. As seamless tubes, but not multicellular or autocellular tracheal tubes, are affected by mutations in wkd and Rab35, this study also establishes an in vivo cell-type-specific requirement for trafficking genes in tube morphogenesis (Schottenfeld-Roames, 2013).

    It is concluded that Wkd and Rab35 regulate polarized growth of seamless tubes, and it is speculated that Wkd and Rab35 direct transport of apical membrane vesicles to the distal tip of terminal cell branches (when equilibrium is shifted towards active Rab35-GTP), or to a central location adjacent to the terminal cell nucleus (when equilibrium is shifted towards inactive Rab35-GDP). Analogous to its previously described roles in targeting vesicles to the immune synapse in T cells, the cytokinetic furrow in Drosophila S2 cells and the neuromuscular junction in motor neurons, Rab35 would promote transport of vesicles from a recycling endosome compartment to the apical membrane. It is further speculated that Breathless-FGFR activation at branch tips may couple terminal cell branching with seamless tube growth within that new branch (Schottenfeld-Roames, 2013).

    Developmental compartments in the larval trachea of Drosophila

    The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 reveals that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein-expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks (Rao, 2015).

    Microtubule-dependent apical restriction of recycling endosomes sustains adherens junctions during morphogenesis of the Drosophila tracheal system

    Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. This study reports a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells showed that MTs were essential during a specific time frame of tracheal cell elongation while the branch extended. In the absence of MTs, one tracheal cell per branch overelongated, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs were crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs. Maintaining E-Cadherin/Par-3 levels at the apical domain required de novo synthesis rather than internalisation and recycling from and to the apical plasma membrane. However, apical targeting of E-Cadherin and Par-3 required functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. The apical enrichment of recycling endosomes was dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, E-Cadherin dynamics and MT requirement differed in remodelling tracheal cells versus planar epithelial cells. Altogether, these results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis (Le Droguen, 2015).

    This study has reveal the importance of the functional interplay between MTs, vesicular trafficking and the control of AJ dynamics in cells undergoing extensive remodelling through collective migration. MT depletion in tracheal cells induces the formation of intracellular dots containing E-Cad and Par-3. Interestingly, these intracellular accumulations are only seen in remodelling tracheal cells and not in planar epithelia. These dots are still detected when endocytosis is affected, showing that they are de novo synthesis route intermediates. Altogether, this suggests that maintaining the correct level of E-Cad/Par-3 at the apical domain requires a continuous supply of newly synthesized proteins, which could be essential for the intensive AJ reorganisation that occurs during cell intercalation and elongation of the tracheal branch (Le Droguen, 2015).

    Using the photo-convertible E-Cad-EosFP in flat epithelium, a previous study showed that E-Cad that is engaged in homophilic interactions at the AJs forms very stable domains. This study has demonstratde that MT depletion does not affect the integrity of this 2D epithelium. In addition, it was shown that the pool of photo-converted E-Cad-EosFP is less stable in tracheal cells than in epidermal cells. Together with a FRAP assay suggesting that AJs are more dynamic in tracheal cells than in epithelial cells, the results highlight a specific fine-tuning of AJ components in tracheal cells undergoing cell movement and cell shape changes in 3D through cell intercalation, cell elongation and thereby organ formation. This fine-tuning is likely to cycle between internalisation, recycling, degradation and de novo synthesis, the latter being MT dependent. When the balance is altered in the absence of MTs, and thus when E-Cad or Par-3 are reduced at AJs, tracheal cells overelongate after completing intercalation. During branch elongation without MTs, the two migrating leading cells generate a pulling force on the following stalk cells, which display a critical reduction in AJ components. Either the tip cell or the base cell of the stalk becomes unable to maintain its integrity in response to this force. Consequently, this tracheal cell overelongates by a factor 1.8, preventing the remaining stalk cells from reaching their average size. As a result, DBs present a single overelongated cell and several underelongated cells (Le Droguen, 2015).

    An overlap was detected between the E-Cad intracellular dots generated in the absence of MTs and recycling endosome vesicles. Interfering with Rab11 function in tracheal cells induces cell overelongation and affects E-Cad and Par-3 distribution at the AJs, as does MT depletion. These results illustrate that the E-Cad de novo synthesis pathway passes through the Rab11-positive recycling endosome compartment. The overlap of E-Cad and recycling endosome markers represents only a small proportion of the total intracellular E-Cad, suggesting a transient residence in this vesicular compartment for this newly synthesized protein. Recent studies conducted in different model systems have revealed that some newly synthesized apical plasma membrane proteins, such as E-Cad and Rhodopsin, leave the trans-Golgi network to cross Rab11-positive recycling endosome compartments before reaching the apical surface). This apical trafficking route is used specifically in tracheal cells and requires the MT network. Quantification of Rab11DN-associated defects upon tracheal branch formation reveals that impairing recycling endosome function has a similar effect to altering the distribution of E-Cad at AJs by depleting the MT network. Moreover, interfering with Rab11 function reduces Par-3 levels at the AJs of tracheal cells. Interestingly, Par-3 does not colocalise with the E-Cad cytoplasmic pool, indicating that functional recycling endosomes are required by Par-3 and E-Cad to assemble as a complex and to be targeted to the apical domain of tracheal cells. However, as E-Cad and Par-3 can be apically targeted in the absence of the other, this suggests that apical targeting of E-Cad and Par-3 can be independent in tracheal cells or that redundant pathways could sustain the localisation of each protein. For example, the Nectin protein Echinoid is required for Par-3 localisation at AJs in shg mutant cells. Moreover, the apical distribution of PI(4,5)P2 in the Drosophila follicular epithelium sustains Par-3 apical anchorage at the plasma membrane. Furthermore, Par-3-independent localisation of E-Cad has been observed (Le Droguen, 2015).

    Dynamic MTs in the ectoderm locally upregulate AJ turnover through RhoA activity. RhoA stabilises cellular contacts through acto-myosin regulation. In tracheal cells, MT depletion does not alter actin distribution. Moreover, tracheal cells mutant for the Myosin light chain zipper (zip) and also expressing a dominant-negative form of Zip do not present obvious defects in E-Cad distribution at stage 14. By contrast, MT depletion induces the cytoplasmic accumulation of E-Cad and Par-3 in tracheal cells only and not in ectodermal cells at the same developmental stage. Thus far, cytoplasmic accumulation of E-Cad and Par-3 has only been observed after colchicine-induced MT depolymerisation during polarity establishment at embryo cellularisation, when AJs are extremely dynamic and vesicular trafficking is strongly active. This study demonstrated that the E-Cad distribution in tracheal cells is more sensitive to MT depolymerisation and to Rab11DN overexpression than that in the overlying ectodermal cells. The comparison of the maximum recovery of the E-Cad signal in a FRAP assay in tracheal cells and in ectodermal cells, together with the differences in stability of the photo-converted E-Cad-EosFP between these two tissues, suggest that AJs are more dynamic in tracheae. It is thus conceivable that tracheal cells, which undergo extensive cell shape changes through collective cell migration, require more dynamic AJs as sustained by an efficient targeting of E-Cad. These discrepancies between ectodermal and tracheal epithelia underline the importance of investigating MT function in different morphogenetic contexts under different constraints (Le Droguen, 2015).

    This study has demonstrated that the MT minus-end motor Dynein is essential for the restricted localisation of recycling endosomes in a developing organism. The Dynein requirement for the apical enrichment of recycling endosomes is in agreement with the MT minus ends being anchored at the apical plasma membrane. The asymmetric distribution of recycling endosome vesicles has been observed in various differentiated cell types, especially during cell division. Vesicles are found enriched either in the apical domain or at the microtubule-organising centre (MTOC; i.e. the centrosome) during mitosis. Indeed, in vivo, Dynein physically interacts with Nuf. During metaphase, Dynein is required for the maintenance of Nuf at the centrosome. This study demonstrates that Dynein is also required for the apical distribution of recycling endosomes in non-dividing tracheal cells. In a context in which Dynein function is altered, Nuf-positive recycling endosome vesicles are dispersed but colocalise with E-Cad and Par-3 intracellular dots, indicating that the recycling endosome compartment remains functional for the assembly of such a complex (Le Droguen, 2015).

    A previous study has characterised the relocalisation of the MTOC in tracheal cells; MTs are nucleated and anchored at the apical domain just above the AJs. It has also been demonstrated that such MT organisation is crucial for tracheal morphogenesis. Non-centrosomal MT organisation occurs in many differentiated cell types but the functional relevance of such an organisation is still poorly understood. It will be informative to investigate whether such non-centrosomal MT organisation provides a means to regulate epithelial remodelling by controlling the apical enrichment of recycling endosomes and thus AJ dynamics (Le Droguen, 2015).

    Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes

    Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. This study identified a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. It is proposed that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells (Hosono, 2015).

    The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity

    Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. This study shows that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network (Caviglia, 2013).

    This work has investigated how the choice between the two types of specialized tip cells in the tracheal system is controlled. The transcriptional repressor Aop plays a key role in linking tracheal tip cell fate selection with branch identity. First, a novel tube morphogenesis phenotype is described in aop mutants, which is due to the massive mis-specification of regular epithelial cells into specialized tracheal tip cells. aop is specifically required for controlling tracheal cell fate, whereas aop, like pnt, is dispensable for primary tracheal branching, thus uncoupling roles of RTK signaling in cell fate specification and cell motility. The finding that tracheal branching morphogenesis proceeds normally in the presence of excess tip cell-like cells suggests that collective cell migration is surprisingly robust and that mis-specified cells apparently do not impede the guided migration of the tracheal primordium. Second, it was demonstrated that in the absence of inhibitors of MAPK signaling (aop and sty), all tracheal cells are competent to assume either TC or FC fate. The transcriptional repressor Aop globally blocks both TC and FC differentiation, but high-levels of MAPK signaling in tip cells relieve Aop-mediated inhibition, thus permitting differentiation. Third, the results suggest that in the DT region Aop limits FC induction through a distinct mechanism by antagonizing Wg signaling in addition to MAPK signaling. Conversely, in the other branches, Aop limits TC differentiation by blocking MAPK-dependent activation of Pnt. Fourth, it was shown that the region-specific choice between the two cell fates in the DT is determined by Wg signaling and by the selector gene salm. Based on these results, a model is proposed in which a single repressor, Aop, integrates MAPK and Wg signals to couple tip cell fate selection with branch identity. High levels of Bnl signaling trigger Pnt activation and Aop degradation in tracheal tip cells. It is proposed that in the DT, unlike in other tracheal cells, MAPK-induced degradation of Aop releases inhibition of Wg signaling. This is consistent with recent work showing an inhibitory effect of Aop on Wg signaling, possibly through direct interaction of Aop and Arm, or through Aop-mediated transcriptional repression of Wg pathway component. The current work extends the evidence for this unexpected intersection between two major conserved signaling pathways, suggesting that this function of Aop is likely to be more widespread than previously appreciated. The findings also provide an explanation for the puzzling observation that, in pnt mutants, TCs are lost, while FCs become ectopically specified. As pnt is required for expression of the feedback inhibitor sty, loss of pnt is expected to lead to MAPK pathway activation and consequently to increased Aop degradation. This would release Aop-mediated repression of Wg signaling, resulting in extra FCs, whereas TCs are absent because of the lack of pnt-dependent induction. This suggests that excessive FC specification in the DT of aop and sty mutants is mainly due to deregulated Wg signaling, rather than to de-repression of pnt-dependent MAPK target genes. Consistent with this notion, it was shown that pnt is not required for Delta and Dys expression in tracheal cells, although constitutively active AopACT represses their expression (Caviglia, 2013).

    The results further show that salm function constrains the fate that is chosen by cells when released from the Aop inhibitory block. MAPK signaling triggers Aop degradation in all tip cells, but only in the absence of salm does this signal lead to TC induction. In salm-expressing cells, degradation of Aop releases Wg signaling, resulting in FC specification. Thus, salm biases the choice between two morphologically different types of seamless tubes. This is reminiscent of the role of salm in switching between different cell types in the peripheral nervous system and in muscles. salm expression is sufficient to repress TC formation. The genetic results, consistent with biochemical data showing that Salm acts as a transcriptional repressor, suggest that salm promotes FC fate by repressing genes involved in TC development. However, salm is not sufficient to overcome the requirement for Wg signaling in FC induction, indicating that Wg does not act solely via salm to induce FC fate. Indeed, FC induction requires genes whose expression is independent of salm (esg, dys). In addition, it is proposed that a feedback loop between Wg signaling and salm expression maintains levels of Wg signaling in the DT sufficiently high to induce FC fate. Taken together, these results suggest that the default specialized tip cell fate, and possibly an ancestral tracheal cell state, is TC fate. Although FCs and TCs differ in their morphology, they share a unique topology as seamless unicellular tubes. FCs and TCs might therefore represent variations of a prototypical seamless tube cell type. Salm might modify cellular morphology by repressing TC genes, including DSRF, which mediates cell elongation and shape change. Intriguingly, Wg-dependent salm expression in the DT of dipterans correlates with a shift towards FC as the specialized fate adopted by the tip cells of this branch. This study has shown that salm expression inhibits TC fate, while promoting the formation of a multicellular main tracheal tube by inhibiting cell intercalation. It is therefore tempting to speculate that the salm-dependent switch from a branching towards an anastomosing tip cell type in the DT may have evolved with the acquisition in higher insects of a main tube that connects separate tracheal primordia to generate a tubular network. It will be of great interest to identify the relevant target genes that mediate the effect of Salm on tube morphology and tip cell fate (Caviglia, 2013).

