Drosophila E-cadherin is essential for proper germ cell-soma interaction during gonad morphogenesis

In most animal species, germ cells require intimate contact with specialized somatic cells in the gonad for their proper development. We have analyzed the establishment of germ cell-soma interaction during embryonic gonad formation in Drosophila melanogaster, and find that somatic cells undergo dramatic changes in cell shape and individually ensheath germ cells as the gonad coalesces. Germ cell ensheathment is independent of other aspects of gonad formation, indicating that separate morphogenic processes are at work during gonadogenesis. The cell-cell adhesion molecule Drosophila E-cadherin is essential both for germ cell ensheathment and gonad compaction, and is upregulated in the somatic gonad at the time of gonad formation. Our data indicate that differential cell adhesion contributes to cell sorting and the formation of proper gonad architecture. In addition, we find that Fear of Intimacy, a novel transmembrane protein, is also required for both germ cell ensheathment and gonad compaction. E-cadherin expression in the gonad is dramatically decreased in fear of intimacy mutants, indicating that Fear of Intimacy may be a regulator of E-cadherin expression or function.     

Drosophila fear of intimacy encodes a Zrt/IRT-like protein (ZIP) family zinc transporter functionally related to mammalian ZIP proteins

Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm; however, little is known about their in vivo roles. Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila. Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI. Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes.     

Wingless and its signalling pathway have common and separable functions during tracheal development

The Drosophila tracheal tree consists of a tubular network of epithelial branches that constitutes the respiratory system. Groups of tracheal cells migrate towards stereotyped directions while they acquire specific tracheal fates. This work shows that the wingless/WNT signalling pathway is needed within the tracheal cells for the formation of the dorsal trunk and for fusion of the branches. These functions are achieved through the regulation of target genes, such as spalt in the dorsal trunk and escargot in the fusion cells. The pathway also aids tracheal invagination and helps guide the ganglionic branch. Moreover the wingless/WNT pathway displays antagonistic interactions with the DPP (decapentaplegic) pathway, which regulates branching along the dorsoventral axis. Remarkably, the wingless gene itself, acting through its canonical pathway, seems not to be absolutely required for all these tracheal functions. However, the artificial overexpression of wingless in tracheal cells mimics the overexpression of a constitutively activated armadillo protein. The results suggest that another gene product, possibly a WNT, could help to trigger the wingless cascade in the developing tracheae.     

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.     

Tracheal development in Drosophila melanogaster as a model system for studying the development of a branched organ

The development of the tracheal system of Drosophila melanogaster represents a paradigm for studying the molecular mechanisms involved in the formation of a branched tubular network. Tracheogenesis has been characterized at the morphological, cellular and genetic level and a series of successive, but linked events have been described as the basis for the formation of the complex network of tubules which extend over the entire organism. Tracheal cells stop to divide early in the process of tracheogenesis and the formation of the interconnected network requires highly controlled cell migration events and cell shape changes. A number of genes involved in these two processes have been identified but in order to obtain a more complete view of branching morphogenesis, many more genes carrying essential functions have to be isolated and characterized. Here, we provide a progress report on our attempts to identify further genes expressed in the tracheal system. We show that empty spiracles (ems), a head gap gene, is required for the formation of a specific tracheal branch, the visceral branch. We also identified a Sulfotransferase and a Multiple Inositol Polyphosphate phosphatase that are strongly upregulated in tracheal cells and discuss their possible involvement in tracheal development.     

The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein

The development of the mature insect trachea requires a complex series of cellular events, including tracheal cell specification, cell migration, tubule branching, and tubule fusion. Here we describe the identification of the Drosophila melanogaster dysfusion gene, which encodes a novel basic helix-loop-helix (bHLH)-PAS protein conserved between Caenorhabditis elegans, insects, and humans, and controls tracheal fusion events. The Dysfusion protein functions as a heterodimer with the Tango bHLH-PAS protein in vivo to form a putative DNA-binding complex. The dysfusion gene is expressed in a variety of embryonic cell types, including tracheal-fusion, leading-edge, foregut atrium cells, nervous system, hindgut, and anal pad cells. RNAi experiments indicate that dysfusion is required for dorsal branch, lateral trunk, and ganglionic branch fusion but not for fusion of the dorsal trunk. The escargot gene, which is also expressed in fusion cells and is required for tracheal fusion, precedes dysfusion expression. Analysis of escargot mutants indicates a complex pattern of dysfusion regulation, such that dysfusion expression is dependent on escargot in the dorsal and ganglionic branches but not the dorsal trunk. Early in tracheal development, the Trachealess bHLH-PAS protein is present at uniformly high levels in all tracheal cells, but since the levels of Dysfusion rise in wild-type fusion cells, the levels of Trachealess in fusion cells decline. The downregulation of Trachealess is dependent on dysfusion function. These results suggest the possibility that competitive interactions between basic helix-loop-helix-PAS proteins (Dysfusion, Trachealess, and possibly Similar) may be important for the proper development of the trachea.     

Distinct sites in E-cadherin regulate different steps in Drosophila tracheal tube fusion

We have investigated how E-cadherin controls the elaboration of adherens junction associated cytoskeletal structures crucial for assembling tubular networks. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Shot, a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to beta-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly.     

The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion

Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short Stop (shot). In these fusion cells, the apical surface determinant Discs Lost (Dlt) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led us to investigate whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. We therefore propose that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization.     

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.     

The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis

In the developing tracheal system of Drosophila melanogaster, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic (Dpp) signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, we find that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth.     

C. elegans as a model system to study the function of the COG complex in animal development

The conserved oligomeric Golgi (COG) complex is an octameric protein complex associated with the Golgi apparatus and is required for proper sorting and glycosylation of Golgi resident enzymes and secreted proteins. Although COG complex function has been extensively studied at the cellular and subcellular levels, its role in animal development mostly remains unknown. Recently, mutations in the components of the COG complex were found to cause abnormal gonad morphogenesis in Caenorhabditis elegans. In C. elegans, the COG complex acts in the glycosylation of an ADAM (a disintegrin and metalloprotease) family protein, MIG-17, which directs migration of gonadal distal tip cells to lead gonad morphogenesis. This is the first link between the COG complex and the function of an ADAM protease that is directly involved in organ morphogenesis, demonstrating the potential of C. elegans as a model system to study COG function in animal development.     

The hindsight gene is required for epithelial maintenance and differentiation of the tracheal system in Drosophila

During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues. Here we show that the Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt) mutants tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. Starting at midembryogenesis, however, the tracheal epithelium collapses or expands to give rise to sacs of tissue. While a subset of hnt mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of the epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distribution of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells. While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia is unique to hnt mutants. These data suggest that HNT is a tissue-specific factor that regulates maintenance of the tracheal epithelium as well as differentiation of taenidia.     

A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster

The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes.     

Dpp and Notch specify the fusion cell fate in the dorsal branches of the Drosophila trachea.

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. We report that Dpp signaling is also required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling is Delta. In conditional Notch loss of function mutants additional tracheal cells adopt the fusion cell fate and ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch(ts1) mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches.     

Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila

During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion.