Airway maturation in Drosophila
Katarína Tiklová
Wenner-Gren Institute Department of Developmental Biology Stockholm University 2011
Doctoral dissertation 2011 Department of Developmental Biology Wenner-Gren Institute Stockholm University Stockholm, Sweden
The cover shows a scanning electron micrograph of a Drosophila embryo merged with the embryonic trachea visualized by the luminal antigen 2A12.
Printed by US-AB, Stockholm, Sweden 2011 ISBN (978-91-7447-338-4) Katarína Tiklová, 2011
2
ʺMan cannot discover new oceans unless he has the courage to lose sight of the shore.ʺ Andre Gide
To my family
3 ABSTRACT
Tubes are a fundamental unit of organ design. Most of our major organs like the lung, kidney and vasculature are composed primarily of tubes. To identify fundamental biological principles of tubular organ formation we used the respiratory organ of Drosophila melanogaster, the trachea. This work dissects embryonic trachea maturation. Three precise epithelial transitions occur during airway maturation. A secretion burst deposits proteins into the lumen; then luminal material is cleared and finally liquid is removed. We identified the cellular mechanisms behind these transitions. Sar1 and γCOP are required for protein secretion, matrix assembly and tube expansion. Rab5-dependent endocytic activity internalizes and clears luminal contents. The data show how programmed transitions in cellular activities form functional airways, and may reflect a general mechanism in respiratory organ morphogenesis. We further focused on tube size regulation. We identified Melanotransferrin, a new component of septate junctions that limits tracheal tube elongation. MTf is a lipid- modified, iron-binding protein attached to epithelial cell membranes, similarly to its human homologue. We show that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. This provides the first genetic model for the functional dissection of MTf in epithelial morphogenesis. In the last part, we describe two genes, which are selectively involved in tube diameter expansion. Obst-A and Gasp are closely related proteins with characteristic chitin-binding domains. They are strongly expressed in the trachea at the time of lumen expansion. The single and double mutants cause a tube diameter reduction, whereas their overexpression leads to its increase. We propose that Obst-A and Gasp organize luminal matrix assembly and thereby regulate the extent of tube diameter expansion.
4 LIST OF PUBLICATIONS
I. Tsarouhas V., Senti K.A., Jayaram S.A., Tiklová K., Hemphälä J., Adler J. and Samakovlis C. Sequential pulses of apical epithelial secretion and endocytosis drive airway dmaturation in Drosophila. Dev Cell. 2007 Aug;13(2):214-25.
II. Jayaram S.A., Senti K.A., Tiklová K., Tsarouhas V., Hemphälä J. and bSamakovlis C. COPI vesicle transport is a common requirement for tube expansion in Drosophila. PLoS One. 2008 Apr 9;3(4):e1964.
III. Tiklová K., Senti K.A., Wang S., Gräslund A. and Samakovlis C. Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila. Nat Cell Biol. 2010 Nov;12(11):1071-7.
IV. Tiklová K., and Samakovlis C. Control of tube diameter expansion by secreted chitin-binding proteins. Manuscript
5 ABBREVIATIONS
AJ Adherens junction ANF Atrial Natriuretic Factor aPKC atypical protein kinase C BM basement membrane bnl branchless btl breathless cont contactin COP coatomer protein complex cora coracle crb crumbs DT dorsal trunk DE-cad Drosophila E-cadherin ECM Extracellular matrix kkv krotzkopf verkehrt knk knickkopf mmy mummy MTf melanotransferrin nrg neuroglian nrx IV neurexin rtv retroactive serp serpentine SJ Septate junction verm vermiform yrt yurt
6 CONTENTS
INTRODUCTION 9 The Drosophila respiratory organ as a model system for epithelial tube morphogenesis 9 Tracheal morphology and development 10 Trachea branching 10 Tube architecture 14 Lumen formation 16 Cell organization 17 a) Apical domain 18 b) AJ complex 19 c) SJ complex 21 d) Basal membrane 24 e) Apical extracellular matrix and tracheal lumen 24 Protein secretion 25 Vesicle transport 27 Control of tracheal tube size 30 AIMS 33 RESULTS AND DISCUSSION 34 Paper I: Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila 34 1) Sar-1 mediated luminal deposition of secreted proteins drives tube expansion 34 2) A Rab-5 dependent endocytic wave clears the lumen of solid material 35 3) The transition from liquid- to gas-filled trachea 36 PaperII: COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila 37 Tube diameter expansion is dependent on COPI vesicles 37 COPI dependent secretion drives the tube diameter expansion 38 Defects in the secretion apparatus underly the tube size defects 39
7 PaperIII: Epithelial Septate Junction Assembly Relies on Melanotransferrin Iron Binding and Endocytosis in Drosophila 40 Melanotransferrin is a new septate junction component 40 Endocytosis is required for functional septate junction formation 41 MTf iron binding is crucial for septate junction assembly 41 Uptake of MTf induces septate junction assembly 42 PaperIV: Control of Tube Diameter Expansion by Secreted Chitin- binding Proteins 43 The Obstructor multigene family 43 Body size, cuticle and tracheal luminal matrix are disrupted in obst-A and gasp mutants 43 Obst-A and Gasp regulate the tracheal tube diameter 44 CONCLUSIONS AND PERSPECTIVES 45 ACKNOWLEDGEMENTS 48 REFERENCES 50
8 INTRODUCTION Tubular organization is a common feature of many tissues. Tubes can also represent a transient phase of organ development. The vertebrate neural tube initially forms as a simple columnar epithelium, but ultimately gives rise to the complex architecture of the brain and spinal cord. Tubular organization is quite useful and can serve many important physiological roles, including: control and delivery of gases, nutrients, waste and hormones. Many respiratory, circulatory, and secretory organs are built of networks of interconnected tubes. Tubular epithelial organs arise from each of the germ layers, ectoderm (e.g. mammary gland), mesoderm (e.g. kidney), and endoderm (e.g. lung). Many of the same cellular and molecular mechanisms are utilized during the formation, elongation and elaboration of endothelial tubes (Ellertsdottir, Lenard et al. 2010). Studies of tube formation in complex vertebrate systems are difficult due to the large number of cells, specialisations along the tubes and variation in size along the tube (Buechner 2002). In order to identify fundamental biological principles for regulation of tube formation it is therefore advantageous to use simpler model systems, which are genetically favourable, such as the respiratory organ of the fruit fly, Drosophila melanogaster.
The Drosophila respiratory organ as a model system for epithelial tube morphogenesis Several features of the tracheal system make it an excellent system for investigating the mechanisms of tubulogenesis and tube size control. First, the tubes are simple in structure. They are an epithelial monolayer and hence lack complexities introduced by the multilayer structure of most vertebrate tubes including blood vessels and the bronchial tubes of the lungs. Second, each tube is composed of only a small number of cells, each of which can be followed during development (Samakovlis, Hacohen et al. 1996). Finally, powerful genetic and genomic approaches can be applied to identify and analyze tubulogenesis genes. Because fundamental mechanisms controlling epithelial tube outgrowth and branching are similar in Drosophila and vertebrates (Metzger and Krasnow 1999), it seems likely that other aspects of the process, including tube formation and size regulation will also be conserved.
9 Tracheal morphology and development The respiratory organ of the fruit fly is called the trachea. The trachea is an air filled network of tubes that spans the entire organism. Air enters the network through specialized openings, called spiracles, and becomes distributed to all branches of the network, ending in narrow capillary-like, blind-ended tubes, which facilitate gas exchange with target tissues. To optimize the air supply, the tracheal branches must extend into all tissues and the tubes have to reach specific dimensions. This is achieved by a two-step process: first, a stereotyped larval tracheal network forms during embryogenesis, later, during larval growth, the network expands by the enlargement of the pre-existing branches and ramification of the blind-ended capillaries that infiltrate the new targets (Manning 1993). The tracheal network is highly stereotyped, both in branch pattern and tube size. In contrast to more complex vertebrate systems, development of tracheal tubes does not involve cell proliferation or cell death, but relies on cell migration and changes in cell shape and cellular junctions (Samakovlis, Hacohen et al. 1996; Beitel and Krasnow 2000). Thus, the Drosophila trachea is well suited for elucidating the molecular and genetic mechanisms that underlie epithelial tube morphogenesis.
Trachea branching Trachea originates from 20 independent units with similar branching pattern, 10 of each side of the embryo. Each tracheal metameric unit (named Tr1-Tr10) is initially an epithelial pocket formed by invagination of epidermal cells during stage 10, and contains approximately 80 cells. Tracheal precursors are specified in the ectoderm by the combined action of two transcription factors, Trachealess (Trh) and Ventral veinless (Vvl) (de Celis, Llimargas et al. 1995; Boube, Llimargas et al. 2000). Trachealess expression identifies the clusters of epidermal tracheal precursor cells and binds to its ubiquitous co-factor Tango (Tgo) (Ohshiro and Saigo 1997). In the next 12 hours and without further cell division the tracheal cells form invaginating pockets and undergo a synchronized program of branch outgrowth and branch fusion to generate a continuous tubular system (Samakovlis, Hacohen et al. 1996). During the branching process cells migrate and change theirs shape and relative positions. Tracheal morphogenesis can be divided into the following phases: invagination, primary branch formation, secondary branch formation, terminal branch formation and branch fusion.
10 During the invagination the epithelial cells of the ectoderm form ingrowing pits towards the underlying mesoderm. After invagination the tracheal pits close, but remain connected to the epidermis through the cells of the spiracular branch. Each pit forms six buds, which will give rise to the primary branches. The six primary branches are termed dorsal trunk anterior (DTa), dorsal trunk posterior (DTp), dorsal branch (DB), visceral branch (VB), lateral trunk anterior (LTa) and lateral trunk posterior/ganglionic branch (LTp/GB) (Fig. 1). This commitment of cells to different branches is determined by the spatial expression of Decapentaplegic (Vincent, Ruberte et al. 1997), Rhomboid (Wappner, Gabay et al. 1997), Wingless (Wappner, Gabay et al. 1997; Chihara and Hayashi 2000; Llimargas 2000) and Hedgehog (Glazer and Shilo 2001) in the early embryo. All primary branches start growing as multicellular tubes. Outgrowth is initiated by the extension of broad cellular protrusions from the tip cells in each bud. DB, GB and VB continue to extend, developing into narrow primary branches. The conversion in the structure of the tubes involves changes in cell contacts and shapes. As primary branches navigate towards the target, a distinct number of cells, typically located at the tip of each primary branch, sprout off and form smaller unicellular branches (Samakovlis, Hacohen et al. 1996). Some of them grow towards target tissues and form terminal branches. The key inducer of tracheal primary branch growth is a member of the secreted fibroblast growth factor (FGF) family, Branchless (Bnl) (Sutherland, Samakovlis et al. 1996). Bnl is temporarily expressed in cell patches around each tracheal sack and as a chemoattractant promotes branch migration towards its source. Bnl activates the tracheal receptor tyrosine kinase Breathless (Btl; a Drosophila FGF receptor) (Klambt, Glazer et al. 1992), and the intracellular protein Downstream-of-FGF- receptor (Dof, Stumps) is specifically required for FGF signal transduction in Drosophila (Vincent, Ruberte et al. 1997; Imam, Sutherland et al. 1999). In mutants for bnl, btl or dof, only rudimentary primary branches form, whereas ectopic expression of Bnl in wild- type embryos can redirect primary branch growth towards its new site of expression. The cells that actively migrate are called the tip cells. Tip cells migrate and pull trailing stalk cells along behind. Cells must communicate with each other to sort into leading tip cells and trailing stalk cells, and this com- munication takes the form of competition. Tip cells are postulated to generate a lateral inhibitory cue proportional to the level of FGFR activity, and thus enforce the
11 follower stalk cell fate upon their neighbors. Notch is required cell autonomously in the stalk cells to restrict tip cell number (Ghabrial and Krasnow 2006). In addition, the Rho-family GTPases, which are instrumental in the regulation of the cytoskeleton during cell migration, have been implicated in primary branching; overexpression of constitutively active Cdc42 in the trachea induces filopodial extensions, whereas expression of a dominant negative form of Cdc42 impedes branch extension (Wolf, Gerlach et al. 2002). The subsequent movement of the cell body and growth of apical cell surface during primary branching appear to require transcriptional gene regulation involving Ribbon (Rib) and Grainyhead (Grh). Rib is a putative transcription factor expressed in all tracheal cells and, although basal filopodia towards the Bnl source still form in rib mutants, cell-body movement and lumen growth are aborted (Bradley and Andrew 2001; Shim, Blake et al. 2001). grh mutants on the other hand, complete branching morphogenesis but show overelongated and tortuous tubes. This defect is accompanied by a selective expansion of the apical membrane of the tracheal cells. As primary branching proceeds, the individual branches grow towards, along and across different cell types, and some, including the DB GB and VB, develop into narrow extended type-II branches. Secondary branching initiates at stage 14, several hours after initiation of primary budding. In each metamere, 23 of the 80 cells of the typical tracheal metamere form unicellular sprouts. During secondary branching, Branchless/Breathless signalling is again used, but in a different manner. In this cases, it determines the acquisition of specific cell fates among the cells at the tip of each primary branch by inducing the expression of pantip markers such as the transcription factor Pointed (Pnt) at stage 13 (Scholz, Deatrick et al. 1993). The Pnt protein plays an essential role in the selection and identity of cells that undergo sprouting. It promotes the next stage of branching by activation of transcription of Drosophila serum response factor (DSRF) and inhibiting the expression of the fusion gene Escargot, in terminal cells (Samakovlis, Manning et al. 1996). Fusion cells expressing transcription factor Escargot (Esg) form the anastomoses that interconnect the hemisegments to the network (tube type III) (Samakovlis, Manning et al. 1996). Terminal cells expressing DSRF continue their journey towards target organs, which they penetrate by forming capillary tubes (tube type IV) (Guillemin, Groppe et al. 1996). Ramification of the terminal branches
12 during larval life is highly variable and regulated by the oxygen-needs of the tissue. Oxygen-deprived cells upregulate the expression of branchless and thereby induce branch migration, similar to VEGF-induced (Vascular endothelial growth factor) chemotaxis during angiogenesis in vertebrates (Shweiki, Itin et al. 1992; Jarecki, Johnson et al. 1999). To form the functional tracheal network, branch fusion events have to interconnect the twenty independent metameric units into a continuous network. During stage 13 the DTa and DTp from neighboring ipsilateral metamers meet and form continuous dorsal trunk. Fusion cells form doughnut-shaped connections that lack autocellular junctions. The fusion event begins as the fusion cells extend broad cytoplasmic processes towards each other. Fusion cells recognize each other and establish an initial contact point. They then move closer to each other broadening their contact surface, and simultaneously forming a new lumen continuous with the primary branch lumen (Samakovlis, Manning et al. 1996). The earliest fusion cell-specific protein identified is the transcription factor Escargot. It is expressed in all five-fusion cells of the tracheal metamere and is required for three of five fusion events to occur (DB, LTa, LTp). During the fusion there is deposition of DE-cadherin (DE-cad) at the contact point between the two fusion partners (Tanaka-Matakatsu, Uemura et al. 1996). DE-cad is upregulated in fusion cells by Esg.
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Figure 1. The Drosophila tracheal system (a) Lateral view of a Drosophila embryo at stage 16 with the anterior part to the left. The tracheal lumen is visualised by the luminal marker 2A12. (b) Each tracheal branch is made up of six primary branches: the dorsal trunk (DT), dorsal branch (DB), visceral branch (VB), spiracular branch (SB), lateral trunk (L T) and ganglionic branch (GB).
Tube architecture The fully developed embryonic trachea consists of a continuous single-layered epithelium of around 1600 cells. The apical cell surface is facing the lumen and the basal surface facing the internal tissues. The luminal surface is lined by a cuticle and function as barrier against dehydration and invading microorganisms. We can distinguish four different tubular types, each of them associated with a distinct cellular architecture, diameter and length (Samakovlis, Hacohen et al. 1996; Uv, Cantera et al. 2003) (Fig. 2). Type I tubes are composed of flattened, wedge- shaped cells connected by intercellular cell junctions (dorsal trunk). The type II tube is also multicellular, but narrower and is represented by the dorsal, visceral and ganglionic branches. Each tracheal cell is placed in a “head to tail” arrangement along the length of these branches. A characteristic feature of these tubes are autocellular
14 junctions that run along the proximal-distal axis of the cells. Type III tubes are represented by the bicellular fusion-branches (anastomoses) that connect the individual metameres to a network. These tubes consist of a pair of doughnut-shaped cells, connected by intercellular junctions. Those tubes are seamless, without intracellular junctions. Type IV tubes are represented by the blind-ended tracheoles. These branches are extended, hollow protrusions emanating from a single terminal cell, and become increasingly narrower along their length. For comparison to other tubular organs, the first tube type is found in lungs, blood vessels and many glandular organs, whereas seamless tubes and tubes with autocellular junctions have been found in the vertebrate vasculature(Wolff and Bar 1972) and the Caenorhabditis elegans excretory cell (Buechner 2002).
Tube type I: multicellular Tube type II: multicellular intercellular junctions intracellular junctions
Tube type III: bicellular Tube type IV: unicellular no junctions no junctions
Figure 2. Tracheal tubular types In type I branches wedge-shaped cells connected by intercellular junctions surround the lumen circumference. Type II branches are formed by cells wrapped around the lumen and sealed with autocellular junctions. The cells are connected to the neighbours by intracellular junctions. Type III branches consists of two doughnut- shaped cells, connected by intercellular junctions. Type IV branches are single cell tubes without junctions. Adapted from (Uv, Cantera et al. 2003).