    The mechanisms of tip cell selection during angiogenesis in vertebrates are beginning to be understood at the molecular level. However, the signals that control the formation of vascular anastomoses by a particular set of tip cells are not known. Intriguingly, the development of secondary lumina in aop mutants is reminiscent of transluminal pillar formation during intussusceptive angiogenesis, which is thought to subdivide an existing vessel without sprouting. Although the cellular basis for this process is not understood, it is conceivable that specialized endothelial cells are involved in transluminal pillar formation. This work provides a paradigm for deciphering how two major signaling pathways crosstalk and are integrated to control cell fate in a developing tubular organ. It will be interesting to see whether similar principles govern tip cell fate choice during tube morphogenesis in vertebrates and invertebrates (Caviglia, 2013).

    Specification of leading and trailing cell features during collective migration in the Drosophila trachea

    The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a FGF homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized; two prototypical types of cytoskeletal organization were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, it was shown that FGF signalling in a single tip cell can trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and how FGF-independent cell features such as adhesion and motility act on coupling the behaviour of trailing and tip cells was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF (Lebreton, 2013).

    Among the tracheal branches from each placode, two grow towards the ventral side of the embryo, one in the anterior and the other in the posterior region of the segment, the lateral trunk anterior (LTa) and the lateral trunk posterior (LTp) respectively. By a combination of migration, intercalation and elongation, the tip cell of the LTp migrates towards the central nervous system (CNS), and the resulting ganglionic branch (GB) connects the CNS to the main tracheal tube. Another cell from the LTp migrates towards the LTa of the adjacent posterior metamere and makes a fusion branch that connects the two LT branches. This study focused on this branch (LTp/GB) because its complex morphology and pattern of migration make it particularly appropriate for analysing the morphology and behaviour of the tip and trailing cell during tracheal collective migration (Lebreton, 2013).

    The FGF signalling pathway is involved in many morphogenetic events requiring collective migration of cell clusters. However, it is not entirely clear whether in these events FGF signalling is directly involved in triggering cell migration, or alternatively if it is required for other processes such as cell determination which only affect cell migration indirectly. Moreover, while FGF might be required it is not clear either whether all the cells or just a subset of those need to directly receive the signal to sustain the migration of the entire cluster. One well-studied case is the role of FGF in the development of the zebra fish lateral line. In that case, FGF appears to be produced by the leading cells which signal to the trailing cells, the cells where FGF signalling is active. Restriction of FGF signalling is thereafter required for the asymmetric expression of the receptors for the chemokines that guide migration (Lebreton, 2013).

    A very different scenario applies in the case of Drosophila tracheal migration. On the one hand, FGF is expressed in groups of cells outside the migrating cluster. On the other hand the results in the LTp/GB indicate that FGF signalling is required and sufficient in the leading cells, and not in the trailing cells, for the migration of the whole cell cluster. Therefore, in spite of its widespread involvement, the mechanisms triggered by FGF signalling in collective migration appear to be quite different (Lebreton, 2013).

    Rho inactivation produced breaks and detachment in the LTp/GB cluster while its constitutive activation led these cells to hold together impairing migration. Likewise, upon Cdc42 inactivation LTp/GB cells were associated by thin extensions associated in some cases with breaks, while upon its constitutive activation, the LTp/GB transient pyramidal organisation did not evolve, or evolved much more slowly, towards branch elongation. However, the phenotypes from each RhoGTPase mutants don't look alike and the detailed analysis suggests that Rho impinges primarily on cell adhesion while Cdc42 does so on cell motility (Lebreton, 2013).

    These results are consistent with previous findings that show a role for Rho in regulating adherens junctions stability and for Cdc42 as the main mediator of filopodia formation. It is noted, however, that Cdc42 was found to exert in the LTp/GB an opposite effect to the one identified in other systems, as Cdc42DN mutants showed more protrusions and were more actin-enriched basally than wild-type cells and Cdc42ACT mutants showed a reduced the motility of LTp/GB (Lebreton, 2013).

    There is an increasing amount of data pointing to the different effect of RhoGTPases in vitro versus in vivo models and also among various cell types. A unidirectional assignment between a specific cellular process in vivo and a single RhoGTPase is probably an oversimplification and this was not the aim of the current study. Rather the study relied on mutant forms of the RhoGTPases to modulate cell features, either individually or collectively, to assess their role in the overall behaviour of the cell cluster. In doing so, the results point to a critical role for a balance between cell adhesion and cell motility for the collective migration of a cell cluster (Lebreton, 2013).

