EGF signalling regulates cell invagination as well as cell migration during formation of tracheal system in Drosophila

 The Drosophila tracheal system is a network of epithelial tubes that arises from the tracheal placodes, lateral clusters of ectodermal cells in ten embryonic segments. The cells of each cluster invaginate and subsequent formation of the tracheal tree occurs by cell migration and fusion of tracheal branches, without cell division. The combined action of the Decapentaplegic (Dpp), Epidermal growth factor (EGF) and breathless/branchless pathways are thought to be responsible for the pattern of tracheal branches. We ask how these transduction pathways regulate cell migration and we analyse the consequences on cell behaviour of the Dpp and EGF pathways. We find that rhomboid (rho) mutant embryos display defects not only in tracheal cell migration but also in tracheal cell invagination unveiling a new role for EGF signalling in the formation of the tracheal system. These results indicate that the transduction pathways that control tracheal cell migration are active in different steps of tracheal formation, beginning at invagination. We discuss how the consecutive steps of tracheal morphogenesis might affect the final branching pattern. Received: 9 October 1998 / Accepted: 5 November 1998     

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. We show here that 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. Our results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

Control of tracheal tubulogenesis by Wingless signaling

The tubular epithelium of the Drosophila tracheal system forms a network with a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching induced by the FGF homolog Branchless, differentiates cells with specialized functions such as fusion cells, which perform target recognition and adhesion during branch fusion, and extends branches toward specific targets. Specification of a unique identity for each primary branch is essential for directed migration, as a defect in either the EGFR or the Dpp pathway leads to a loss of branch identity and the misguidance of tracheal cell migration. Here, we investigate the role of Wingless signaling in the specification of cell and branch identity in the tracheal system. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, including specifying the dorsal trunk through activation of Spalt expression and inducing differentiation of fusion cells in all fusion branches. Moreover, we show that Wingless signaling regulates Notch signaling by stimulating delta expression at the tip of primary branches. These activities of Wingless signaling together specify the shape of the dorsal trunk and other fusion branches.     

Interplay of Notch and FGF signaling restricts cell fate and MAPK activation in the Drosophila trachea.

The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless that activates a receptor tyrosine kinase Breathless and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tip of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Here we show that Notch signaling plays crucial roles in the singling out process of the fusion cell. We found that Notch is activated in tracheal cells by Branchless signaling through stimulation of &Dgr; expression at the tip of tracheal branches and that activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tip of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis.     

Cell fate choices in Drosophila tracheal morphogenesis

The Drosophila tracheal system is a branched tubular structure that supplies air to target tissues. The elaborate tracheal morphology is shaped by two linked inductive processes, one involving the choice of cell fates, and the other a guided cell migration. We will describe the molecular basis for these processes, and the allocation of cell fate decisions to four temporal hierarchies. First, tracheal placodes are specified within the embryonic ectoderm. Subsequently, branch fates are allocated within the tracheal placodes, prior to migration. Localized presentation of the FGF ligand, Branchless, to tracheal cells that express the FGF receptor, Breathless, guides migration. Once cell migration is initiated, distinct cell fates are determined within each migrating branch. Finally, inhibitory feedback mechanisms ensure the correct assignment of these fates. Tracheal cell fate choices are determined by signaling cascades triggered by signals emanating from the tracheal cells, as well as by ligands produced by adjacent tissues.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

Branch-specific migration cues in the Drosophila tracheal system

The Drosophila tracheal system forms by highly stereotyped migration of the tracheal cells, generating an elaborate network of interconnected tubes supplying oxygen to all tissues. A major guiding system in the migration process of all branches is the dynamic and localized expression of Branchless (Bnl), an FGF-like molecule. Bnl triggers the activation of the FGF receptor Breathless (Btl) locally in all tracheal cells. Is this the only guiding cue, or do additional local signals provide distinct inputs to each branch? Several recent papers identify such local signals, relying on contacts with specific cell types and with the matrix encountered by the migrating tracheal branches. In particular, the paper by Boube et al(1) demonstrates a role for PS integrins in promoting migration of a specific tracheal branch.     

The alternative migratory pathways of the Drosophila tracheal cells are associated with distinct subsets of mesodermal cells

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. Here we analyze how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. We find that cells in different branches exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. We also find that alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and propose a model for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints.     

Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF.

The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Here we show that ramification of the fine terminal branches is variable and regulated by oxygen, and that this process is controlled by a local signal or signals produced by oxygen-starved cells. We provide evidence that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression.     

The Notch pathway helps to pattern the tips of the Drosophila tracheal branches by selecting cell fates

The Drosophila tracheal system consists of a stereotyped network of epithelial tubes formed by several tracheal cell types. By the end of embryogenesis, when the general branching pattern is established, some specialised tracheal cells then mediate branch fusion while others extend fine terminal branches. Here evidence is presented that the Notch signalling pathway acts directly in the tracheal cells to distinguish individual fates within groups of equivalent cells. Notch helps to single out those tracheal cells that mediate branch fusion by blocking their neighbours from adopting the same fate. This function of Notch would require the restricted activation of the pathway in specific cells. In addition, and probably later, Notch also acts in the selection of those tracheal cells that extend the terminal branches. Both the localised expression and the mutant phenotypes of Delta, a known ligand for Notch, suggest that Delta may activate Notch to specify cell fates at the tips of the developing tracheal branches.     

A clonal genetic screen for mutants causing defects in larval tracheal morphogenesis in Drosophila

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration.     

Integrin-α5 coordinates assembly of posterior cranial placodes in zebrafish and enhances Fgf-dependent regulation of otic/epibranchial cells

Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes.     

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 are surprisingly variable. Immunohistochemical and electron microscopic studies 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 were analyzed. 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 antennocerebral 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.     

ribbon encodes a novel BTB/POZ protein required for directed cell migration in Drosophila melanogaster

During development, directed cell migration is crucial for achieving proper shape and function of organs. One well-studied example is the embryonic development of the larval tracheal system of Drosophila, in which at least four signaling pathways coordinate cell migration to form an elaborate branched network essential for oxygen delivery throughout the larva. FGF signaling is required for guided migration of all tracheal branches, whereas the DPP, EGF receptor, and Wingless/WNT signaling pathways each mediate the formation of specific subsets of branches. Here, we characterize ribbon, which encodes a BTB/POZ-containing protein required for specific tracheal branch migration. In ribbon mutant tracheae, the dorsal trunk fails to form, and ventral branches are stunted; however, directed migrations of the dorsal and visceral branches are largely unaffected. The dorsal trunk also fails to form when FGF or Wingless/WNT signaling is lost, and we show that ribbon functions downstream of, or parallel to, these pathways to promote anterior-posterior migration. Directed cell migration of the salivary gland and dorsal epidermis are also affected in ribbon mutants, suggesting that conserved mechanisms may be employed to orient cell migrations in multiple tissues during development.