15 Lumen formation Lumen can form by different mechanism, including the wrapping, budding, cavitation, cord hollowing or cell hollowing (Lubarsky and Krasnow 2003). Wrapping occurs when a portion of an epithelial sheet invaginates and curls until the edges of the invaginating region meet and seal, as during neural tube formation in many vertebrates (Colas and Schoenwolf 2001). During budding, a group of cells in an existing epithelial tube (or sheet) migrates out and forms a new tube as the bud extends. The new tube is a direct extension of the original tube. This is how tubes arise during branching morphogenesis of many organs, including the mammalian lung and the major branches of the Drosophila tracheal (respiratory) system (Metzger and Krasnow 1999; Hogan and Kolodziej 2002). In both of these mechanisms, tubes arise from a polarized epithelium. In the other three mechanisms described below, tubes arise from cells that are not epithelial, and the cells polarize as the tubes form. During cavitation, cells organized in a mass create a central cavity by eliminating cells in the center of the mass, as occurs during salivary gland morphogenesis (Borghese 1950; Melnick and Jaskoll 2000). In cord hollowing, a lumen is created de novo between cells in a thin cylindrical cord, without cell loss. Examples include the Caenorhabditis elegans gut (Leung, Hermann et al. 1999), the Drosophila heart and Madin-Darby canine kidney (MDCK) cells (Pollack, Runyan et al. 1998). In cell hollowing, a lumen forms within the cytoplasm of a single cell, spanning the length of the cell. For instance, some capillary endothelial cells (Wolff and Bar 1972). Tubes are generally very small when they form and must grow to achieve their mature size and shape. The expansion process can begin as soon as the lumen forms or can occur at specific times later in development. The tracheal tubes of the tracheal branches are all narrow and of similar diameter when they form. Several hours later, they undergo an expansion that can triple the diameter of some of the branches. Just before expansion begins, multiple vesicle-like structures carrying an apical membrane antigen appear in the cytoplasm. These are presumably membrane vesicles, like those of MDCK and endothelial cells, which target and fuse to the apical surface, driving the apical membrane expansion that occur during lumen growth. As the apical surface expands, the basal surface changes
16 little if at all, demonstrating that lumen expansion is not a process of generalized cell growth but rather a specific growth and remodeling of the apical surface. Two additional expansions later in development increase lumen diameter up to 40 times its original size. Expansion is not accompanied by cell proliferation, only by a large increase in apical surface area and dramatic thinning of the cells. Although apical membrane biogenesis is required for both lumen formation and expansion, it is not sufficient. There must also be a mechanism that drives apical membranes apart to keep the lumen open. Liquid secretion into the lumen may play such a role. Although tracheal tubes function in oxygen transport, liquid accumulates in the lumen when the tubes first form and during each expansion, only to be cleared after each expansion cycle so that oxygen can flow (Manning and Krasnow, 1993).
Cell organization The tracheal epithelium derives from the blastoderm epithelium, which forms by a process known as cellularization. During cellularization the common plasma membrane that surrounds the syncytial embryo becomes subdivided into domains. Subdivided regions or domains fulfill specialized roles in cell organization and physiology. The main subdivisions of the plasma membrane are the apical, lateral and basal domain (Tepass, Tanentzapf et al. 2001). In a tubular organ the apical domain faces the external environment of the lumen, the lateral domain contacts neighboring cells within the epithelial layer, and the basal domain is anchored to the basement membrane. Cohesion between epithelial cells relies pre- dominantly on the zonula adherens, a belt-like adherens junction (AJ) located at the interface between the apical and lateral membrane (Meng and Takeichi 2009). Other junctional complexes limit paracellular diffusion to support the barrier function and the selective permeability of epithelia. In most invertebrates, this function is mediated by the septate junction (SJ), which is most often located basal to the AJ (Tepass, Tanentzapf et al. 2001) (Fig. 3). In chordates, the intercellular space is sealed by tight junctions, which are apical to the zonula adherens (Tsukita, Furuse et al. 2001).
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Figure 3. Epithelial cell structure A schematic drawing of an epithelial cell with intercellular junctions. Right brackets indicate the positions of cuticle, MZ (marginal zone), AJ (adherens junctions), SJ (septate junctions) and basement membrane. Cuticle is composed of epicuticle and procuticle. Adapted from (Tepass, Tanentzapf et al. 2001).
a) Apical domain The apical domain contains several polarity regulators, which form two complexes: Crumbs and Par complex.
The Crumbs complex is build around the transmembrane protein Crumbs (Crb), which binds to Stardust (Sdt; mammalian PALS1), Patj, Lin7, the FERM proteins Moesin and Yurt (Yrt; EBP41L5 in mammals) and βHeavy-Spectrin (Bulgakova and Knust 2009; Tepass 2009). Both Crb and Sdt are essential for the maintenance of epithelial-cell polarity in the embryo (Tepass, Theres et al. 1990; Tepass and Knust 1993; Bachmann, Schneider et al. 2001), whereas other factors either have a more restricted function in epithelial differentiation (e.g. Yrt, Lin7) or their function within the Crb complex needs to be further clarified (e.g. Moesin, Patj, bHeavy-Spectrin) (Laprise and Tepass 2011).
18 The Crumbs complex is concentrated in a distinct region of the apical plasma membrane adjacent to the ZA; this region is called the subapical or marginal region in epithelial cells (Tepass 1996). There are four main components of Par complex: atypical protein kinase C (aPKC), adaptor protein Par6, the small GTPase Cdc42, and the scaffolding protein Bazooka (Baz; the Drosophila homolog of the mammalian protein Par3) (Suzuki and Ohno 2006; Goldstein and Macara 2007). These proteins usually function together. However, Baz can also act independently of aPKC, Par6 and Cdc42 in epithelial polarity. The Par complex is a major determinant of apical-basal polarization and in epithelial cells it is required for apical localization of Crb (Bilder, Schober et al. 2003). There are multiple interactions between members of the Crumbs complex and the Par complex. The PDZ domain of the scaffolding protein Par-6 can bind to Crb (Kempkens, Medina et al. 2006), to the evolutionarily conserved regions of Sdt (Wang, Hurd et al. 2004), and to the PDZ domain of PATJ (Nam and Choi 2003). Both of those complexes are needed both for the establishment of the apical-basal polarity and for formation of AJs (Muller and Wieschaus 1996). b) AJ complex Adherens junctions (AJs) are cell–cell adhesion complexes that provide a defining feature of epithelial tissues and make important contributions to homeostasis (Gumbiner 2005; Halbleib and Nelson 2006). Cadherin adhesion molecules are core AJ components (Takeichi 1991). Most classic cadherins operate as homophilic adhesion receptors to facilitate cell–cell recognition and adhesion. All cadherins contain two or more extracellular cadherin domains. Classic cadherins additionally have a highly conserved cytoplasmic tail that interacts with a defined set of cytoplasmic proteins — the catenins. p120 catenin and β-catenin (called Armadillo in Drosophila melanogaster) bind the cytoplasmic tail of cadherins, and β-catenin binds to α-catenin to form the cadherin– catenin complex (Pokutta and Weis 2007; Nishimura and Takeichi 2009) . A second protein complex, the nectin–afadin complex, can also be an integral part of AJs. Cadherin clustering could be promoted by lateral interactions between cadherin extracellular regions or through cytoplasmic interactions that link cadherins into a supramolecular complex (Troyanovsky 2005; Kovacs and Yap 2008). The catenins
19 make key links from cadherins to actin and microtubules. The relationship is mutual in both cases, actin and microtubules support AJ formation and stability, and AJs can organize actin and microtubules. β-catenin (Armadillo). The cytoplasmic tail of classic cadherins becomes structured only when bound to β-catenin (Huber, Stewart et al. 2001), an association that begins in the endoplasmic reticulum and is required for the effective surface transport of cadherin through the biosynthetic pathway (Chen, Stewart et al. 1999; Lock and Stow 2005). Once at the plasma membrane, the cadherin–β-catenin complex rapidly recruits α-catenin (Bajpai, Correia et al. 2008). p120 catenin. p120 catenin is a positive regulator of cadherin function. The cadherin– p120 catenin interaction counteracts cadherin endocytosis and degradation, and thus enhances the surface abundance of cadherin (Ireton, Davis et al. 2002; Davis, Ireton et al. 2003; Ishiyama, Lee et al. 2010). p120 catenin also links cadherin to microtubules. α-catenin. α-catenin is essential for AJ formation and function and operates at the interface of the cadherin– catenin complex and the actin cytoskeleton (Hirano, Kimoto et al. 1992; Kobielak and Fuchs 2004). How α-catenin acts at AJs is currently not well understood. α-catenin binds to β-catenin and can, at least in vitro, directly bind actin filaments (Rimm, Koslov et al. 1995). There is a general model for the interplay between actin and cadherin–catenin clusters during AJ assembly. First, actin produces cell protrusions that initiate cadherin clustering. Second, actin polymerization and reorganization are coupled to cell contact growth and the aggregation of cadherin–catenin clusters into AJs. Third, multiple actin networks associate with cadherin– catenin clusters at mature AJs to hold them in place.
AJs also associate with microtubules (Stehbens, Akhmanova et al. 2009). In Drosophila, microtubules have a central role in AJ assembly. During cellularization, cells are forced into contact by the invagination of lateral membranes from the embryo surface, forming the first embryonic epithelium (Harris, Sawyer et al. 2009). Cadherin–catenin clusters first form between apical microvilli and then shift to the apicolateral domain to form spot AJs (Tepass and Hartenstein 1994; McGill, McKinley et al. 2009). Bazooka plays a role in relocalization of the cadherin–catenin clusters to the apicolateral domain (Muller and Wieschaus 1996; McGill, McKinley et al. 2009).
20 In addition to forming AJs, cells must disassemble and remodel AJs during tissue morphogenesis. Endocytosis is a major mechanism of AJ remodeling (Delva and Kowalczyk 2009; Wirtz-Peitz and Zallen 2009).
Figure 4. Organization of Adherens junction and its cytoskeletal relationship The main cytoplasmic binding partners of cadherins are p120 catenin, β-catenin and α-catenin. The main cytoplasmic binding partner of nectin is afadin. The relationship of these proteins to the cytoskeleton is shown. Adapted from (Harris and Tepass 2010).
c) SJ complex A second type of insect epithelial junctions is localized on the lateral membrane and is called septate junctions (SJ). SJs first appear midway through embryogenesis, after cellularization is completed, epithelial polarity has been established, and the ZA has formed (Tepass, Tanentzapf et al. 2001). The SJ lies just basal to the ZA in epithelial cells, and within the SJ the membranes of adjacent cells maintain a constant distance of approximately 15 nm. In the pleated SJ (found in ectodermally derived epithelia and the glia sheets), regular arrays of electron-dense septae span the intermembranal space. The septae form circumferential spirals around the cell much like the threads of a screw, and increase the distance that molecules must travel to pass between the apical and basolateral compartments of the epithelial sheet This regular alignment pattern of both the membranes and the septae gives the junction a ladder-like appearance (Tepass and Hartenstein 1994). Smooth SJs, which lack these ladder-like structures, are found only in the midgut and its derivatives. Apart from their role in transepithelial diffusion barrier formation (Lamb, Ward et al. 1998), SJs have been proposed to play a role in cell adhesion (Tepass, Tanentzapf et al. 2001), in the formation of the blood–brain barrier (BBB) (Schwabe, Bainton et al.
21 2005), control of tracheal tube size (Wu and Beitel 2004) and apical secretion of chitin deacetylases (Wang, Jayaram et al. 2006). SJs may play also a role in the maintenance of cell polarity, because loss of the Dlg component in the imaginal cells results in the loss of polarity (Woods and Bryant 1991). However SJs are not required for the initial polarization of the epithelial cells in the embryos as evidenced by the normal epithelial polarization in embryos that lack SJ genes, such as mega (Behr, Riedel et al. 2003) and nrv2 (Paul, Ternet et al. 2003). Insect SJs have a function analogous to vertebrate tight junctions. TJs also form a diffusion barrier that prevents water and solute exchange across epithelia (Tepass, Tanentzapf et al. 2001; Tsukita, Furuse et al. 2001). Tight junctions are located apical of the adherens junctions, whereas SJs are basal. Despite morphological and molecular differences between SJs and TJs (Anderson 2001; Tepass, Tanentzapf et al. 2001), the identifications of two claudin-like Drosophila SJ proteins, Sinuous (Sinu) and Megatrachea (Mega), suggests that the barrier functions of septate and tight junctions could have a common biochemical basis and evolutionary origin (Behr, Riedel et al. 2003; Wu and Beitel 2004). Identified SJ components are summarized in Table 1. Among these is the Lethal giant larvae/Discs large/Scribble (Lgl/Dlg/Scrib) complex (Tepass, Tanentzapf et al. 2001), which has an early role in establishing apical/basal polarity. The SJ components have been recently subdivided to the core complex formed by at least 8 members: ATPα, Cora, Mega, Nrg, Nrv2, Nrx IV, Sinu and Vari. The SJ proteins are initially highly mobile on the lateral membrane at stage 12, but in stage 13 they form the stable core complex (Oshima and Fehon 2011). Loss of SJ components dramatically affects the mobility of SJ core complex (Laval, Bel et al. 2008; Oshima and Fehon 2011). Lys-6 family proteins (Boudin, Crooked, Coiled, Crimpled), which are essential for SJ formation and maintenance but do not localize to the SJ, have also a function in the formation of the SJ core complex. Loss of them increases the mobility. The endocytosis and the apical-basal polarity Lgl-group proteins do not affect the mobility of the SJ core components. This divides the SJ assembly to two processes: the formation of SJ core complex and its localization to the apico-lateral membrane. Endocytosis and Lgl group proteins play function in the localization part (Oshima and Fehon 2011).
22
Gene product Protein domains Function References
Drosophila Vertebrate Drosophila Vertebrate Na+/K+ ATPase Na+/K+ ATPase P-type ATPase Ion pump, SJ formation Ion transport, TJ formation (Genova and Fehon 2003; Rajasekaran, Hu et al. 2003) Boudin Not known TFP-domain, 10 cysteins SJ formation Not known (Hijazi, Masson et al. 2009) Coiled Not known TFP-domain, 12 cysteins SJ formation Not known (Nilton, Oshima et al. 2010) Contactin Contactin Ig, FNIII Cell-adhesion, SJ Paranodal TJ formation (Boyle, Berglund et al. 2001; Faivre- formation Sarrailh, Banerjee et al. 2004) Coracle Band 4.1 FERM, Actin binding SJ formation Hereditary elliptocytosis (Fehon, Dawson et al. 1994; Shi, Afzal et al. 1999; Ohara, Yamakawa et al. 2000) Crimpled Not known TFP-domain, 10 cysteins SJ formation Not known (Nilton, Oshima et al. 2010) Crooked Not known TFP-domain, 10 cysteins SJ formation Not known (Nilton, Oshima et al. 2010) Disc Large hDlg PDZ, SH3, GUK SJ formation, tumor Tumor suppressor, linker (Woods and Bryant 1991; Cavatorta, supressor Fumero et al. 2004) Fasciclin II NCAMs Ig, FNIII SJ formation Cell adhesion (Hemphala, Uv et al. 2003; Tonning, Hemphala et al. 2005) Fasciclin III Not known Ig SJ formation Not known (Snow, Bieber et al. 1989) Gliotactin Neuroligin 3 Cholinesterase-like SJ formation Cell-cell-interactions (Auld, Fetter et al. 1995; Philibert, Winfield et al. 2000) Lachesin Not known Ig SJ formation Not known (Llimargas, Strigini et al. 2004) Lethal giant larva hLgls WD40 Tumor supressor Tumor suppressor, linker (Strand, Raska et al. 1994; Grifoni, Garoia et al. 2004) Megatrachea Claudins PDZ SJ formation TJ formation (Behr, Riedel et al. 2003; Miyamoto, Morita et al. 2005) Melanotransferrin Melanotransferrin iron binding SJ formation (Tiklova, Senti et al. 2010) Neurexin IV Caspr/Paranodin EGF, Discoidin, LamininG SJ formation Paranodal SJ formation (Baumgartner, Littleton et al. 1996; Bhat, Rios et al. 2001) Neuroglian NF155 Ig, FNIII SJ formation Not known (Charles, Tait et al. 2002; Genova and Fehon 2003; Faivre-Sarrailh, Banerjee et al. 2004) Scribble hScribble PDZ, LRR SJ formation, tumor Tumor suppressor, linker (Lamb, Ward et al. 1998; Bilder and Perrimon 2000; Dow, Brumby et al. supressor 2003) Sinuous Claudins PDZ SJ formation TJ formation (Wu, Schulte et al. 2004; Miyamoto, Morita et al. 2005) Varicose PALS2 HOOK, L27, PDZ, GUK, SJ formation Not known (Wu, Yu et al. 2007) SH3
Table 1. List of Septate junction related molecule
23 d) Basal membrane Most information about the basal membrane of epithelial cells and ECM, which is in contact with basal membrane, comes from oocyte follicle cells and cell cultures studies. Interactions between the epithelial cells and BM are mediated by a variety of cell surface receptors including integrins and Dystroglycan (Yurchenco, Amenta et al. 2004). The basement membrane (BM) is a highly specialized form of extracellular matrix found on the basal side of polarized animal epithelia (Quondamatteo 2002). The BM is composed primarily of the secreted glycoproteins Laminin, Collagen IV, and Nidogen and the heparan sulfate proteoglycan Perlecan. Evidence from genetic analyses in model organisms and experiments in cultured cells has indicated an important role for the BM in generating epithelial cell polarity (O'Brien, Zegers et al. 2002; Li, Edgar et al. 2003). The accumulation of BM on the basal side of the epithelium is crucial to convey the positional information to maintain a polarized architecture. e) Apical extracellular matrix and tracheal lumen The apical surface of the tracheal cells is covered by the cuticle, which is deposited during late embryogenesis. The cuticle is an ECM produced by the epidermis at its apical site and is characterized by a layered organization. The first step in cuticle deposition is the formation of an outer impermeable envelope. The envelope protects the tissue from dehydration. Subsequently, the middle protein- rich epicuticle responsible for cuticle stiffness is formed and, finally, an innermost procuticle loaded with lamellar linear chitin is assembled to confer stability and elasticity to the cuticle (Locke 2001). Tracheal cuticle forms prominent ridges that are called taenidial folds and are projected inside the lumen. They form annular rings or run a helical course around the tracheal lumen. The taenidial pattern can differ between branches and even along the length of the branch. Taenidia are believed to allow the tubes to be flexible while simultaneously providing strength against collapse. Taenidial spacing expands as the tube elongate (Glasheen, Robbins et al. 2010). Next to the taenidial chitin, there is also a transient chitin accumulation inside the growing tracheal tubes. Already at the stage of tube expansion the tracheal lumen
24 contains chitin fibers running along the length of the tubes. The uniform growth of this tracheal luminal filament has been shown to assist the coordinated tracheal tube dilation (Tonning, Hemphala et al. 2005). At the end of embryogenesis chitin fibers start loosing their continuity (Moussian, Seifarth et al. 2006) and the luminal contents will be endocytosed (Tsarouhas, Senti et al. 2007). At stage 17 the lumen of the trachea is completely cleared and the trachea will fill with air. Chitin is synthesized by chitin synthase, which is called Krotzkopf verkehrt (kkv) in flies. Kkv is a transmembrane enzyme that links cytosolic UDP-N-actylglucosamine (UDP-N-GlcNAc) into long GlcNAc polymers (chitin) and extrudes them across the plasma membrane (Tonning, Hemphala et al. 2005). After chitin is synthesized it has to be arrange to stereotypic structure. An essential chitin-organizing factor is Knickkopf (Knk) that is inserted via a GPI-anchor in the apical plasma membrane. The proposed function of Knk is the release of chitin fibers from the chitin synthase complex (Moussian, Tang et al. 2006). Another membrane-inserted protein needed for correct chitin organization is Retroactive (Rtv). Rtv may bind chitin and assist orienting chitin microfibrils at the surface of chitin producing cell (Moussian, Soding et al. 2005). Chitin is also modified by chitin deacetylases Serpentine (Serp) and Vermiform (Verm), which are secreted molecules (Luschnig, Batz et al. 2006; Wang, Jayaram et al. 2006). Presumably the modification of chitin including deacetylation is a prerequisite for its abilities to interact with luminal proteins and to form filaments, as the phenotypes of serp and verm double mutant and knk or rtv mutants are similar.