    The results support the following model for the specification, features and behaviour of leading cells in the migration of the LTp/GB branch. Upon signalling from the FGF pathway, tip cells reorganise their cytoskeleton features (actin enrichment at the basal membrane, small apical surface and an apicobasal polarity along the proximo-distal axis), thereby enabling them to acquire leading behaviour. Indeed, FGF can induce migratory capacity to the whole cluster by signalling only the tip cells, where a dynamic transition between states of Rac activity is needed to acquire a leading role. How the behaviour of tip cells leads collective migration thereafter depends on the features of the cells in the cluster, which are determined by various regulators (among these, the RhoGTPases) which act, at least in part, in an FGF-independent manner. Ultimately, the balance between individual cell properties such as cell adhesion, motility and apicobasal polarity will (1) determine the net movement of the overall cell bodies or alternatively changes in cell shape in terms of elongation, (2) control the migratory speed and (3) define whether cells will migrate individually or in clusters and whether clusters will bifurcate in different paths. The combined effect of the changes promoting leading cell behaviour and modulation of cell features is likely to be a widely exploited mechanism in collective migration. In particular, the actual balance between these cell features may dictate the specifics of each migratory process and, consequently, the final shape of the tissues and organs they contribute to generate (Lebreton, 2013).

    Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophil

    Tissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, this study shows that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, it was demonstrated that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule (see Tip-Cell-Dependent Anchorage of Anterior Tubules to Alary Muscles). Tip cell anchorage antagonizes forward-directed, TGF-beta-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function (Weavers, 2013; see Graphical Abstract).

    As the embryonic renal tubules assume their mature shape they interact with other tissues, responding to Dpp guidance cues as they take up their characteristic positions in the body cavity (Bunt, 2010). This study shows that, in addition, a single cell at the distal end of each renal tubule makes specific transitory, and finally long-term, contacts with target tissues. These cells express a distinctive pattern of genes and show characteristic exploratory activity, which is crucial for the stereotypical looped shape and position of the tubules in the body cavity. In turn, these features have profound consequences for the efficacy of fluid homeostasis in the whole animal (Weavers, 2013).

    It is suggested that the elongation and forward extension of the tubules result from the combined effects of cell rearrangements that lengthen the tubule and the response of kink region cells to regional Dpp guidance cues. The evidence indicates that the tip cells act as anchors, through their interactions with alary muscles, so that tubules are tethered at both ends, the proximal end being attached through ureters to the hindgut. These attachments perform two functions: they stabilize the looped architecture, maintaining the kink close to the tubule midpoint, and they limit forward and ventral movement to ensure the stereotypical tubule arrangement in the body cavity (Weavers, 2013).

    If tip cell contact with the alary muscles is lost, the kink 'unravels,' shifting distalward, and the tubule as a whole extends too far into the anterior, with the distal region lying more ventrally close to the Dpp-expressing gastric caeca. Confirming the existence of a forward tractive force responsible for tip cell detachment are the distortion of transient alary muscle targets before the tip cell detaches and the characteristic 'recoil' seen when the tip cell is ablated. Evidence that this results from the response to guidance cues is the failure of tip cells to detach from their first alary muscle contact (A5/A6) in the absence of the midgut Dpp guidance cue and the more anterior location of the kink region (close to the gastric caeca) in tubules where the tip cell stalk is greatly extended, for example when the activity of RhoA is repressed. The critical nature of the balance between these forward and restraining influences is also revealed when adhesion between the tip cells and alary muscles is increased by manipulating tip cell number or adhesive strength. In each case, the tip cells remain attached to alary muscles posterior to their normal final contacts, and this results in more posterior positioning of the whole tubule. Together, these results strongly suggest that tip cells detach because the forward movement of tubules overcomes the adhesive strength of their early transient contacts (Weavers, 2013).

    The final tip cell/alary muscle target is highly reproducible, suggesting recognition through segmental identity, the A3/A4 target being the first encountered by the tip cell that expresses Ubx. However, altering Ubx expression in alary muscles has no effect on the final tip cell contact. Instead, it appears that tip cells adhere to each alary muscle they contact, and the final target depends on the balance between forward tubule movement and the strength of tip cell/target adhesion. Consistent with this view, when all the normal muscle targets are ablated, tip cells can make stable contacts with the A2/A3 alary muscle (Weavers, 2013).

