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

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

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

A matrix metalloproteinase mediates airway remodeling in Drosophila

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

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 Drosophil


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).

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).

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).

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).

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).



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genes expressed in trachea and spiracles

Genes involved in organ development

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