Protein secretion The analysis of many mutants in chitin biosythesis indicates that the complex intraluminal matrix acts as a mold for tube growth. How are all those matrix components delivered to the right place to form the functional trachea? The synthesis of all proteins begins on ribosomes in the cytosol, except for the few that are synthesized on the ribosomes of mitochondria. Their subsequent fate depends on their amino acid sequence, which can contain sorting signals that direct their delivery to specific locations. Transmembrane and secreted proteins have signal sequences mostly at the N-terminus. When the ER signal sequence is synthesized, it directs the ribosome to the ER. The ER membrane contains translocators, which form
25 pores in the membrane through which the poplypeptide is translocated into the ER lumen. As they are synthesized, these proteins translocate into the ER lumen, where they are glycosylated and acquire their three dimensional folding. Misfolded proteins are identified and retrotranslocated to the cytosol, where they become degraded by the proteasome. The vesicles containing the properly folded proteins then enter the Golgi apparatus. In the Golgi apparatus, the glycosylation of the proteins is modified and further posttranslational modifications may occur. Transport between the ER and Golgi compartments is facilitated by specialized vesicles. These vesicles are surrounded by distinctive coating proteins called coatomer protein complex-I (COPI) and COPII. COPII mediates anterograde transport from the ER to either the ER-Golgi intermediate compartment (ERGIC) or the Golgi complex (Barlowe, Orci et al. 1994), while COPI, is involved in intra-Golgi transport and retrograde transport from the Golgi to the ER (Letourneur, Gaynor et al. 1994) (Fig. 5). The core COPII components are the small Ras-like GTPase Sar1, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex (Barlowe, Orci et al. 1994). The COPII coat assembles by the stepwise deposition of Sar1GTP, Sec23/Sec24, and
Sec13/Sec31 onto sites, where newly synthesized proteins exit from the ER. Sar1GTP together with Sec23/Sec24 forms pre-budding complex (Lederkremer, Cheng et al. 2001). Sec23 makes direct contact with Sar1GTP (Bi, Corpina et al. 2002), while
Sec24 participates in cargo recognition. Sec23 stimulates the GTP hydrolysis activity of Sar1 (Yoshihisa, Barlowe et al. 1993). This activity of Sec23 as a GTPase- activating protein (GAP) is increased approximately ten-fold by addition of
Sec13/Sec31 (Antonny, Madden et al. 2001). The majority of cargo proteins are actively concentrated in COPII-coated vesicles prior to export from the ER (Malkus, Jiang et al. 2002). Most transmembrane cargo proteins exit the ER by binding directly to COPII (Aridor, Weissman et al. 1998; Kuehn, Herrmann et al. 1998), but some transmembrane and most soluble cargo proteins bind indirectly to COPII through transmembrane export receptors (Muniz, Nuoffer et al. 2000; Powers and Barlowe 2002). Export receptors leave the ER together with their ligands, unload their cargo into the acceptor compartment, and recycle back to the ER. The sorting signals recognized by the COPII coat are found in
26 the cytosolic domains of transmembrane cargo proteins. These signals are quite diverse (Barlowe 2003). COPI vesicles are involved in intra-Golgi transport and retrograde transport. Their assembly involves activation and membrane recruitment of GTPase ADP ribosylation factor (Arf) (Donaldson, Cassel et al. 1992). During COPI coat assembly; ArfGTP simultaneously recruits the membrane-proximal βγδζ and the membrane-distal αβ’ε subcomplexes (Hara-Kuge, Kuge et al. 1994), in contrast to the stepwise assembly of COPII, COPI entry requires specific signals in the cytosolic domains of transmembrane cargo proteins. These signals function to retrieve proteins from the ERGIC or the Golgi complex to the ER (Cosson and Letourneur 1994). Once the proteins reach the trans-Golgi network (TGN), they are again sorted into vesicles by intrinsic sorting motifs and cytoplasmic adaptor complexes, and are transported along cytoskeletal elements directly to the apical or basolateral plasma membrane. The adaptor complex crucial for the transport between the Golgi, the plasma membrane and endosomes is called the adaptor protein (AP)–clathrin complex (the AP–clathrin complex). Four AP complexes (AP-1–4) have been identified, and these localize to different membrane compartments between the Golgi complex, the plasma membrane and endosomes (Robinson 2004). Each AP complex is composed of four subunits. One subunit mediates the binding to the target membrane, the other one recruits clathrin through the clathrin binding sequence (Brodsky, Chen et al. 2001), the third subunit is responsible for cargo recognition and the last subunit is involved in the stabilization of the complex (Collins, McCoy et al. 2002).
Vesicle transport Each vesicle transport event can be divided into four essential steps that include vesicle budding, transport, tethering, and fusion (Bonifacino and Glick 2004). Vesicle budding is mediated by protein coats (Kirchhausen 2000; Bonifacino and Lippincott-Schwartz 2003). Protein coats are dynamic structures that cycle on and off membranes. They are recruited from the cytosol onto donor membranes by small GTPases that regulate their assembly (Springer, Spang et al. 1999). Coats deform flat membranes into round buds, which lead to the release of coated vesicles. Coat proteins
27 also participate in cargo selection through the recognition of sorting signals present in the cytoplasmic domain of transmembrane cargo proteins. Clathrin was the first coat to be identified (Pearse 1975). After budding, vesicles are transported to their final destination by diffusion or by motor-mediated transport along a cytoskeletal track (microtubules or actin). The molecular motors kinesin, dynein, and myosin have all been implicated in this process (Hammer and Wu 2002; Matanis, Akhmanova et al. 2002). The third step in vesicle-mediated membrane traffic is tethering. Tethering is a term used to describe the initial interaction between a vesicle and its target membrane. It precedes the pairing of transmembrane SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on apposing membranes, an event that leads to membrane fusion (Sollner, Whiteheart et al. 1993). Together with Rabs, small GTPases of the Ras superfamily, tethers play a critical role in determining the specificity of vesicle targeting. Rabs continuously cycle between the cytosol and membranes. In the cytosol, the GDP-bound form of the Rab is complexed with GDI (guanine nucleotide dissociation inhibitor). Rabs are recruited to membranes with the aid of a GDF (GDI displacement factor) (Dirac-Svejstrup, Sumizawa et al. 1997). The membrane-bound Rab is then activated by a specific GEF (guanine nucleotide exchange factor) through the exchange of GDP for GTP (Ullrich, Horiuchi et al. 1994). Rabs in their GTP-bound form appear to facilitate the recruitment of tethers to specific locations. Activation of the Rab is terminated when GTP hydrolysis is stimulated by a specific GAP (GTPase- activating protein) (Rybin, Ullrich et al. 1996). To date more than 60 Rab proteins have been identified in mammalian cells. The last step in vesicle-mediated transport is the fusion of the vesicle with its target membrane. Fusion is thought to occur by the pairing of SNAREs, a family of membrane proteins that are related to three different neuronal proteins: synaptobrevin, syntaxin, and SNAP-25. A SNARE on a transport vesicle (v-SNARE) pairs with its cognate SNARE-binding partner (t-SNARE) on the appropriate target membrane (Sollner, Whiteheart et al. 1993). Protein trafficking is crucial for building the polarized cells. Like other transmembrane proteins, cadherins are synthesized in the ER, modified in the Golgi, and trafficked to the basolateral cell surface by the exocytosis. Once at adherens junctions, cadherins
28 can be endocytosed into Rab5-positive early endosomes, and either recycled back to adherens junctions via Rab11-positive recycling endosomes, or sent via the Rab7- and Hrs-positive multivesicular body to lysosomes for destruction (Duncan and Peifer 2008). When the exocyst function is reduced, cadherin accumulates in Rab11 recycling endosomes (Langevin, Morgan et al. 2005). This is an example how important is the protein trafficking through the right vesicles, to get the functional cell with proper junctional complex.
Figure 5. Post-translational pathway for protein trafficking to the plasma membrane After synthesis in the endoplasmic reticulum (ER), membrane proteins are sorted into vesicles by the coatomer protein complex-II (COPII) machinery and delivered to the Golgi complex by vesicle-tethering and SNARE machineries. Intra-Golgi transport and retrograde transport from the Golgi to the ER are regulated by the COPI machinery. At the trans-Golgi network (TGN), proteins are sorted into different vesicles by intrinsic sorting motifs and cytoplasmic adaptor complexes, and are transported along cytoskeletal elements to the plasma membrane or different compartments. Clathrin coats are heterogeneous and contain different adaptor and accessory proteins at different membranes. Additional coats or coat-like complexes exist but are not represented in this figure. EE - early endosome, RE – recycling endosome, LE –late endosome, SG – secretory granule, PM – plasma membrane. Adapted from (Bonifacino and Glick 2004).
29 Control of tracheal tube size An important step in trachea tube morphogenesis is the acquisition of the proper length and diameter. Tube size is critical for organ function therefore it must be controlled not only during tube formation but also throughout development in order to maintain functionality as the animal grows. The diameter and length of the tracheal branches are controlled independently. The tube diameter expansion occurs step-wise, whereas the length of the branches increases continuously through development. Tube diameter grows at three brief periods of development, during which the branches expand to their characteristic sizes (Beitel and Krasnow 2000). These sizes are genetically programmed, and some of the genes in the program have been identified. Cell number does not directly determine tracheal tube size. There is not a correlation between the size of a tracheal tube and the number of cells in it, and altering cell number using cell cycle mutations did not affect tube size. Furthermore, the dramatic expansions of the tracheal tubes in the embryo and larva occurred without changing cell number, and tube sizes were altered in tube expansion mutants without affecting cell number. Tracheal cells in a given branch must collectively adjust their shapes to form a tube of the specified size. One idea is that the program dictates branch size by controlling the amount of apical membrane biogenesis and secretion for each branch. To this group belongs transcription factor Grh. In grh mutants, excessive apical membrane forms, resulting in elongated and convoluted tubes, whereas tracheal over expression of Grh prevents lumen extension (Hemphala, Uv et al. 2003). The next group of genes involved in tube size regulation is the genes involved in luminal matrix formation. The matrix is transiently secreted into the lumen, and is composed of fibrillar chitin. Mutations of genes involved in chitin biogenesis and assembly (krotzkopf verkehrt, mummy/cystic, knickkopf, retroactive) result in irregular diametric expansion of the tracheal tubes with local constrictions and dilations (Araujo, Aslam et al. 2005; Tonning, Hemphala et al. 2005; Moussian, Tang et al. 2006). In addition, these mutants develop overelongated tracheal branches. Also two putative chitin deacetylases, Vermiform and Serpentine, which are secreted into the lumen and assumed to modify chitin structure, regulate tracheal length (Luschnig, Batz et al. 2006; Wang, Jayaram et al. 2006). This suggest that the chitin matrix
30 provides either a physical scaffold for the underlying epithelia or it sends signals to the tracheal cells to adjust tube shape and size. Another group of tube size mutants includes genes encoding septate junction components. These mutants develop over-elongated tubes with irregular cell shapes. Most of the septate junction mutants show impaired secretion of Vermiform and Serpentine and a defect in the assembly of the luminal chitin filaments. Given that verm and serp mutants also show tortuous tubes, the function of SJ proteins in tube size control may be to facilitate the apical secretion of chitin modifying enzymes. An additional mechanism by which SJ proteins may control tube size is, through the regulation of apical polarity proteins such as Crumbs and aPKC (Wu and Beitel 2004). The reduction in the levels of the apical membrane determinant Crumbs suppresses the tube length defects in cora mutants. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Similarly, mutations in yurt and scribble cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing the activity of Crumbs suppresses the length defects in yurt but not in scribble mutants, suggesting that Yurt acts by negatively regulating Crumbs. Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway, where Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, Paul et al. 2010). The tight control of tube expansion suggests that tubes can sense their size and inhibit expansion when a specified size is reached. The sensor and output pathway have to be still identified in the tracheal system.
31
Figure 6. Model of the pathways involved in tube size regulation by SJ proteins Interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. This mechanism acts independently of and in parallel to a pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. There can be one more mechanism regulating the tube size, acting through Scrib. scrib mutants have long trachea with normal Verm levels and the defects are not suppressed by loss of Crb, like in cora and yrt mutants. Adapted from (Laprise, Paul et al. 2010).
32 AIMS The aim of the work described in this thesis was to: 1) Dissect respiratory organ maturation in the embryonic trachea 2) Identify genes, which are required for tracheal tube size control 3) Address the mechanism of septate junction assembly
33 RESULTS AND DISCUSSION
Paper I: Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila To gain insight into Drosophila tracheal maturation, we used a combination of live imaging and genetic analysis. Those methods allowed us to analyze the diametric expansion of the narrow tracheal branches to their final size and the final clearance of the tubes from matrix and liquid at the end of embryogenesis. We used live imaging of secreted GFP-tagged proteins and analysis of mutants with defects in gas filling. These experiments revealed the distinct steps of airway maturation: 1) secretion burst followed by tube diameter expansion, 2) clearance of solid luminal material, 3) replacement of luminal liquid by gas.
1) Sar-1 mediated luminal deposition of secreted proteins, drives tube expansion ANF-GFP (rat Atrial Natriuretic Factor fused to GFP) was used as a heterologous sand Gasp-GFP as an endogenous secreted marker. Both of them were predominantly localized in the cytoplasm at early stage 13. At late stage 13 there was a sudden secretion burst that deposited those markers into the tracheal lumen. 30 minutes after the secretion burst tube diameter began to expand. We found that the small GTPase Sar1 is required for efficient secretion into the tracheal lumen. In the absence of Sar1 luminal secretion of ANF-GFP, Gasp-GFP, Gasp and other endogenous proteins like 2A12 and Verm was incomplete and secreted proteins remained in the cytoplasm. The consequence of incomplete secretion was the failure of tube diameter expansion. Sar1 regulates COPII vesicles budding from the ER to the Golgi complex (Bonifacino and Glick 2004; Abrams and Andrew 2005). sar1 mutant showed disrupted ER structures and loss of Golgi structures. To test if there is general requirement of COPII complex, two additional COPII coat subunits sec13 and sec23 were analyzed for protein secretion and tube expansion phenotypes (Abrams and Andrew 2005). Both mutants had the same phenotypes like sar1 mutant. This showed the requirement of secretion apparatus for efficient protein secretion, which consequently drives tube
34 expansion. We proposed three ways by which the secretion burst drives tube expansion: 1) by deposition of apical membrane, 2) by deposition of a secreted expanding matrix that molds the growth of the tubes 3) by targeting of apical channels or other regulators that may inflate the lumen. The secretory burst depends on COPII complex, which can be important for transport of unknown regulators of tube expansion, or add additional membrane to the growing luminal surface. The other hypothesis is that the luminal deposition of chitin binding proteins during the secretory burst can organize the construction and swelling of luminal matrix, which in turn induces lumen diameter expansion.