    From the time that they are specified, tip cells show distinctive patterns of gene expression, morphology, and behavior critical to their ability to make alary muscle contacts; they form dynamic filopodia, which explore the alary muscle surface, remain denuded of the BM that envelops the rest of the tubule, and express cell-adhesion proteins, including Neuromusculin (Nrm) and integrins. Protrusive activity depends on the rapid turnover of actin, mediated by regulators such as Rac GTPase and the actin-capping protein Enabled, which are active in tip cells (Weavers, 2013).

    BM deposition basally around the tip cells severely inhibits protrusive behavior, and tip cells therefore employ multiple mechanisms to ensure that they remain denuded. These include the absence of expression of factors that promote BM deposition and stabilization (hemocyte attractants, BM components, or receptors), the removal by transcytosis of any BM that is deposited, and expression of MMP1, which is able to cleave matrix components. MMP1 is expressed late during tubule elongation and the protein is localized apically in tip cells, suggesting that its function might be to degrade transcytosed BM proteins (Weavers, 2013).

    Protrusive exploratory behavior results in adhesive contacts made possible through tip cell expression of Nrm and integrins. Nrm is a homophilic cell-adhesion molecule of the Ig-domain superfamily. The binding partner for tip cell Nrm is unclear, as alary muscles do not express it. However, driving nrm expression in alary muscles induces strong adhesion, resulting in tip cells remaining bound to their first target in A5/A6. It is possible that Nrm in tip cells normally makes heterophilic associations with Ig domain-containing proteins such as Dumbfounded (Kirre), which is expressed in alary muscles and is sufficient, when overexpressed, to induce more posterior target adhesio (Weavers, 2013).

    Tip cells express integrins, and complexes accumulate as each target contact is made, but initially they do not lead to long-term adhesion. It is suggested that the strength of adhesion increases with successive contacts, either through increased expression of integrins and their associated factors or by regulated adhesive complex turnover, as shown in other tissues. Once the final tip cell contact is made, BM accumulates around the tip cellalary muscle surface, increasing the concentration of integrin ligands at the junction. The accompanying decline in the protrusive activity of the tip cell could also result from integrin-mediated adhesion, which is known to reduce levels of the actin-capping protein Enabled. This sequence of events parallels the mechanism by which elongated myotubes and tendon cells establish their myotendinous junctions (Weavers, 2013).

    Once the anterior tubule tip cells make their final alary muscle contact, they remain attached throughout development into adult life. Such interaction of excretory tubule tips with muscles is a common feature of renal systems in insects, either with alary muscles or with fine striated muscles that spiral along the tubule. Muscle contacts increase tubule movement, maximizing the effectiveness of excretion, by increasing hemolymph sampling and enhancing tubule flow. Similar contacts are found outside the arthropods; the flame cells that cap planarian protonephridial tubes develop prominent filopodia and interact with nearby muscle fibers, providing anchorage, thought to be important during branching morphogenesis in this system (Weavers, 2013).

    Tip cells or groups of cells at the distal tips of outgrowing epithelial tubes act as organizers in tubular systems, from the migrating Dictyostelium slug to the branching epithelial scaffolds of human organs. As in fly renal tubules, these distinctive cells regulate cell division and guided tubule extension, and in mammalian systems they control branching morphogenesis (Weavers, 2013).

    However, in distinct contrast to the role of tip cells in the morphogenesis of these systems, the tip cells of the anterior renal tubules play no role in leading outgrowth. Instead, they act to counteract outgrowth, and importantly this leads to the development of a looped tubular structure both by tethering the distal tips of tubules close to their proximal junction with the ureter and by maintaining the tightness of the tubule kink region. Looped tubular structures are relatively uncommon; a tubule tree as in the lung, pancreas, or liver or an anastomosing network as in the vascular system is more frequently seen. However, a striking example of looped tubules is found in the mammalian kidney, where the distal and proximal convoluted tubules together with the loop of Henle connect the tubule tip (at the glomerulus) to the collecting duct (close to the site of urine outflow). Looping of both the nephron and its vascular supply creates a countercurrent system that maximizes the efficiency of ion and fluid homeostasis. Such exchange systems also occur in insects with specialized diets or those living in dry conditions. Countercurrent exchange has not been demonstrated in Drosophila melanogaster tubules, where it is more likely that the looped tubule structure is important for effective hemolymph sampling (Weavers, 2013).