2) A Rab-5 dependent endocytic wave clears the lumen of solid material At stage 16 the dorsal trunk lumen contains multiple proteins and a chitinous cable, which have been deposited during the secretion phase. To follow the fate of the luminal proteins, we used live imaging of ANF-GFP and Gasp-GFP. At stage 16 the lumen contained high levels of ANF-GFP. At mid-stage 17 luminal ANF-GFP rapidly decreased to very low levels. The same was observed for Gasp-GFP, which was first accumulated in the lumen and than was removed similarly to ANF-GFP. We also tested if the endogenous protein Gasp and the fluid-phase marker dexran were taken up into tracheal cells during the clearance. For the dextran we established a live endocytosis assay. Dextran was injected inside the hemocoel of stage 13 embryos, which SJs are not formed yet, and accumulated inside the tracheal lumen. Both endogenous Gasp and dextran were cleared from the DT lumen. This confirmed the ANF-GFP and Gasp-GFP results. To prove that the dextran detected inside the cell during the luminal clearance is coming from lumen and not from the hemocoel, we injected rhodamine-labeled dextran into the hemocoel of late stage-16 embryos. At stage 16 paracellular barriers are formed and prevent the luminal access of the 10 kDa dextran (Lamb, Ward et al. 1998). We did not find any dextran inside the tracheal cells at stage 17. This demonstrates that the transient intracellular dextran puncta in embryos preloaded at stage 13 indeed derive from the lumen. Transmission Electron Microscopy also showed that the luminal chitinous cable disappeared within the interval of ANF-GFP clearance.
35 What is the mechanism of protein clearance? We found that Rab5, dynamin and clathrin are required to remove the luminal contents, indicating that the endocytosis is required for this process. Dynamin encodes the GTPase that clips Clathrin-coated pits off the membrane to form endocytic vesicles (van der Bliek and Meyerowitz 1991). Rab5 encodes GTPase that regulated this process (Bucci, Parton et al. 1992). In all three mutants ANF-GFP, Gasp-GFP, endogenous Gasp, Verm, 10 kDa dextran and chitin have been retained inside the tracheal tubes. In the wild type situation the material, which is endocytosed during the wave of endocytosis, colocalized with the endocytic markers inside tracheal cells. The most pronounced colocalization was with clathrin coated vesicles, late endosomes and lysosomal markers. The lysosomal localization indicated that the endocytosed proteins from the tracheal lumen might be degraded inside the tracheal cells during the protein clearance. In rab5 mutant we observed defects in those endocytic compartments during the period of protein clearance but the secretion burst and the diameter expansion have been not affected. The apically localized dotted pattern of Clathrin heavy chain was lost, GFP-FYVE endosomal apical vesicles were absent and also the Rab7 late endosomal structures were decreased in intensity. Those defects argue for role of endocytosis in direct internalization of luminal material.
3) The transition from liquid- to gas-filled trachea After the protein clearance step, the liquid has to be removed from the lumen to allow the trachea to be gas filled and become functional. To follow this process, late stage 17 embryos have been recorded by live imaging. In a stereotypic way, a bubble of a so far unknown gas appeared in one of the dorsal trunk metamers (4-6) and quickly expanded first posteriorly and then anteriorly. Within 10 min, the entire trachea was filled with gas. Approximately 80 min later the mature larva hatched. The tracheal maturation steps 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 and provides an example of how pulses of epithelial activity drive distinct developmental events to form functional tracheal tube.
36 PaperII: COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila The second paper follows the studies from the first paper, where we analyzed the function of anterograde COPII transport in the tracheal formation. COPII anterograde transport mediates luminal deposition of secreted proteins, what drives tube expansion. Here we focused our analysis on the retrograde transport which is mediated by COPI vesicles. COPII vesicles bud from the ER and shuttle nascent secreted proteins to the Golgi apparatus. COPI coated vesicles retrieve escaped luminal ER enzymes and recycling cargo adaptor proteins from the Golgi back to the ER (Bonifacino and Glick 2004). The coatomer complex of COPI vesicles is composed of two different layers. The tetrameric subcomplex of the inner layer is formed from four subunits: β, δ, γ, ζ. The β- and γ- subunits bind to active Arf1-GTP, γCOP also binds to p23. The outer trimeric coat layer contains three subunits: α, β’, ε (Bethune, Wieland et al. 2006).
Tube diameter expansion is dependent on COPI vesicles In the P-element screen for tracheal tube size mutants we identified the γCOPP1 mutant. By imprecise excision we generated a null allele, which we used for our analysis. In γCOP mutants the tracheal dorsal trunk diameter was reduced by~50% compared to wild type embryos. To follow the diameter expansion defects in time we imaged live wild type and γCOP mutant embryos expressing btl>GFP-CAAX. Wild type embryos initiate tracheal tube diameter expansion at stage 14. γCOP mutant embryos already showed a narrower tube diameter at stage 14 and later expanded their tubular diameter with a slower rate compared to wild type. γCOP is also expressed in other ectodermally derived tissues: salivary glands and epidermis from stage 11, hindgut and foregut from stage 16. To see if γCOP is required for diameter expansion in other tubular organs, we measured tube diameter of salivary glands. Like in tracheal tubes, the salivary glands of γCOP mutants were thinner compared to wild type. Both trachea- and salivary glands- tube diameter phenotype can be rescued by re-expression of γCOP in the mutants. Those results
37 suggest that γCOP is crucial for tube expansion in tubular organs and there is a common cellular mechanism, which expands tubular organs.
COPI dependent secretion drives the tube diameter expansion Diametric expansion is mediated by the secretory burst of luminal proteins at stage 13 in tracheal tube (Tsarouhas, Senti et al. 2007). We tested if the tracheal diameter defects in γCOP mutant are caused by defects in luminal protein secretion. Three tested secreted proteins 2A12, Gasp and Verm were localized to the tracheal lumen in wild type embryos at stage 15, but strongly retained inside tracheal cells in γCOP mutants. Those proteins are binding and modifying the luminal chitin. Transmission electron microscopy and luminal chitin visualization in the tracheal tube revealed the change of chitin structure in γCOP mutant. The chitin was denser in mutant compared to the wild type embryos at stage 16. The salivary gland lumen is also filled with a luminal matrix. The composition of this matrix is not well known; we know that it contains glycans with a single N-acetyl- Galactosamine O- linked to Serine or Threonine (Tian and Ten Hagen 2007), which can be visualized by antibodies recognizing the Tn antigen. First, the Tn antigen is localized inside the cells, followed by luminal localization at stage 12 and 13, when the salivary gland diameter expansion occurred. γCOP mutants showed reduced cellular accumulation and luminal deposition of the antigen. Similar defects were detected in sar1 mutants. Transmission electron microscopy analysis confirmed the change of salivary gland luminal matrix in γCOP mutant, where the material was deformed and abnormally dense. Those results suggest the requirement of COPI and COPII for efficient luminal deposition and diameter expansion. COPI vesicle coat consists next to the γCOP of another six subunits. We tested if another COPI vesicle coat subunit: δCOP, plays similar function in the protein secretion and tube expansion. Both Verm and Gasp retained in the tracheal cells in δCOP mutant and tube expansion failed both in tracheal- and salivary gland tube. The secretion defects of COPI vesicle mutants can be indirectly caused by defects in epithelial polarization. The tested apical membrane, adherens- and septate- junctions
38 were not affected in those mutants arguing that the tube diameter reduction is due to insufficient secretion of luminal components.
Defects in the secretion apparatus underly the tube size defects We examined the cellular localization of γCOP in Drosophila cells and the integrity of the secretion apparatus in γCOP mutant. A γCOP antibody showed punctuate cytoplasmic signal in S2 cells. Those punctuated structures were identified as ER and cis-Golgi units by co-localization of γCOP with ER markers KDEL (the ER retention signal found in many ER proteins) and Calreticulin, and cis-Golgi markers GM130 and Lava lamp. Because of the localization of γCOP in the ER and cis-Golgi, we analyzed those structures in γCOP mutant. γCOP mutant embryos showed a substantial reduction of the ER markers: KDEL, Calreticulin, the transmembrane adaptor, p23 and a cis-Golgi marker, Lava lamp both in the trachea and salivary glands. TEM analysis additionally revealed bloated ER structures in the γCOP mutants. Those defects are indicative of an activated unfolded protein response (UPR). The UPR activation was further confirmed by the presence of processed xbp1 transcripts in γCOP mutants. Our analysis indicated that COPI vesicles are required for ER integrity. Once the ER integrity and vesicle transport are disrupted, the luminal proteins cannot be secreted leading to a tube diameter reduction. The same cellular defects were detected in the mutants affecting COPII vesicles (Tsarouhas, Senti et al. 2007). The double mutant of COPII and COPI components: sar1, γCOP did not show any additive phenotype in the trachea, indicating that anterograde COPII- and retrograde COPI-mediated transports are equally important to maintain both ER and Golgi structures. The bidirectional ER- Golgi traffic is essential for secretion and assembly of luminal matrixes during tube expansion.
39 PaperIII: Epithelial Septate Junction Assembly Relies on Melanotransferrin Iron Binding and Endocytosis in Drosophila Analysis of Drosophila mutants revealed two groups of tracheal tube length mutants. One group of mutants relates to apical membrane growth and includes two transcription factors: ribbon (rib) and grainy head (grh). rib mutants fail to extend the tracheal lumens, as the apical membrane growth is limited (Shim, Blake et al. 2001), grh mutants have excessive apical membrane, resulting in elongated and convoluted tubes, whereas tracheal over-expression of Grh prevents lumen extension as in rib mutants (Hemphala, Uv et al. 2003). Another group of tube size mutants, disrupt genes encoding septate junction components. The tubes of these mutants become overelongated and convoluted with irregular cell shapes. The first septate junction mutant discovered to have a tracheal tube size phenotype was ATPα, which lacks a functional α-subunit of the Na+/K+ ATPase (Hemphala, Uv et al. 2003). Subsequently, mutations in other 14 genes have been found which showed a similar tracheal tube size phenotype. Much has been learned about the components of the septate junctions, but the mechanism of their assembly into functional junctions was unknown.
Melanotransferrin is a new septate junction component Melanotransferrin (MTf) is expressed in all ectodermal epithelial tissues and is phylogenetically conserved between mammals and Drosophila. MTf mutants have overelongated tracheal tubes, resembling the defects of mutants lacking septate junctions. Expression of the Drosophila or the mouse MTf in the trachea rescued the tracheal phenotype of mutant Drosophila embryos arguing that the function of the protein is conserved. Further analysis of the cellular phenotypes of MTf mutant embryos showed that the septate junction epithelial barrier function was disrupted, the secretion of Vermiform failed and that septate junction components were mislocalized. To confirm our hypothesis that MTf is a new septate junction component, we raised antibodies against the MTf protein. MTf colocalized with other septate junction markers. Its localization was dependent on other septate junction components since
40 MTf was spread basolaterally in cora and ATPα mutants. Finally, we showed that MTf forms complexes with Nrx, Cont and Nrg during late embryogenesis. We concluded that MTf is a septate junction component required for junction integrity.
Endocytosis is required for functional septate junction formation During our analysis of MTf function we found that MTf and other septate junction components were localized along the lateral and basal epithelial cell membranes and partially in the vesicular structures inside the cell during stage 13, before septate junctions form. In contrast to stage 13, there is restricted apicolateral localization at stage 16. We wanted to know how are septate junctions assembled into functional junctions and what is the mechanism that drives their formation. Substantial portion of MTf already formed complexes with Cont and Nrx at early stages. MTf and another septate junction component, Nrg colocalized with endosomal markers, and were also co-purifying with Rab5 early- and Rab11 recycling-endosomes at stage 13. At stage 16 when septate junctions were fully functional, we did not detect this co-localization. The potential role of endocytosis in septate junction formation was confirmed when the septate junction components were found mislocalized in endocytic mutants (like clathrin heavy chain, dynamin, and in embryos expressing dominant negative forms of rab5 and rab11). The results suggested that septate junction components are relocalized from basolateral to the apicolateral membrane through early and recycling endosomes.
MTf iron binding is crucial for septate junction assembly To elucidate the function of MTf in septate junction assembly, we examined its conserved motifs. By using phoshatidylinositol-phospholipase Cγ we confirmed that MTf is a GPI-linked membrane protein. The significance of the GPI-modification motif was tested in over-expression experiments by expressing MTf with deleted GPI- motif in MTf mutant embryos. This construct rescued septate junction defects in MTf mutants, suggesting that GPI-motif is not essential for MTf function.
41 MTf has two iron binding domains with conserved residues, which were expected to mediate the iron binding. The iron binding capacity of MTf was tested by two different methods: atomic absorption spectroscopy and electron paramagnetic resonance. Both methods confirmed the iron binding of wild type MTf. The mutations of the conserved residues Y231F in the N-terminal lobe reduced iron binding, whereas the Y533F in the C-terminal lobe abolished it. Expression of the Y231F construct rescued the MTf mutant phenotypes including the septate junctions defects, but the iron-deficient Y533F construct did not rescue those defects. This indicates that iron binding is critical for MTf function and septate junction maturation. We tested if iron homeostasis is defective in MTf mutants, by monitoring ferritin levels. Ferritin is an iron storage molecule and its levels indicate aberrant iron concentrations (Hentze, Muckenthaler et al. 2004). MTf mutants did not show changes in ferritin levels suggesting that MTf has not a major role in iron uptake; its iron binding is specifically required for septate junction maturation.
Uptake of MTf induces septate junction assembly Requirement of endocytosis for septate junction formation, made us to investigate if endocytosis of iron bound MTf is sufficient to induce septate junction assembly in epithelial cells lacking MTf. Wild type MTf, MTfΔGPI and MTfY533FΔGPI were expressed in epidermal stripes of MTf mutant using en-GAL4 driver. We could detect MTfΔGPI also in cells not expressing en-GAL4, but not the wild type MTf. Cells, lacking MTf, were binding and internalizing exogenous secreted MTfΔGPI. In those cells MTfΔGPI localized along the lateral membranes and in recycling endosomes, suggesting that it is endocytosed. The iron depleted form of MTf the MTfY533FΔGPI was not detected in non expressing en-GAL4 cells, indicating that MTf internalization requires iron bound MTf. MTfΔGPI, which localized in the en-GAL4 not expressing MTf mutant cells, also rescued the Nrx mislocalization in those cells. The results indicate that endocytosis of the iron binding form of MTf is sufficient to induce septate junction assembly in MTf mutant cells.
42 PaperIV: Control of Tube Diameter Expansion by Secreted Chitin- binding Proteins
The Obstructor multigene family Despite that tube size is critical for the organ function, the cellular mechanisms of tube expansion are not clear. Tracheal tube expansion involves apical secretion and deposition of luminal matrix, but the mechanistic role and the nature of the forces involved are not clear. In our studies we analyzed two members of the Obstructor multigene family, Obst-A and Gasp. Phylogenetic analysis showed, that Obst-A and Gasp are the most closely related genes among the members of the Obstructor gene family. Also their protein motif arrangement is the same: a signal peptide, followed by three chitin-binding domains type 2. This suggests that Obst-A and Gasp can have similar function. Obst and Gasp are expressed in the trachea, at the time of diametric tube expansion and cuticle deposition; therefore we analyzed their function in cuticle formation and tracheal maturation. The Gasp expression pattern is very similar to the pattern detected with the widely used 2A12 antibody, which recognizes the unknown antigen. Gasp and the unknown antigen are first localized in the cells and become later secreted into the lumen. The similarity made us to test if the 2A12 antibody shows staining in gasp mutant embryos. We did not detect any signal both by 2A12 and Gasp antibodies in the mutants. When we ectopically expressed Gasp protein in the dorsal part of the hindgut, and found that both 2A12 and Gasp antibodies could recognize this protein. This argues that the unknown antigen which 2A12 antibody recognizes is Gasp.
Body size, cuticle and tracheal luminal matrix are disrupted in obst-A and gasp mutants For our analysis we used null mutants of both genes. We found that single obst-A and gasp mutants showed reduced larval length: 8% in obst-A mutant and 14% in gasp mutant. The phenotype became enhanced in the obst-A, gasp double mutant to 25% length reduction compared to the wild type. The enhanced defects of obst-A, gasp
43 double mutants were also detected when we examined cuticle preparations of first instar larva. Single mutants showed normal cuticular structures but the cuticles of the double mutant were dilated. Those results suggest that Obst-A and Gasp have overlapping partially redundant functions. The presence of the chitin binding domains in Obst-A and Gasp proteins led us to examine the luminal and taenidial chitin in the trachea. Labeling with a chitin-binding probe revealed that the single mutants showed a reduced luminal chitin cable compared to the wild type embryos. In double mutants the reduction in intensity of the chitin labeling was more severe than in the single mutants. We also detected a reduction in the luminal stainings for the chitin-modifying enzyme Verm. This showed that the luminal chitinal matrix is defective in obst-A, gasp mutants. Do also the taenidia, which contain chitin, display any phenotypes? We analyzed the mutant taenidia and their chitin containing procuticular structure by TEM. This revealed an irregular shape and size of the taenidial folds and a defective procuticular layer in the obst-A, gasp double mutant. Collectively, the structure of both luminal chitin and taenidial procuticle are affected in the mutants lacking Obst-A and Gasp. These two chitin-binding proteins may help to form and assemble the chitin filaments into higher order structures.