    In the development of the mammalian nephron, as in fly renal tubules, both the site of connection to the ureter and the tubule tip, the renal corpuscle, are established early in organ development so that tubule extension, by both cell proliferation and rearrangements, occurs between these fixed points. It will be interesting to discover whether similar tissue interactions stabilize the position of the developing glomerulus, and so play a prominent role in maintaining the looped structure as kidney tubules extend, resulting in the final intricate and regular array of nephrons apparent in the mature mammalian kidney (Weavers, 2013).

    Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

    Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

    The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

    The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

    The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

    Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

    To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

    The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

    To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

    sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

    This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

    These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

    A dynamic microtubule cytoskeleton directs medial actomyosin function during tube formation

    The cytoskeleton is a major determinant of cell-shape changes that drive the formation of complex tissues during development. Important roles for actomyosin during tissue morphogenesis have been identified, but the role of the microtubule cytoskeleton is less clear. This study shows that during tubulogenesis of the salivary glands in the fly embryo, the microtubule cytoskeleton undergoes major rearrangements, including a 90° change in alignment relative to the apicobasal axis, loss of centrosomal attachment, and apical stabilization. Disruption of the microtubule cytoskeleton leads to failure of apical constriction in placodal cells fated to invaginate. This failure is due to loss of an apical medial actomyosin network whose pulsatile behavior in wild-type embryos drives the apical constriction of the cells. The medial actomyosin network interacts with the minus ends of acentrosomal microtubule bundles through the cytolinker protein Shot, and disruption of Shot also impairs apical constriction (Booth, 2014).

    Compensatory branching morphogenesis of stalk cells in the Drosophila trachea

    Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. It was determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase); both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, this study found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. This study has thus identified a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor (Francis, 2015).

    Microtubule-dependent balanced cell contraction and luminal-matrix modification accelerate epithelial tube fusion

    Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. This study shows that as adhesion sites form between fusion cells, myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. It was demonstrated that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. Also, retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. The study proposes that microtubule bundles connecting these cell-cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion (Kato, 2016). 

    Previous studies showed that F-actin-enriched cell protrusions form in the tip of migrating tracheal branches. This study showed that tracheal FCs form polarized microtubule bundles oriented towards the leading edge of the migrating cells. The function of these microtubules is twofold: to concentrate E-cadherin to the newly contacted cell interface and to initiate the formation of new adherens junctions. The E-cadherin that accumulated at the new cell interface is not recycled from the cell surface, but is instead drawn from a newly synthesized pool and recruited preferentially to the FC contact site, and not to existing adherens junctions between FCs and stalk cells. It is speculated that the forward reorientation of the MTOC and the polarization of the microtubule plus ends towards the leading edge underlie the preferential deposition of E-cadherin at the FC contact site. One possible mechanism of preferential deposition is considered, in which microtubules transport endosomes containing E-cadherin towards the contact site, and attempts were made to test this model by imaging vesicular trafficking of the complex containing E-cadherin-GFP and other adherens junction components. No definitive evidence for this model was found. However, the Golgi apparatus was found to shift forward, towards the FC contact site, and that an E-cadherin-GFP signal increased in the plasma membrane before becoming concentrated at the contact site. Based on these observations, a model is favored in which the relocalization of the Golgi apparatus near the leading edge of the FC provides a source for E-cadherin that is deposited locally in the plasma membrane and the trans association of E-cadherin between the FCs nucleates a further concentration of E-cadherin via cis clustering. MTOC components have been shown to be located apically in stalk cells and the microtubule function is required for the apical assembly of adherens junction proteins Par-3 and E-cadherin through regulation of recycling endosomes. This mechanism appears different from FCs, as assembly of new adherens junction occurs in the cell interface enriched with microtubule plus ends opposite to the centriole located in the proximal side (Kato, 2016).

    A second microtubule function was discovered in this study, which was to equalize the contraction in FC pairs after contact. The coordinated contraction in FC pairs pulls two FC-stalk cell junctions simultaneously towards the FC contact site. The contractile force comes from a myosin-driven process; the microtubules may serve as a 'ratchet' to fix the length of the FCs after each round of contraction. When microtubules were inhibited, the FCs relaxed to their original length after contracting, which delayed the overall fusion process. When microtubules were destabilized, branch fusion proceeded, albeit with delays and imbalances, and fusion was eventually completed. Even in this condition, the conversion of the FC cells into a torus shape occurred simultaneously, suggesting that there is a mechanism to coordinate the fusion event in FC pairs. The proposed ratchet-like function of microtubule must in some way be linked to the contractile activity of myosin. One good candidate molecule for coupling the actomyosin contraction to the microtubule function is Short-stop (Shot), which belongs to the conserved spectraplakin family of cytoskeletal proteins, and was shown to be required for tracheal branch fusion. The involvement of microtubules and Shot in the ratchet-like mechanisms observed in several contraction-dependent morphogenetic events should be tested in the future (Kato, 2016).