Obst-A and Gasp regulate the tracheal tube diameter Both Obst-A and Gasp are secreted into the tracheal lumen, during diameter tube expansion. Is luminal diameter expansion influenced by the amount of secreted proteins and chitin structure? To address this question, we measured the tracheal tube diameter both in obst-A and gasp single mutants, and obst-A, gasp double mutant. We labeled the apical and lateral membranes with Crumbs and α-Spectrin. Both obst-A and gasp single mutants showed a minor reduction in DT diameter. obst-A mutant 5% and gasp mutant 10% reduction compared to the wild type embryos at stage 16. This phenotype became enhanced in the double mutant to 15%. This suggests, that Obst-A and Gasp proteins are required for tube diameter expansion. The two proteins presumably modify the structure of the luminal chitin filament and provide a mold or an inflating force for tube expansion.
44 To test if an increase in luminal levels of chitin binding proteins may influence tube expansion, we overexpressed obst-A or gasp in the trachea. The measurements showed a minor increase of tube diameter, 7% in Obst-A and 13% in Gasp overexpressed embryos. This supports the idea that the tube expansion can be regulated by the amounts of secreted proteins.
CONCLUSIONS AND PERSPECTIVES In our work we made several important findings. In paper I, we dissected the process of airway maturation and identified three precise epithelial transitions: deposition of proteins into the tracheal lumen which we named secretion burst, this is followed by luminal protein clearance and shortly thereafter by luminal liquid clearance. We also identified genes, which are required for those transitions. Sar1 (paper I) and γCOP (paper II) are required for secretion burst and Rab5 (paper I) for protein clearance. All three transitions are prerequisites for gas filling and functional trachea. We still have to find out the genes, which drive the third step of trachea maturation the luminal liquid clearance, which is followed by gas filling. To identify such genes we could perform screens for mutants that fail in liquid clearance without showing defects in the secretion burst and the protein clearance processes. This way we will specifically find genes regulating only gas filling process. There is a class of proteins that likely regulate the liquid clearance and gas filling process, the epithelial sodium channels (ENaCs). In the lung the influx of sodium through these channels drives water from the lumen into the epithelial cells. RNAi inactivation of Drosophila homologs leads to liquid clearance phenotypes. This suggested that a similar process could be involved in the embryonic liquid clearance of the airways. The three epithelial transitions are highly coordinated and occur at specific time points. What is regulating this sequence in such precise manner? Probably there are some signaling pathways activated at every step of the maturation. The secretion burst can be activated by signals from the apical membrane. Once the apical membrane reaches a certain size, which can be monitored by the apical cytoskeleton, the lumen needs to also expand. A sudden secretion of luminal proteins drives this expansion. Once the lumen reaches certain size, we believe that ECM sensors on the apical membrane initiate clearance of the luminal material to allow the tube to be gas filled. In this case again the apical membrane triggers the luminal
45 clearance, by sensing the tube size and activating the Rab5-dependent endocytic activity, which internalizes and clears luminal contents. An important step in testing these models is the identification of tube size regulators. Here, we identified mutants in MTf (paper III), which show overelongated tracheal tubes. Melanotransferrin is in complex with other septate junction components and is required to form the epithelial barrier. Melanotransferrin binds iron and the iron binding is required for septate junction assembly. We explored the mechanism by which septate junctions assemble to functional complex. The assembly relies on endocytosis and apicolateral recycling of septate junction components. First, septate junction components are localized along the basolateral membrane where they form complexes, later they are endocytosed and relocalized to apicolateral region, where they form mature functional junctions. The relocalization of septate junction components is iron dependent. We speculate that the iron triggers the SJ complex formation and later has a function in stabilization of the complex. Once the iron binds to MTf, MTf can increase its affinity to other SJ molecules and this initiates the formation of the complex. The assembled complex can trigger endocytosis and its re-localization to apicolateral membrane. The targeting to apicolateral membrane can be guided by signals from AJs or apical membrane. The next possibility is the presence of specific SNARES at the vesicles and targeted membrane domain. The identification of such targeting molecules could be accomplished by the isolation and biochemical analysis of the vesicles containing SJ proteins. We also found that mouse Melanotransferrin can replace the Drosophila Melanotransferrin function. It will be of a great interest to investigate if the tight junctions, the homolog of septate junctions, assemble in a similar fashion like septate junctions. The diametric expansion of the tubes during development is critically important for airway function. In our studies we found several genes, which are required for tube diameter expansion. During the secretion burst, Sar1 and γCOP facilitate protein transport from ER to Golgi and vice versa. In both sar1 and γCOP mutants secretion is disrupted, which causes narrow tracheal tubes. It has been shown that sec24, a cargo-binding subunit of the COPII complex can support secretion and local tube expansion in a cell-autonomous fashion. Another class of genes, which encode regulators of tube expansion, is Obst-A and Gasp, which we identified in paper IV. Those genes belong to Obstructor multigene family, are
46 strongly expressed in the trachea and encode proteins that are secreted into the tracheal lumen during the secretion burst. Impairment of their secretion leads to tracheal diameter reduction. We showed that secreted molecules are regulating tube expansion, which could be based on the generation of intra-luminal forces. The next step would be to measure the intra-luminal forces and find the receptors, which respond to those forces. The forces could be measured by visualization of the apical cytoskeleton and its localized changes. Forces are probably generated by luminal chitin, which expands after being modified. It could be possible to reconstitute chitin modification in vitro, by applying chitin-modifying proteins on chitin. This will allow testing directly if the chitin-modifying proteins play a role in inducing changes in the biophysical properties of the polymers and in local force generation.
47 ACKNOWLEDGMENTS
Finally I am here! There are so many people who made this work possible and I would like to say Thanks! It will be difficult to remember everyone, but I will at least try.
Thanks to Christos for giving me an opportunity to do a PhD in his group, for the guidance and support during all those years. Thank you for always keeping the doors open, for all the advices - scientific and nonscientific.
Thanks to Astrid Gräslund and Torbjörn Astlind for the help with my publication in very crucial moments.
Thanks to past and present members of the lab: Satish for being such great lab-mate, office-mate, but mostly a great friend. The fun, all the trips and all the discussions we used to have made my PhD and time outside the lab more enjoyable! Kirsten to you I have also special thanks, for teaching me the science, especially the fly genetics and for being always so enthusiastic, helpful and ready to discuss everything. Fergal also big thanks to you, it was just two years what we overlapped, but I really enjoyed your presence, all the talks and fun we had. Also thanks for all the lunches we had after Oliver was born. Meenakshi, thank you so much, especially for the warm welcome during the really first days in the lab and in Sweden. The first year was special also because of you! Nafiseh thank you for being always optimistic, supportive and for all the good advices. Naumi thank you for all the talks, help with Rufus and being a friend mostly in the last part of PhD. Nicole it was nice when you have been around and all the talks across the lab bench, thank you for it. Shenqiu you have been my teacher when I joined, thank you so much for the things you thought me and for your big optimism. Vasilios, to you I want to thanks mostly for the microscop help and the statistics, Chie thanks for being nice office-mate and for being ready to listen my talking, Badrul thanks for all the help, big thanks also to all the others lab members: Liqun, Ryo, Yi, Michael, Oliviano, Souli, Maria, Fatemeh, Annika, Martin, Johanna, Nikos, Peggy, Ryan, Kostas, Zhi, Didier, Jeremy, Dimitrios, Kassianni.
48 Thanks to the present and former members at Devbio: Mattias, Stefan, Bhumica (thanks for the nice company in and out of the lab and all the advices), Dana (it was nice to have you around, and speak the same language), Emad, Naghmeh, Filip, Ann, Olle, Per-Henrik, Olga, Katrin, Lili, Musarat, Qi, Helen, Mattias, Tobias, Jiang, Feng, Sidney, Andreas.
Thanks to Monika for taking care about the flies and lab, being a nice company and special thanks for the big help with Rufus! Thanks to Inger for the best EM help one can wish, with the excellent sectioning and always being ready to try new things. Thanks for everything you thought me. Thanks to Magdalena for all the administrative work of a great fast-speed quality. Thanks also to Anna-Leena for the administrative work.
Thanks now to family and friends for time spent away from lab. Thanks to all my friends in Sweden, without you it would be not easy to survive all those years in a foreign country: Olga, Yasar, Camilla, Vivek, Vasilij, Vova, Saleem, Attia, Aleks, Uliana, Roy, Sohini, Vanessa, Niklas, Thomas, Bea, Branka, Andrea, Wei, Anders, Anna, Olivia, Natalija, Raul, Svetlana, Denisa, Feri, Katka, Jana.
Thanks also to my friends in Slovakia for keeping in touch all those years and being a big support whenever I needed: the endless talks on skype with Zuzka H. and Zuzka H., Marcela, Slava, Janko, Katka, Vlado, Jozef.
Thanks to my family in Slovakia, especially to my mother, father and sister for all your support and help over so many years. Living faraway has never been easy. Last, but foremost Thanks to Pawel, for your endless love, support and patience during the good and bad times and for our biggest treasure Oliver!!!
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58 Developmental Cell Article
Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila
Vasilios Tsarouhas,1,2 Kirsten-Andre´ Senti,1,2 Satish Arcot Jayaram,1 Katarı´na Tiklova´ ,1 Johanna Hempha¨ la¨ ,1 Jeremy Adler,1 and Christos Samakovlis1,* 1 Wenner-Gren Institute, Stockholm University, Department of Developmental Biology, Svante Arrheniusva¨ g 16, Arrheniuslab E3, S-10691 Stockholm, Sweden 2 These authors contributed equally to this work. *Correspondence: [email protected] DOI 10.1016/j.devcel.2007.06.008
SUMMARY Polycystic Kidney Disease. Conversely, stenoses, the abnormal narrowing of blood vessels and other tubular The development of air-filled respiratory organs organs, are associated with ischemias and organ obstruc- is crucial for survival at birth. We used a combi- tions (Lubarsky and Krasnow, 2003; Sadler, 1985). nation of live imaging and genetic analysis to While the early steps of differentiation, lumen formation, dissect respiratory organ maturation in the and branch patterning begin to be elucidated in several embryonic Drosophila trachea. We found that tubular organs (Affolter et al., 2003; Hogan and Kolodziej, tracheal tube maturation entails three precise 2002), we only have scarce glimpses into the cellular events of lumen expansion and tubular organ maturation. epithelial transitions. Initially, a secretion burst De novo lumen formation can be induced in three-dimen- deposits proteins into the lumen. Solid luminal sional cultures of MDCK cells. Recent studies in this sys- material is then rapidly cleared from the tubes, tem revealed that PTEN activation, apical cell membrane and shortly thereafter liquid is removed. To polarization, and Cdc42 activation are key events in lumen elucidate the cellular mechanisms behind these formation in vitro (Martin-Belmonte et al., 2007). In zebra- transitions, we identified gas-filling-deficient fish embryos and cultured human endothelial cells, capil- mutants showing narrow or protein-clogged lary vessels form through the coalescence and growth of tubes. These mutations either disrupt endo- intracellular pinocytic vesicles (Kamei et al., 2006). These plasmatic reticulum-to-Golgi vesicle transport tubular vacuoles then fuse with the plasma membranes to or endocytosis. First, Sar1 is required for pro- form a continuous extracellular lumen. Salivary gland tein secretion, luminal matrix assembly, and extension in Drosophila requires the transcriptional upre- gulation of the apical membrane determinant Crumbs diametric tube expansion. Subsequently, a (Crb) (Myat and Andrew, 2002), but the cellular mecha- sharp pulse of Rab5-dependent endocytic nism leading to gland expansion remains unclear. activity rapidly internalizes and clears luminal The epithelial cells of the Drosophila tracheal network contents. The coordination of luminal matrix form tubes of different sizes and cellular architecture, secretion and endocytosis may be a general and they provide a genetically amenable system for the mechanism in tubular organ morphogenesis investigation of branched tubular organ morphogenesis. and maturation. Tracheal development begins during the second half of embryogenesis when 20 metameric placodes invaginate from the epidermis. Through a series of stereotypic INTRODUCTION branching and fusion events, the tracheal epithelial cells generate a tubular network extending branches to all Branched tubular organs are essential for oxygen and embryonic tissues. In contrast to the wealth of knowledge nutrient transport. Such organs include the blood circula- about tracheal patterning and branching (Ghabrial et al., tory system, the lung and kidney in mammals, and the 2003; Uv et al., 2003), the later events of morphogenesis tracheal respiratory system in insects. The optimal flow and tube maturation into functional airways have yet to of transported fluids depends on the uniform length and be elucidated. As the nascent, liquid-filled tracheal net- diameter of the constituting tubes in the network. Alter- work develops, the epithelial cells deposit an apical chitin- ations in the distinct tube shapes and sizes cause ous matrix into the lumen. The assembly of this intralumi- pronounced defects in animal physiology and lead to nal polysaccharide cable coordinates uniform tube growth serious pathological conditions. For example, tube over- (reviewed by Swanson and Beitel, 2006). Two luminal, growth and cyst formation in the collecting duct are putative chitin deacetylases, Vermiform (Verm) and Ser- intimately linked to the pathology of Autosomal Dominant pentine (Serp), are selectively required for termination of
214 Developmental Cell 13, 214–225, August 2007 ª2007 Elsevier Inc. Developmental Cell Epithelial Transitions Drive Airway Maturation
Figure 1. Identification of Four Distinct Events in Tracheal Tube Maturation (A and B) Selected images from two time-lapse confocal movies of live wild-type btl > ANF-GFP embryos showing the (A) luminal deposition burst of ANF-GFP at 10.5 hr after egg laying (AEL) and the (B) luminal ANF-GFP clearance at 19 hr (green) (Movies S1 and S2). (C and D) (C) Deposition and (D) clearance of Gasp-GFP in btl > Gasp-GFP embryos (green) (Movies S3 and S4). (E) Two selected images from a wide-field movie illustrating the liquid clearance/gas filling of a wild-type embryo. The upper panel shows initiation of the gas filling (first gas bubble), while the lower panel shows its completion, when all tracheal branches are gas filled (Movie S5). (F) Summary of recordings showing the maturation events of embryos expressing btl > ANF-GFP in real time (n = 237). Tracheal cells were marked by the (A) btl-mRFP1-moe or (B–D) btl > myr-mRFP1 transgenes (magenta). SEM denotes the standard error of means. Scale bars are 10 mm in (A)–(D) and 20 mm in (E). branch elongation. The analysis of verm and serp mutants RESULTS indicates that modifications in the rigidity of the matrix are sensed by the surrounding epithelium to restrict tube Live Imaging of Airway Maturation length (Luschnig et al., 2006; Wang et al., 2006). What We established a live-imaging approach to analyze the drives the diametric expansion of the emerging narrow maturation of the Drosophila tracheal network. Using branches to their final size? How are the matrix- and btl-GAL4, we coexpressed heterologous or endogenous liquid-filled tracheal tubes cleared at the end of embryo- GFP-tagged secreted molecules along with cytoplasmic genesis? RFP reporters to label tracheal cells (Shiga et al., 1996). Here, we use live imaging of secreted GFP-tagged Live embryos were then imaged by wide-field or confocal proteins to identify the cellular mechanisms transforming microscopy. the tracheal tubes into a functional respiratory organ. We ANF-GFP (rat Atrial Natriuretic Factor fused to GFP) characterize the precise sequence and cellular dynamics serves as a functionally inert, heterologous secretion of a secretory and an endocytic pulse that precede the marker in Drosophila (Rao et al., 2001). We recorded the rapid liquid clearance and gas filling of the network. Anal- development of the dorsal trunk (DT) of embryos express- ysis of mutants with defects in gas filling reveals three ing btl > ANF-GFP (btl-GAL4 UAS-ANF-GFP) and btl-RFP- distinct but functionally connected steps of airway matu- moesin (moe)(Ribeiro et al., 2004) to visualize tracheal ration. Sar1-mediated luminal deposition of secreted cells (Figure 1A; see Movie S1 in the Supplemental Data proteins is tightly coupled with the expansion of the intra- available with this article online). Initially, during early luminal matrix and tube diameter. Subsequently, a Rab5- stage 13, ANF-GFP predominantly localized in the cyto- dependent endocytotic wave frees the lumen of solid plasm, and the apical concentration of RFP-moe demar- material within 30 min. We show that the precise coordina- cated the narrow lumen of the nascent tracheal tubes. In tion of secretory and endocytotic activities first direct tube a sudden burst, ANF-GFP was deposited into the lumen, diameter growth and then ensure lumen clearance to and its luminal levels increased abruptly within 15 min. generate functional airways. As luminal secretion continued, cytoplasmic ANF-GFP
Developmental Cell 13, 214–225, August 2007 ª2007 Elsevier Inc. 215 Developmental Cell Epithelial Transitions Drive Airway Maturation
ance (Figure S1). Thus, ANF-GFP faithfully reports the rapid and massive deposition of endogenous luminal con- tent at stage 13 and its complete removal at mid-stage 17. To visualize the transition from liquid- to gas-filled trachea, we recorded late stage-17 embryos (Figure 1E; Movie S5). In a stereotypic sequence, a bubble of a so far unknown gas appears stochastically in one of the DT central metameres (4–6) and quickly expands first poste- riorly and then anteriorly. The bubble then spreads through the posterior anastomosis to the contralateral DT, where it again rapidly extends anteriorly. Within 10 min, the entire tracheal tree fills with gas. Approxi- mately 80 min later, the mature larva hatches. To embed the single steps in a developmental frame- work, we recorded multiple embryos expressing btl > Figure 2. sar1 Is Required for Efficient Luminal Secretion of ANF-GFP and timed each of the morphological changes. Proteins (A–I) Confocal sections of the DT of (A, D, and G) wild-type, (B, E, and All transitions occurred robustly within a defined, precise H) the sar1P1 mutant, and (C, F, and I) btl > sar1.1; sar1P1 rescued time interval (Figure 1F; Experimental Procedures). embryos. All embryos expressed btl > GFP-CAAX (magenta). Embryos Taken together, our live-imaging approach identifies were stained for the luminal antigens (A–C) 2A12, (D–F) Verm, and (G–I) three dramatic and dynamic changes in luminal morphol- Gasp (green). sar1P1 mutant embryos show strong intracellular reten- ogy that divide tracheal maturation into precisely timed tion of luminal markers. Tracheal expression of sar1 largely rescues steps. A secretory burst deposits large quantities of lumi- the luminal secretion phenotype of sar1P1 mutant embryos. Scale nal proteins into the tracheal lumen and tightly precedes bars are 10 mm. tube diameter expansion. The tracheal epithelium then clears these proteins and liquid by two sequential pulse- rapidly passed through dynamic, large vesicular struc- like events generating functional, mature airways. What tures. Half an hour after the secretion burst, tube diameter are the underlying cellular and molecular mechanisms began to expand, shown by the RFP-moe labeling. By executing the rapid transitions? What is the functional early stage 16, the ANF-GFP signal resided mostly in the relevance of the transitions for airway maturation? We lumen, and, concomitantly, diametric expansion ended. addressed those questions by analyzing mutants with Thus, ANF-GFP reports a sudden luminal deposition defects in gas filling. event. At stage 16, the DT lumen contains multiple proteins and a chitinous cable (Devine et al., 2005; Tonning et al., sar1 Is Required for Efficient Secretion 2005). To follow the fate of the luminal proteins, we imaged into the Tracheal Lumen embryos expressing ANF-GFP and the membrane marker We identified two strong, hypomorphic sar1 alleles in myr-RFP in the trachea from stage 16 onward (Figure 1B; screens for mutants with tracheal tube defects (see Exper- Movie S2). Initially, high levels of ANF-GFP filled the imental Procedures and Figure S2). sar1 encodes a small lumen. At mid-stage 17, luminal ANF-GFP rapidly declined GTPase that regulates COPII vesicle budding from the within 24 min to very low levels. In contrast, the cytoplas- endoplasmic reticulum (ER) to the Golgi apparatus mic ANF-GFP levels remained constant and low. There- (reviewed in Bonifacino and Glick, 2004). In wild-type fore, the dramatic drop in luminal ANF-GFP intensity high- embryos, the bulk of luminal markers 2A12, Verm, and lights a wave of luminal protein clearance. Gasp has been deposited inside the DT lumen by stage How does the dynamic ANF-GFP deposition and clear- 15 (Figures 2A, 2D, and 2G). However, in zygotic sar1P1 ance relate to endogenous luminal markers? Live imaging mutants (hereafter referred to as sar1), luminal secretion of embryos expressing the luminal protein Gasp tagged to of 2A12, Verm, and Gasp was incomplete. The tracheal GFP (Barry et al., 1999) shows that Gasp-GFP first accu- cells outlined by GFP-CAAX partially retained those mulates in the lumen and is then removed similarly to markers in the cytoplasm (Figures 2B, 2E, and 2H). sar1 ANF-GFP (Figures 1C and 1D; Movies S3 and S4). The zygotic mutant embryos normally deposited the early dynamic localization of GFP-tagged variants of Verm or luminal marker Piopio by stage 13 (Jazwinska et al., Serp was identical (data not shown). Furthermore, analy- 2003). Luminal chitin was also detected in sar1 mutants sis of fixed embryos shows that endogenous Verm and at stage 15. However, the luminal cable was narrow, Gasp are first deposited and then cleared from the tra- more dense (Figure S3), and distorted compared to wild- cheal lumen very much like ANF-GFP (Figure S1). Finally, type (Figures 3E and 3F). To test if the sar1 secretory phe- we fixed btl > ANF-GFP embryos at set times before and notype in the trachea is cell autonomous, we reexpressed after the luminal ANF-GFP clearance (18 hr and 20 hr after Sar1 specifically in the trachea of sar1 mutants by using egg laying [AEL]) for Transmission Electron Microscopy btl-GAL4. Such embryos showed largely restored secre- (TEM). Also, this assay showed that the luminal chitinous tion of 2A12, Verm, and Gasp (Figures 2C, 2F, and 2I). cable disappeared within the interval of ANF-GFP clear- Thus, we conclude that tracheal sar1 is required for the
216 Developmental Cell 13, 214–225, August 2007 ª2007 Elsevier Inc. Developmental Cell Epithelial Transitions Drive Airway Maturation
Figure 3. Defective ER and Golgi in sar1P1 Embryos P1 (A–D0) Confocal sections of stage-14 (A–B0) wild-type and (C–D0) sar1 mutant embryos expressing btl > GFP-CAAX (magenta). Embryos were stained with anti-KDEL for ER (white in [A] and [C] and green in [A0] and [C0]) and with anti-gp120 for Golgi (white in [B] and [D] and green in [B0] P1 and [D0]). sar1 mutant embryos show a (C and C0) disruption of ER and a (D and D0) marked decrease of the Golgi staining in the tracheal cells. (E and F) TEM sections of DT of late stage-16 (E) wild-type and (F) sar1P1 embryos. (E) Wild-type tracheal cells show a tubular organization of the rough ER, tightly lined by dark ribosomes (white arrowheads). (F) In sar1P1 mutants, tracheal cells show a disrupted and bloated rough ER organization (orange arrowheads). Black arrowheads mark the luminal electron-dense extracellular matrix. (F) Note its distortion in the mutant.
Scale bars are 10 mm in (A)–(D0) and 1 mm in (E) and (F). efficient secretion of luminal markers, which are predicted opment, zygotic Sar1 is required for efficient luminal to associate with the growing intraluminal chitin matrix. secretion.
Zygotic sar1 Mutants Have Reduced Sar1 Function Sar1 Localizes to the ER and Is Required for ER in the Trachea and Golgi Integrity sar1 mRNA has been reported to be abundantly mater- Given the conserved role of Sar1 in vesicle budding from nally contributed (Zhu et al., 2005). At later stages, zygotic the ER, we determined its subcellular localization in the expression of sar1 mRNA is initiated in multiple epithelial trachea by using anti-Sar1 antibodies. Sar1 localizes tissues (Abrams and Andrew, 2005). To monitor Sar1 zy- predominantly to the ER (marked by the PDI-GFP trap) gotic expression in the trachea, we used a Sar1-GFP pro- (Figure S2). Continuous COPII-mediated transport from tein trap line (Wilhelm et al., 2005). Embryos carrying only the ER is required to maintain the Golgi apparatus and paternally derived Sar1-GFP show a strong zygotic ex- ER structure (Altan-Bonnet et al., 2004; Yamanushi pression of GFP in the trachea (Figure S2). We also used et al., 1996). To test if zygotic loss of Sar1 compromises an anti-Sar1 antibody to analyze Sar1 expression in the the integrity of the ER and Golgi in tracheal cells, we trachea of wild-type, zygotic sar1P1, and sar1EP3575D28 stained sar1 mutant embryos with antibodies against null mutant embryos (Zhu et al., 2005)(Figure S2). Both zy- KDEL (marking the ER lumen) and gp120 (highlighting gotic mutants showed a clear reduction, but not complete Golgi structures). In sar1 mutant embryos, we observed elimination, of Sar1 expression in the trachea. To test the a strongly disrupted ER structure and loss of Golgi staining effects of a more complete inactivation of Sar1, we gener- in DT cells at stage 14 (Figures 3A–3D). Additionally, TEM ated transgenic flies expressing a dominant-negative of stage-16 wild-type and sar1 mutant embryos showed sar1T38N form in the trachea (Kuge et al., 1994). In btl > a grossly bloated rough ER structure in DT tracheal cells sar1T38N-expressing embryos, we observed early defects (Figures 3E and 3F). Consistent with its functions in yeast in tracheal branching and epithelial integrity as well as and vertebrates, Drosophila Sar1 localizes to the ER and is a complete block in Verm secretion (Figure S2). In contrast not only required for efficient luminal protein secretion, but to btl > sar1T38N-expressing embryos, zygotic sar1P1 also for the integrity of the early secretory apparatus. mutant embryos show normal early tracheogenesis with no defects in branching morphogenesis and epithelial Zygotic sar1 Is Selectively Required for Luminal integrity (Figure S3). Diameter Expansion In summary, tracheal expression of Sar1 is markedly To analyze tracheal maturation defects in sar1 mutant reduced in zygotic sar1 mutant embryos. While maternally embryos, we generated and imaged sar1 strains that carry supplied Sar1 is sufficient to support early tracheal devel- either btl > ANF-GFP btl-mRFP-moe or btl > Gasp-GFP
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Figure 4. sar1 Is Selectively Required for Tube Diameter Expansion (A and B) Frames from two time-lapse confocal movies showing the luminal secretion burst (0 and 55 min) and diametic expansion (225 min) in (A) wild-type and (B) sar1P1 embryos expressing btl > ANF-GFP (green) and btl-mRFP1-moe (magenta) (Movie S7). (C and D) TEMs of DT cross-sections from late stage-16 (C) wild-type and (D) sar1P1 mutant embryos. (E–G) y-z confocal projections of the DT at metamere 6 in late stage-16 (E) wild-type, (F) sar1P1, and (G) btl > sar1.1; sar P11 rescued embryos. (H and I) The DT diameter expansion rates at metamere 6 in live embryos. Diameter growth in sar1P1 mutants is significantly reduced compared to wild-type embryos (p < 0.001). Tube elongation rates in sar1P1 are indistin- guishable from wild-type. Bars are means ± SEM. (J) sar1P1 embryos are liquid-clearance defec- tive. Transgenic expression of Sar1 specifically in the trachea of sar1P1 embryos significantly rescued the liquid-clearance phenotype (p < 0.001). Bars are means ± SEM. Scale bars are 10 mm in (A), (B), and (E)–(G) and 1 mm in (C) and (D).
(Movies S6 and S7). In sar1 mutants, luminal deposition of pleted luminal liquid clearance, suggesting that efficient both ANF-GFP and Gasp-GFP is reduced (Figures 4A and tracheal secretion and the integrity of the secretory appa- 4B; Figure S4). Like endogenous Gasp in the mutants, ratus are prerequisites for later tube maturation steps Gasp-GFP was clearly retained in the cytoplasm of sar1 (Figure 4J). Taken together, the above-described results embryos. ANF-GFP was also retained in the tracheal cells show that tracheal Sar1 is selectively required for tube of sar1 mutants, but to a lesser extent. Strikingly, sar1 diameter expansion. Additionally, subsequent luminal mutants failed to fully expand the luminal diameter of protein and liquid clearance fail to occur in sar1 mutants. the DT outlined by the apical RFP-moe localization (Figure 4B). We quantified this defect by measuring dia- metric growth rates in metamere 6 for wild-type (n = 11) Mutants in COPII Subunits Phenocopy sar1 and sar1 mutant (n = 7) embryos. While early lumen expan- Secretion and Diameter Phenotypes sion commences in parallel in both genotypes, the later di- Do the tracheal defects of sar1 reflect a general require- ametric growth of sar1 mutants falls significantly behind ment for the COPII complex in luminal secretion and diam- compared to wild-type embryos. The DT lumen in sar1 eter expansion? To test this, we analyzed lethal P element mutants reaches only an average of 70% (4.05 mm ± 0.2 insertion alleles disrupting two additional COPII coat sub- SEM) of the wild-type diameter (5.75 mm ± 0.2) at early units, sec13 and sec23 (Abrams and Andrew, 2005). We stage 16 (Figure 4H). We detected identical diametric stained mutant sec13 and sec23 embryos for luminal growth defects in fixed sar1 mutant embryos expressing Gasp and for Crb and a-Spectrin to highlight tracheal btl > GFP-CAAX by analysis of confocal yz sections or cells. At stage 15, embryos of both mutants show a clear TEM (Figures 4C–4F). Reexpression of sar1 in the trachea cellular retention of Gasp (Figure S5). Furthermore, stage- of sar1 mutant embryos not only rescued secretion, but 16 sec13 and sec23 embryos show significantly narrower also the lumen diameter phenotype at stage 16 DT tubes when compared to wild-type. The average diam- (Figure 4G). In contrast to the diametric growth defects, eter of the DT branches in metamere 6 was 4.8 mm (±0.2 DT tube elongation in sar1 embryos was indistinguishable mm) and 4.4 mm (±0.1 mm) in fixed sec13 and sec23 from that in wild-type (Figure 4I). This demonstrates dis- embryos, respectively, compared to 6.3 mm (±0.1 mm) in tinct genetic requirements for tube diameter and length wild-type (Figure S5). Therefore, sec13 and sec23 mutants growth. It also reveals that the sar1 DT luminal volume rea- phenocopy sar1. The phenotypic analysis of three inde- ches less than half of the wild-type volume. Prolonged live pendent mutations disrupting ER-to-Golgi transport imaging showed that sar1 mutants are also completely de- thus provides a strong correlation between deficits in ficient in luminal protein and liquid clearance (Figure 4J; luminal protein secretion and tube diameter expansion Figure S4). Up to 80% of the rescued embryos also com- (Figure 7A).