    It is interesting to note that balancing of the force applied to the E-cadherin conjugated cell interface via microtubule plus ends is similar in configuration to the mitotic spindle, where microtubule plus ends attached to the kinetochore of each of paired sister chromosomes applies pulling force to each spindle pole. The equal number of cadherin-catenin complex in each side of the FC interface associated with trans-conjugated E-cadherin pairs in the FC interface may provide a platform for microtubule plus end attachment for generating balanced contractile force (Kato, 2016).

    When FCs are fully contracted, the plasma membrane of the two adherens junctions in each FC are connected in a single burst and cell pairs are converted simultaneously into a torus shape. This process requires ADP-ribosylation factor-like 3 (Arl3) GTPase, which associates with the microtubules and intracellular vesicles concentrated at the FC contact site. This study showed that Arl3 is required for directed Serp trafficking, and that GFP-Rab9 overexpression overrides the requirement for Arl3. It is proposed that the microtubule-dependent transport of the Golgi apparatus and endosomes facilitates the concentration of Rab9 and Arl3 at the FC contact site, where they act together to increase the concentration of Serp in the lumen. The deacetylation of chitin converts it to the more hydrophilic form chitosan. The increase in water absorption by chitosan would cause the luminal matrix gel to swell, simultaneously pushing the plasma membranes of the FC interface closer to the plasma membrane of the FC-stalk cell interface so that the membrane-fusion machinery triggers the conversion of the paired FCs into a torus shape (Kato, 2016).

    A number of issues remain to be explained. Lumen formation in FCs was clearly detected in arl3 mutants, but not in the normal context. This is probably because very small lumen is sufficient to trigger fusion of wild-type FCs. Although Arl3 is absolutely required for fusion, Rab9 and Serp are not, suggesting that the proposed luminal-matrix swelling due to chitin deacetylation is not the sole mechanism of plasma membrane fusion, and additional Arl3-regulated process of fusion control must exist. Moreover, additional Rab9 cargo that acts together with Serp to rescue the arl3 mutants is predicted. To uncover the entire fusion process, it will be necessary to search for additional Arl3 and Rab9 targets, and to analyse FC-specific membrane trafficking and secretion (Kato, 2016).

    Multigenerational effects of rearing atmospheric oxygen level on the tracheal dimensions and diffusing capacities of pupal and adult Drosophila melanogaster

    Insects are small relative to vertebrates, and were larger in the Paleozoic when atmospheric oxygen levels were higher. The safety margin for oxygen delivery does not increase in larger insects, due to an increased mass-specific investment in the tracheal system and a greater use of convection in larger insects. Prior studies have shown that the dimensions and number of tracheal system branches varies inversely with rearing oxygen in embryonic and larval insects. This study tested whether rearing in 10, 21, or 40 kPa atmospheric oxygen atmospheres for 5-7 generations affected the tracheal dimensions and diffusing capacities of pupal and adult Drosophila. Abdominal tracheae and pupal snorkel tracheae showed weak responses to oxygen, while leg tracheae showed strong, but imperfect compensatory changes. The diffusing capacity of leg tracheae appears closely matched to predicted oxygen transport needs by diffusion, perhaps explaining the consistent and significant responses of these tracheae to rearing oxygen. The reduced investment in tracheal structure in insects reared in higher oxygen levels may be important for conserving tissue oxygen partial pressure and may provide an important mechanism for insects to invest only the space and materials necessary into respiratory structure (Klok, 2016).

    Failure to burrow and tunnel reveals roles for jim lovell in the growth and endoreplication of the Drosophila larval tracheae

    The Drosophila protein Jim Lovell (Lov) is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system this study identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. These phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, lov was stablished to be normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death lov tracheal knockdown was shown to result in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. The role of lov in the tracheae that initiates this sequence of events was investigated. When lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together these findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior (Zhou, 2016).

    Centrosome amplification increases single-cell branching in post-mitotic cells

    Centrosome amplification is a hallmark of cancer, although how this process affects tumorigenesis is still not understood. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. This study analyzed the effects of centrosome amplification in post-mitotic cells during single-cell branching. It was shown that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. Mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. Rca1 and CycA post-mitotic cells have supernumerary centrosomes and other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. The study proposes that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects (Ricolo, 2016).


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    genes expressed in trachea and spiracles

    Genes involved in organ development

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