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Luminal Protein Clearance Depends on Tracheal Rab5 Activity and Endocytosis Anticipating that the wave of luminal protein clearance may depend on endocytic uptake, we screened several mutations with known defects in endocytosis. To directly assess luminal protein clearance, we generated strains of endocytic mutants carrying btl > ANF-GFP and ana- lyzed them live. We found that clathrin heavy chain1 (chc1), shibirets2 (shits2 at the restrictive temperature of 2 32C), and rab5 null mutant embryos all display profound defects both in luminal protein and liquid clearance (Figure S6). Chc is the heavy chain of the Clathrin coat (Bazinet et al., 1993), and shi codes for the Dynamin GTPase that clips Clathrin-coated pits off the membrane to form endocytic vesicles (van der Bliek and Meyerowitz, 1991). rab5 encodes for a small GTPase that is a key reg- ulator of early endocytosis (Wucherpfennig et al., 2003). We focused our further analysis on rab5, since rab52 showed the most penetrant defects in protein clearance (Figure S6). To follow luminal protein clearance live, we imaged wild-type and rab52 mutant embryos expressing ANF-GFP or Gasp-GFP. rab52 mutant embryos failed to eliminate both luminal GFP markers (Figures 5A–5D; Movies S8–S10). In contrast to wild-type, rab52 mutants also retained substantial quantities of endogenous luminal Gasp, Verm, chitin, and 2A12 antigen inside the tracheal tubes (Figures 5E–5H; Figure S6). When we expressed GFP-rab5 specifically in the trachea of rab5 mutant embryos, luminal clearance was rescued significantly Figure 5. Luminal Protein Clearance Requires Rab5-Medi- (Figure 5I). Our analysis thus shows that rab5 is required ated Endocytosis in the trachea for proper clearance of luminal proteins (A and B) Selected frames from a time-lapse confocal movie showing 1 ts2 and chitin. The additional analysis of chc and shi the clearance of ANF-GFP (green) in a live (A) wild-type and a (B) rab52 mutant embryos argues that not only Rab5, but endocyto- embryo. DT tracheal cells were marked by btl > myr-mRFP1 (ma- sis in general, is required for the clearance of luminal genta). Wild-type embryos clear ANF-GFP from the lumen while 2 solids. rab5 embryos retain it (Movie S8). (C and D) Wide-field images from a time-lapse movie of a (C) wild-type and a (D) rab52 mutant embryo expressing Gasp tagged with GFP (btl > Normal Secretory Capacity and Epithelial Integrity, Gasp-GFP) (Movies S9 and S10). rab52 embryos retain Gasp-GFP but Defective Endosomal Structures, in rab5 Mutants inside the lumen. We examined whether the clearance phenotypes of rab5 (E–H) Confocal sections of DT in late stage-17 (E and F) wild-type and mutants may be secondary to disruptions of either secre- (G and H) rab52 mutant embryos stained with (E and G) Verm and (F 2 tion (as for sar1) or loss of epithelial integrity. Apart from and H) Gasp (white). rab5 mutant embryos retain endogenous luminal a minor and partially penetrant tube elongation pheno- antigens at late stage 17. 2 (I) Plot showing the percentage of liquid-clearance defects in wild- type, rab5 mutants develop an essentially wild-type type, rab52, and rab52; btl > GFP-rab5 embryos. There is a significant tracheal network. They show normal secretion of luminal reduction of defects in rab52; btl > GFP-rab5 (rescue embryos) com- markers at stage 15, as well as intact tracheal epithelial pared to rab52 embryos (p < 0.001, two-tailed t test). Bars represent polarity at stage 16. We also analyzed DTs of late stage- means ± SEM. 16 rab52 mutant embryos by TEM. These embryos display Scale bars are 10 mm. a wild-type cellular ultrastructure and apical luminal lining in the DT tubes (Figure S7). Finally, we performed a dextran injection assay that showed that rab52 mutant embryos 2x-FYVE, recycling endosomes (RE) by using GFP- have intact paracellular junctions (Figure S9). Thus, Rab11, and late endosomes (LE) by using GFP-Rab7 zygotic rab5 mutant embryos show an overall normal (Emery et al., 2005; Wucherpfennig et al., 2003). For the tracheal development and secretion. latter three markers, the analysis of live and fixed embryos We tested the integrity of several distinct endosomal yielded identical results. compartments in rab52 mutants. We visualized Clathrin- We found no changes between wild-type and rab52 coated vesicles (CCV) by using anti-Chc antibodies in mutant embryos in the predominantly apical localization fixed embryos. We also used GFP-tagged reporters of GFP-Rab11-positive REs (Figure S8). The remaining expressed in the trachea to analyze early endosomes endocytic markers were also unaffected during tracheal (EE) and multivesicular bodies (MVB) by using GFP-myc- development in rab52 mutant embryos up to early stage
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17. However, at mid-stage 17, during the period of luminal We analyzed the distribution of intracellular Gasp protein clearance, the cellular distribution of Chc, GFP- puncta relative to endosomal markers in the trachea of FYVE, and GFP-Rab7 was clearly aberrant in rab52 mutant fixed embryos. We tested the following genotypes of embryos. embyos for colocalization: btl > Clathrin light chain-GFP In wild-type mid-stage-17 embryos, the majority of Chc (Clc-GFP) for CCVs, btl > GFP-Rab5 for EEs, btl > GFP- was localized apically in a discrete, dotted pattern. In the Rab7 for LEs, and btl > GFP-LAMP1 (GFP-Lysosomal DT of rab52 mutants, this discrete localization of Chc was Associated Membrane Protein 1) for lysosomes (Chang lost (Figure S8). Live imaging of btl > GFP-FYVE embryos et al., 2002; Pulipparacharuvil et al., 2005). While intracel- at mid-stage 17 reveals the dynamic movement of large, lular Gasp staining only occasionally colocalized with GFP-FYVE-positive apical structures in the tracheal cells GFP-Rab5-positive endosomes, Gasp puncta more in a background of small endosomal vesicles. Strikingly, consistently colocalized with Clc-GFP, GFP-Rab7, and GFP-FYVE labeling of those apical structures was absent GFP-LAMP1 staining (Figure S9). Thus, considerable por- in rab52 mutants, leaving few, small and randomly distrib- tions of intracellular Gasp staining coincide with CCVs, uted vesicles (Figure S8; Movie S11). The lack of the LEs, and lysosomal markers, suggesting that Gasp may extensive GFP-FYVE-positive structures in the mutants traverse those endosomal compartments. The lysosomal may either reflect the function of Rab5 generating the localization of intracellular Gasp indicates that Gasp may FYVE ligand phosphatidylinositol-3-phosphate or a direct be degraded inside tracheal cells during clearance of solid role of Rab5 in endosomal assembly and growth (Christo- luminal material. foridis et al., 1999). In wild-type, GFP-Rab7 highlights late While the detection of endogenous Gasp in endosomal endosomal structures spread along the apicobasal axis of structures during stage 17 argues for a direct role of endo- the tracheal cells. In most rab5 mutant embryos, the abun- cytosis in tube clearance, the localization of intracellular dance of GFP-Rab7 endosomes was decreased or com- Gasp puncta to specific endosomal compartments was pletely lost (Figure S8). relatively infrequent. To visualize the cellular dynamics Taken together, rab5 mutants show normal epithelial de- during luminal protein clearance directly, we established velopment and secretion in the trachea, but aberrant a live endocytosis assay in the trachea by using the localization and abundance of Chc-, GFP-FYVE-, and fluid-phase marker dextran (Swanson, 1989). We injected GFP-Rab7-positive structures at mid-stage 17. This 10 kDa dextran into the hemocoel of stage-13 to stage-14 suggests that Rab5 either directly mediates or indirectly embryos, which lack the paracellular barrier function pro- facilitates the formation and intracellular organization of vided by fully assembled septate junctions (Tepass and CCVs and LEs. The manifestation of endocytic phenotypes Hartenstein, 1994). In these experiments, the fluorescent in the trachea of rab52 mutants precisely correlates with the dextran consistently accumulated inside the tracheal lu- initiation of solid luminal material clearance in the wild-type. men of wild-type embryos (Figure 6D). Taking advantage of this assay, we preloaded the trachea of btl > ANF- The Tracheal Epithelium Directly Internalizes GFP embryos with rhodamine-labeled dextran and live and Clears Luminal Material by Endocytosis imaged ANF-GFP along with the injected marker. Both ANF- Our mutant analysis shows that endocytosis is required GFP and dextran were cleared from the DT lumen simulta- for luminal material clearance. Protein trafficking through neously (Movie S12), suggesting that exogenous luminal endocytic compartments may either directly absorb and dextran is cleared similarly to other luminal constituents. clear luminal proteins or indirectly facilitate the extinction Is dextran taken up into tracheal cells during luminal of luminal material. If the tracheal epithelium directly protein clearance? We injected 10 kDa dextran into early internalizes luminal material by endocytosis, we expect embryos carrying the tracheal btl > GFP-CAAX marker that: (1) tracheal cells take up luminal markers, and that and analyzed them. We initially detected only few intracel- (2) luminal markers colocalize with endosomal structures lular puncta in such embryos at stage 16 (Figure 6H and inside the tracheal cells during tube clearance. data not shown). However, from early to mid-stage 17 To test if endogenous proteins are taken up into tracheal and before the drop in luminal fluorescent intensity, the cells during luminal solid clearance, we used an amplified number of rhodamine-dextran puncta distinctly increased staining protocol that allows for the detection of the lumi- inside tracheal cells (Figure 6D, 0 min and 28 min). Upon nal marker Gasp in mid-stage-17 embryos. gasp mutants luminal clearance completion (late stage 17), the count were devoid of any Gasp staining throughout tracheal of intracellular dextran puncta was diminished to basal development (Figure S9; K.T. and C.S., in preparation). levels (Figure 6D, 50 min). Quantification of intracellular In early stage-16 embryos marked by btl > GFP-CAAX, dextran puncta demonstrates a dynamic peak of dextran Gasp localized almost completely inside the lumen of internalization just before and during luminal protein clear- the DT (Figure 6A). From early to mid-stage 17, a large ance (Figure 6H). number of intracellular Gasp puncta became apparent in Since not all of the injected dextran is taken up into the tracheal cells (Figure 6B; Figure S9). At late stage 17, the tracheal lumen, the detected intracellular dextran the remaining Gasp was mainly localized along the apical signals may derive either from the lumen or the hemocoel. tube lining (Figure 6C). Thus, intracellular Gasp levels To test the origin of intracellular dextran, we injected transiently increase before and during the period of lumi- rhodamine-labeled dextran into the hemocoel of late nal protein clearance. stage-16 embryos. These embryos display fully developed
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Figure 6. Trachea Cells Directly Remove Luminal Material through Endocytosis
(A–C0) (A–C) Confocal xy sections and yz pro- jections of DT of wild-type embryos expressing btl > GFP-CAAX at (A) stage 16, (B) mid-stage 17, and (C) late stage 17. yz confocal sections correspond to the DT position indicated by thin, vertical lines in xy sections. Embryos were stained with anti-Gasp (green) and anti-
GFP (magenta). (A0–C0) Gasp staining (white) of the images in (A), (B), and (C). The number of intracellular Gasp puncta (arrowheads) in- creases during the period of luminal protein clearance at mid-stage 17 and decreases af- terwards. (D and E) Frames from a time-lapse confocal analysis before (0 min), during (28 min), and af- ter (50 min) protein clearance of a dextran-pre- loaded (D) wild-type and a (E) rab52 live embryo expressing btl > GFP-CAAX and btl > GFP- CD8, respectively. Intracellular puncta of lumi- nal dextran are present just before (0 min) and during (28 min) luminal protein clearance in wild-type (arrowheads), but not in rab52 mu- tant, embryos. Luminal dextran clearance is defective in rab52 embryos (n = 11).
(F–G0) Images from confocal time-lapse analy- sis of dextran-injected embryos expressing the endosomal markers (F) btl > GFP-Rab7 and (G) btl > GFP-FYVE, just before protein clearance. A proportion of intracellular dextran puncta localize to GFP-Rab7- or GFP-FYVE-positive endosomes (arrowheads). (H) The dynamics of endocytosed tracheal puncta during late embryonic development. A significant increase in the number of internal- ized dextan occurs before luminal protein clearance (17–19 hr AEL). The green arrow indi- cates the time of luminal clearance. Values rep- resent mean and standard error from five inde- pendent experiments. For all developmental time points n = 24, except for 13 hr AEL, where n = 8. The asterisk denotes p < 0.01 by Stu- dent’s t test comparing the data sets between two time points. Bars represent means ± SEM. Scale bars are 10 mm.
paracellular barriers that prevent the luminal access of the luminal protein clearance (Figure 6E). Thus, tracheal cells 10 kDa dextran (Lamb et al., 1998). Importantly, we did not internalize the preloaded dextran from the lumen in find any fluorescent signal inside the tracheal cells at mid- a Rab5-dependent manner exactly before and during stage 17 in such embryos (Figure S9). This demonstrates the peak of luminal solid clearance. that the transient intracellular dextran puncta in embryos To test if intracellular dextran dots localize to endo- preloaded at stage 13 indeed derive from the lumen. We somes, we expressed GFP-Rab5, GFP-2x-FYVE, and next asked if dextran internalization requires Rab5-depen- GFP-Rab7 in the trachea under the control of btl-GAL4. dent endocytosis. We injected rhodamine-labeled dextran Embryos of each genotype were preloaded with 10 kDa into rab52 mutant embryos with tracheal cells labeled by rhodamine-labeled dextran and analyzed live during its btl > CD8-GFP. A total of 56% of rab52 mutant embryos luminal clearance. While localization of dextran to GFP- (n = 11) failed to show the characteristic transient peak Rab5-marked EEs was very rare (data not shown, 1.3%, of intracellular dextran accumulation during the period of n = 8 embryos), the proportion of dextran puncta localizing
Developmental Cell 13, 214–225, August 2007 ª2007 Elsevier Inc. 221 Developmental Cell Epithelial Transitions Drive Airway Maturation
DISCUSSION
Our live-imaging approach defines the developmental dynamics of functional tracheal maturation. At the organ level, we identified three sequential and rapid develop- mental transitions: (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 imag- ing 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 com- parison between wild-type and mutant embryos further highlights the dynamic nature of epithelial activity during each pulse. We identified several mutations that selectively disrupt distinct cellular functions and concurrently interrupt the maturation process at specific steps. This clearly demon- strates the significance of phenotypic transitions in epithe- lial organ maturation and establishes that secretion is required for luminal diameter expansion and endocytosis for solid luminal material clearance.
The Secretory Burst and Tube Diameter Expansion The sudden initiation of an apical secretory burst tightly Figure 7. Distinct Pulses of Secretion and Endocytosis Drive precedes diametric tube expansion. The completion of Tracheal Airway Maturation both events depends on components of the COPII com- (A and B) Schematic illustration of a cross-section through a DT at (A) plex, further suggesting that the massive luminal secretion stage 14 and at (B) early stage 17. (A) The ER, Golgi, and post-Golgi is functionally linked to diametric growth. How does apical vesicles carry luminal proteins (green) into the lumen. COPII vesicular transport from the ER to the Golgi is required for efficient luminal secre- secretion provide a driving force in tube diameter expan- tion and diameter expansion. (B) After diametric expansion, tracheal sion? In mammalian lung development, the distending cells internalize and clear luminal content (green) by Rab5-dependent internal pressure of the luminal liquid on the epithelium ex- endocytosis. pands the lung volume and stimulates growth. ClÀ chan- (C) Schematic model of airway maturation based on live-imaging and nels in the epithelium actively transport ClÀ ions into lumi- mutant analysis. The solid luminal content is indicated in green. The nal liquid. The resulting osmotic differential then forces initial secretion burst extends into a massive, continued secretion pulse (orange) of chitin-binding proteins that fill the lumen. The water to enter the lung lumen, driving its expansion (Olver COPII-dependent secretion pulse is essential for luminal matrix as- et al., 2004). By analogy, the tracheal apical exocytic burst sembly and tube diameter expansion. At early stage 17, the tracheal may insert protein regulators such as ion channels into the epithelium activates a pulse of endocytic activity (blue) that internalizes apical cell membrane or add additional membrane to the and removes solid contents from the lumen. The endocytosis pulse growing luminal surface. Since the ER is a crucial cellular requires the function of Rab5, Clathrin, and Dynamin. The two pulses compartment for intracellular traffic and lipogenesis, its represent peaks of cellular activity above the background of basal cellular secretion and endocytosis, which, for simplicity, are not shown disruption in sar1 mutants may disrupt the efficient trans- in the model. port of so far unknown specific regulators or essential apical membrane addition required for diametric expan- sion. Alternatively, secreted chitin-binding proteins (ChB) may direct an increase of intraluminal pressure and tube to GFP-FYVE-positive EE and MVBs, and to GFP-Rab7- dilation. Overexpression of the chitin-binding proteins marked LEs, was 8.0% and 31%, respectively (n = 5) Serp-GFP or Gasp-GFP was insufficient to alter the dia- (Figure 6F). metric growth rate of the tubes, suggesting that lumen In summary, our data show that endogenous Gasp and diameter expansion is insensitive to increased amounts injected rhodamine-labeled dextran are internalized by of any of the known luminal proteins (unpublished data). tracheal cells before and during the clearance interval. In sar1 mutants, the secretion of at least two chitin-binding Both markers localize to endosomal structures in tracheal proteins, Gasp and Verm, is reduced. Chitin, however, is cells (Figure 7B). We conclude that the tracheal epithelium deposited in seemingly normal quantities, but assembles directly removes luminal Gasp protein and the preloaded into an aberrantly narrow and dense chitinous cable. dextran polymers by a transient, Rab5-dependent, This phenotype suggests that the correct ratio between endocytic wave. chitin and multiple interacting proteins may be required
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for the correct assembly of the luminal cable. Interestingly, dextran in rab5 mutants provides further loss-of-function sar1, sec13, and sec23 mutant embryos form a severely evidence for Rab5 function in fluid-phase endocytosis in defective and weak epidermal cuticle (Abrams and vivo. The above-described arguments lead us to specu- Andrew, 2005). The luminal deposition of ChB proteins late that additional Rab5-regulated endocytic mecha- during the tracheal secretory burst may orchestrate the nisms most likely cooperate with CME in the clearance construction and swelling of a functional matrix, which, of solid luminal material. in turn, induces lumen diameter dilation. While we favor How is liquid cleared from the lumen? While we still this later hypothesis, we cannot exclude that other mech- know very little about this fascinating process (Kallis, anisms, either separately or in combination with the dilat- 1939), some developmental and mechanistic arguments ing luminal cable, drive luminal expansion (Figure 7A). suggest that this last maturation step is mechanistically distinct. First, the interval of luminal liquid clearance is Endocytotic Clearance of Luminal Material clearly distinct from the period of endocytic clearance of During tube expansion, massive amounts of luminal mate- solids. Second, the dynamic internalization of dextran rial, including the chitinous cable, fill the tracheal tubes. and the abundance of GFP-marked endocytic structures We found that Dynamin, Clathrin, and the tracheal function decline before liquid clearance. Finally, our assessment of Rab5 are required to rapidly remove luminal contents, of liquid clearance further suggests that it requires a dis- indicating that endocytosis is required for this process. tinct cellular mechanism (K.-A.S. and V.T., unpublished Several lines of evidence argue that the tracheal epithe- data). lium activates Rab5-dependent endocytosis to directly Viewing the entire process of airway maturation in internalize luminal material. First, the tracheal cells of conjunction, some general conclusions may be drawn. rab5 mutants show defects in multiple endocytic compart- First, the three epithelial pulses are highly defined by their ments. These phenotypes of rab5 mutants become appar- sequence and exact timing, suggesting that they may be ent during the developmental period matching the interval triggered by intrinsic or external cues. Second, the analy- of luminal material clearance in wild-type embryos. Sec- sis of mutants that selectively reduce the amplitude of the ond, tracheal cells internalize two luminal markers, the secretory or endocyic pulses demonstrates the require- endogenously encoded Gasp and the dextran reporter, ment for each epithelial transition in the completion of exactly prior and during luminal protein clearance. The the entire maturation process. These pulses are induced number of intracellular dextran puncta reaches its peak in the background of basal secretory and endocytic activ- during the clearance process and ceases shortly thereaf- ities that operate throughout development. Third, specific ter. Lastly, intracellular puncta of both Gasp and dextran cellular activities exactly precede each morphological colocalize with defined endocytic markers inside tracheal transition. Finally, the separate transitions are interdepen- cells. The colocalization of Gasp and dextran with GFP- dent in a sequential manner. Efficient secretion is a prereq- Rab7 and of Gasp with GFP-LAMP1 suggests that the uisite for the endocytic wave. Similarly, protein endocy- luminal material may be degraded inside tracheal cells. tosis is a condition for luminal liquid clearance. This Taken together, our data show that the tracheal epithelium suggests a hierarchical coupling of the initiation of each activates a massive wave of endocytosis to clear the pulse to completion of the previous one in a strict develop- tubes (Figure 7B). mental sequence (Figure 7C). Endocytic routes are defined by the nature of the inter- Our study provides a striking example of how pulses of nalized cargoes and the engaged endocytic compart- epithelial activity drive distinct developmental events and ments (Conner and Schmid, 2003). What may be the mold the nascent tracheal lumen into an air delivery tube. features of the endocytic mechanisms mediating the Our findings are likely to be relevant beyond the scope of clearance of luminal material? The phenotype of chc tracheal development. The uniform growth of salivary mutants and the presence of intracellular Gasp in CCVs gland tubes in flies and the excretory canal and amphid indicate that luminal clearance at least partly relies on Cla- channel lumen in worms also require the assembly of a thrin-mediated endocytosis (CME). In addition to CME, luminal matrix for uniform tube growth (Abrams et al., Dynamin and Rab5 have also been implicated in other 2006; Perens and Shaham, 2005). Luminal material is routes of endocytosis, suggesting that multiple endocytic also transiently present during early developmental stages mechanisms may be operational in tracheal maturation. in the distal nephric ducts of lamprey (Youson, 1984). The nature of the endocytosed luminal material provides Thus, the coordinated, timely deposition and removal of an additional perspective. While cognate uptake recep- transient luminal matrices may represent a general mech- tors may exist for specific cargos such as Gasp, Verm, anism in tubulogenesis. and Serp, the heterologous ANF-GFP, degraded chitin, and the fluid-phase marker dextran may be cleared by either fluid-phase internalization or multifunctional scav- EXPERIMENTAL PROCEDURES enger receptors. Interestingly, Rab5 can regulate fluid- Genetics phase internalization in cultured cells by stimulating The P element alleles l(3)s009124 (sar1P1 in text) and l(3)05712 fail to macro-pinocytosis and the activation of Rabankyrin-5 complement the nulls sar1EP3575D28, D71 and the GFP trap sar1CA07674. (Bucci et al., 1992; Schnatwinkel et al., 2004; Stenmark Insertion sites were determined by plasmid rescue and PCR. Precise et al., 1994). The defective tracheal internalization of excision of sar1P1 reverted tracheal phenotypes. The COPII subunit
Developmental Cell 13, 214–225, August 2007 ª2007 Elsevier Inc. 223 Developmental Cell Epithelial Transitions Drive Airway Maturation
mutations are l(3)01031 (sec13) and l(3)j13C8 (sec23). For more strain points, 15 s apart, were estimated for each embryo. The measurements information, see the Supplemental Data. were assembled with AxioVision 4.5. Intracelllar puncta of dextran were assessed from Z stacks of confocal sections (DT metamere 7). Molecular Biology PCR subcloning was used to generate pET28a His-Gasp (14–189 aa) Supplemental Data for recombinant protein expression and pUAST-Gasp-GFP construc- Supplemental Data include 12 movies, 9 figures, Supplemental Exper- tion from clones RH12464 and RH10284, respectively. Purified imental Procedures, and Supplemental References and are available at recombinant Gasp was used to immunize guinea pigs. pUAST-sar1 http://www.developmentalcell.com/cgi/content/full/13/2/214/DC1/. and pUAST-sar1T38N were generated by subcloning of RE74312 and PCR mutagenesis. ACKNOWLEDGMENTS
Immunostaining, TEM, and Dextran Injections We would like to thank David L. Deitcher, Marcos Gonza´ lez-Gaita´ n, Immunohistochemistry, TEM and 10 kDa dextran (Molecular Probes) Markus Affolter, Maria Leptin, Rick Fehon, Alain Debec, Ju¨ rgen Kno- injections was performed as described (Lamb et al., 1998; Wang blich, Helmuth Kra¨ mer, Allan Spradling, Greg Beitel, the Bloomington et al., 2006). Additional antibodies used were: guinea pig anti-Coracle Drosophila Stock Center, the Drosophila Genomics Resource Center (from R.G. Fehon), mouse mAb anti-gp120/Golgi (clone7H6D7C2, (DGRC; Indiana), and the Developmental Studies Hybridoma Bank Calbiochem), mouse mAb anti-KDEL (clone10C3, Nordic Biosite), (DSHB; Iowa) for fly strains, clones, and antibodies. We would like to goat anti-Clathrin (Sigma), chicken anti-Human Sar1 (cprosci Inc.), thank Monika Bjo¨ rk and Inger Granell for expert technical assistance. and guinea pig anti-Gasp. DmSar1 protein is 70% identical to HsSar1. We are grateful to members of the Samakovlis lab, Harald Stenmark, Anti-Gasp staining was amplified using a secondary goat anti-guinea Elisabeth Knust, and Gabriele Senti for discussions and critical com- pig and tertiary donkey anti-goat antibodies. ments on the manuscript. K.-A.S. acknowledges a European Molecu- lar Biology Organization long-term fellowship. This work was funded Live Imaging by grants of National Research Council (VR), SSF, and WCN to C.S. For wide-field live imaging, dechorionated embryos were mounted on a gas-permeable membrane stretched over two silicon bars on the top Received: April 23, 2007 of a slide. Embryos were imaged on an Axioplan2 microscope by using Revised: June 15, 2007 either a x20/0.5NA Plan-Neofluar or a x63/1.3NA C-Apochromat Accepted: June 18, 2007 water-immersion objective and an attached CCD camera controlled Published: August 6, 2007 by the AxioVision software 4.5. 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Developmental Cell 13
Supplemental Data
Sequential Pulses of Apical
Epithelial Secretion and Endocytosis
Drive Airway Maturation in Drosophila
Vasilios Tsarouhas, Kirsten-André Senti, Satish Arcot Jayaram, Katarína Tiklová, Johanna Hemphälä, Jeremy Adler, Christos Samakovlis
Figure S1. Luminal Deposition and Clearance of ANF-GFP Matches Endogenous Verm and Gasp (A-H) Confocal micrographs of embryos expressing btl>ANF-GFP stained for GFP (green) (A)-(H), Verm (A)-(D) and Gasp (E)-(H) (magenta). ANF-GFP and Verm (A) and Gasp (E) are largely cytoplasmic prior to the secretory burst at stage 13 (A) and (E). Shortly thereafter at late stage 13, all three markers are detected in the lumen (B) and (F). At late stage 16, the markers are found predominantly in the lumen and along the apical
lining of trachea (C) and (G). At late stage 17, ANF-GFP, Verm and Gasp are absent from the luminal cavity, but remain localized to the apical lining of the tubes (D) and (H). Minor-moderate levels of ANF-GFP are present in the cytoplasm of the tracheal cells throughout late stage 16 till mid stage 17 (C), (D), (G), (H). (I and J) TEM sections of DT of a wild type embryo expressing btl>ANF-GFP. Embryos were fixed before the anticipated ANF-GFP clearance (I) and after disappearance of luminal fluorescence in (J). The electron dense luminal cable structure is removed during the luminal protein clearance. Scale bars 10 µm (A)-(H) and 1 µm (I), (J).
Figure S2. Sar1 Expression and Function in Different sar1 Mutant Conditions (A) Genomic organisation of sar1 locus and position of P element insertions. (B-C) Confocal micrographs of wild type (B) and btl>sar1DN embryos (C) stained with anti-Verm (green) and anti-GFP stainings. Trachea development is completely arrested in sar1DN before the formation of primary branches (white arrowhead) in (D).
(D-D’) Confocal section of an embryo with only paternally contributed sar1GFP-trap stained with anti-GFP (white) in (D) and (green) in (D’) and anti-Crb in (D’) (magenta). Tracheal cells show strong zygotic expression of Sar1. (E-G’) Confocal sections of Stage 15 wild type (E), (E’), sar1P1 (F), (F’) and zygotic null sar1EP3575∆28 embryos (G), (G’) expressing btl>GFP-CAAX stained for Sar1 and GFP. (E-G) show Sar1 staining alone (white) (E’-G’) represent merged images of Sar1 (green) and GFP staining (magenta). (H and H’) Sar1 localizes to the periphery of the ER lumen marked GFP staining of PDI- GFP trap embryos. Sar1 is labelled white in (H) and magenta in (H’). The ER lumen is visualized by anti-GFP staining in (H’) (green). Zygotic Sar1 expression was strongly reduced mutants of both sar1 alleles (F) and (G), suggesting that sar1P1 represents a strong hypomorph (F). Scale bars, 10 µm
Figure S3. sar1P1 Mutant Embryos Show Normal Epithelial Organization (A-L) Confocal micrographs show DTs of wild type (A), (C), (E)-(H) and sar1P1 mutant embryos (B), (D), (I)-(L). All micrographs are single confocal sections except (G) and (K), which represent projections. Trachea cells were labeled by the transgenic expression of btl>GFP-CAAX. (A-C) Localization of Piopio (green) at stage 13 (A), (B) and of chitin (white) at stage 15 (C), (D) Insets in (C) and (D) represent yz projections of (C) and (D). There are no luminal deposition defects or any excessive cellular accumulation apparent for either of
these luminal markers in sar1P1 embryos compared to wild type. Note that the chitinous cable is narrower and more dense in sar1 mutants (D) compared to wild type (C). (E-L) Epithelial organization assessed from embryos at early stage 16 by following staining for the apical marker Crb (E), (I) (green), adherens junction marker E-cad (F), (J) (green), projections of (F) and (J) are shown in (G) and (K) (white) and septate junction marker coracle (H), (L) (green). None of the stainings show discernable phenotypes in the localization or organization of epithelial markers in sar1P1 embryos. Further, no defects in the epithelial cell arrangements (like irregular cellular intercalation) were detected (G, K). Scale bars 10 µm.
Figure S4. sar1 Is Required for Gasp-GFP Secretion and ANF-GFP Clearance (A and B) Frames of widefield movies of a wild type (A) and sar1P1 mutant (B) embryos expressing btl>Gasp-GFP. While wild type embryos secrete Gasp-GFP efficiently and expand the luminal diameter normally, sar1P1 mutant embryos partially retain Gasp-GFP intracellularly (0 min and onwards) and fail to expand luminal diameter (90-180 min). Imaging started 10 hours AEL and acquired every 3 min for 5 hours. For simplicity the initiation of luminal secretion is presented as time 0 in the figure.
(C and D) Frames from widefield movies showing stage 17 wild type (C) and sar1P1 mutant embryos expressing btl>ANF-GFP (D) (white). Wild type embryos clear ANF- GFP from the lumen, while sar1P1 mutant embryos retain it. Scale bars 10 µm.
Figure S5. sec13 and sec23 Mutants Show Defects in Secretion and Luminal Diameter Expansion (A-F) Confocal sections of the DT of wild type (A), (B), sec13 (C), (E) and sec23 embryos (D), (F). Embryos were stained with anti-Gasp (green) and a mixture of anti-Crb and anti-α-Spectrin to show tracheal cells (magenta). sec13 and sec23 mutant embryos
retained a considerable amount of Gasp inside tracheal cells at stage 15 (C) and (E). At early stage 16, sec13 and sec23 (D), (F) embryos show narrower DT lumina when compared to wild type (B). (G) Lumen diameter of DT at metamer 6 was measured in wild type, sec13 and sec23 embryos at stage 16 using Crb staining as an apical cell marker. Embryos of both mutants show a significant reduced lumen diameter when compared to wild type embryos. Bars are means ± s.e.m. * denotes statistical significant differences at p< 0.0001 by a Student’s t test. Scale bars 10 µm
Figure S6. shits2 and chc1 Mutant Embryos Fail to Clear ANF-GFP and Luminal Liquid rab52 is Required for Clearance of Endogenous Luminal Proteins (A-C) Widefield microscope images of live embryos expressing btl>ANF-GFP depict a wild type (A), a shibirets2 (shits2) at restrictive temperature 32°C from 13 h to 16 h (B) and
a clathrin heavy chain1 (chc1) mutant embryo (C). shits2 and chc1 show strong defects in the luminal clearance of ANF-GFP at mid stage 17. (D) Quantification of the luminal protein and liquid clearance defects for wild type, rab52, shits2 and chc1. (E-H) Confocal sections of DT in late stage 17 wild type (E), (F) and rab52 mutant embryos (G), (H) stained with 2A12 (E), (G) and Chitin binding probe (CBP) (F), (H) (white). rab52 mutant embryos retain endogenous luminal antigens at late 17. Scale bars 10 µm.
Figure S7. rab52 Mutants Show Normal Luminal Marker Secretion and Epithelial Organization (A-O) Confocal sections show the DT of wild type (A)-(D), (I)-(K) and rab52 mutant embryos expressing btl>CD8-GFP (E)-(H), (M)-(O). Staining against GFP is shown in magenta in all panels. To analyze the secretion pulse, stage 15 embryos were stained for 2A12 (A), (E), Verm (B), (F), Gasp (C), (G) (green) and Chitin binding probe (white) (D), (H). rab52 mutants show no discernable defects in luminal secretion or luminal chitin deposition. To assess epithelial organization, we stained early stage 16 embryos for the apical domain marker Crb (I), (M), the AJ marker E-Cad (J), (N) and SJ marker Coracle (K), (O) all shown in green. None of the analysed epithelial markers showed any
apparent mislocalization in rab52 mutant embryos. However, a partially penetrant frequency of mild tube elongation phenotypes in rab52 mutant DT was noticed. (L-P) TEM of DT in late stage 16 wild type (L) and rab52 mutant (P) embryos shows that both genotypes display indistinguishable apical lining and overall cellular ultrastructure. Scale bars 10 µm (A)-(O) and 1 µm (L), (P).
Figure S8. Rab5 Is Required for the Integrity of Endosomal Compartments in Trachea Cells (A and B) Confocal sections show the DT of wild type (A) and rab52 (B) mutant embryos stained with anti-Chc. In wild type embryos, Chc staining is localised apically (A) while in rab52 mutant embryos, the discrete Chc localisation is lost at mid stage 17. (C and D) Images from a widefield time-lapse movie showing the endosomal structures of the tracheal cells in wild type (C) and rab52 embryos (D) expressing GFP-FYVE (white) at mid stage 17. Loss of Rab5 results in drastic reduction of the endosomal labeling while diffuse cytoplasmic accumulation of GFP-FYVE is detected (movie S11). (E - H) Confocal sections of the DT of wild type (E), (G) and rab52 (F), (H) mutant embryos expressing btl>GFP-Rab7 (E), (F) and btl>GFP-Rab11 (G), (H) stained with anti-GFP. rab5 mutant embryos showed a reduced number of GFP-Rab7 positive endosomal puncta when compared to the wild type. No changes were detected between
wild type and rab52 mutant embryos for the predominantly apically localised GFP- Rab11-positive REs irrespective of embryonic stage. (I) Quantifications of the diameter of the GFP-FYVE positive endosomal puncta at 13 h AEL and 18.30 h AEL for wild type and rab52 embryos. A significant reduction of the size of the endosomal structures was detected. * indicates statistically significant differences between rab52 and wild type embryos (p<0.001). At least 80 tracheal cells were examined for each developmental time point. (J) Plot comparing the number of intracellular GFP-Rab7 positive puncta for metamere 7 between live wild type and rab52 embryos. About 80% of the analyzed wild type embryos exhibited moderate to high number of GFP-Rab7 positive puncta. Only 30% of the rab52 embryos had moderate number of GFP-Rab7 positive structures while none of the analyzed rab52 embryos showed high number of puncta. The number of observed intracellular puncta were organized into the following categories: low (0-5 puncta), moderate (6-15 puncta) and high (>15 puncta). Scale bars 10 µm.
Figure S9. A Proportion of Intracellular Gasp Puncta Transiently Colocalize with Specific Endosomal markers (A-B) Confocal micrographs of early stage 17 wild type (A) and gasp mutant embryos (B) stained for Gasp (green) and a mixture of anti-Crb and anti-α -Spectrin to highlight tracheal cells (magenta). y-z confocal sections correspond to the DT position indicated by white vertical lines in xy sections. (A’-B’) displays Gasp staining (white) of the images (A) and (B). (C-E) xy confocal sections, xz, and yz projections of DT of early stage 17 embryos expressing btl>Clc-GFP (C), btl>GFP-Rab7 (D), and btl>GFP-LAMP1 (E) stained for Gasp (green) and GFP (magenta). White vertical and horizontal thin lines indicate the plain of xz and yz confocal projections and each indicate a Gasp dot localizing to a marked endosomal structure. Additional co-localising puncta are marked by orange
arrowheads. A subset of intracellular Gasp puncta co-localize with Clc-GFP (CCV), GFP-Rab7 (LE) and GFP-LAMP1 (lysosomes). (F) Quantification of the number of Gasp puncta localizing to a specific endosomal marker relative to the total intracellular Gasp puncta per confocal section. For each marker four confocal sections were analysed from a total of four embryos. Error bars denote s.e.m. (G) Confocal micrograph of the DT of a live, early stage 17 embryo expressing btl>GFP- CAAX (green) that was pre-injected with dextran (magenta) at stage 16. No intracellular dextran puncta can be seen, indicating that intracellular puncta may not derive from basal internalization. (H) DT of a live, early stage 17 rab52 mutant embryo expressing btl>CD8-GFP (green) that was injected with dextran (magenta) at stage 16. rab52 mutant embryos show intact paracellular junctions. Scale bars, 10 µm.
Supplemental Experimental Procedures
btl-GAL4 (on 2nd) and UAS-ANF-GFP (on X) were mobilised to generate additional
autosomal insertions that were meiotically recombined with different GFP transgenes.
Fixed and live mutant embryos were identified using appropriate balancers (Casso et al.,
2000; Halfon et al., 2002; Le et al., 2006). Additional insertions used were UAS-CD8-
GFP (Lee and Luo, 2001), UAS-GFP-rab5, UAS-2xmyc-FYVE-GFP, UAS-GFP-rab11,
UAS-clc-GFP, GFP-trap for PDI (on 3rd) (Bobinnec et al., 2003), UAS-GFP-CAAX,
UAS-GFP-rab7, and UAS- GFP-LAMP1 (on 2nd), btl-mRFP1-moe and UAS-myr-
mRFP1 (on both on 2nd and 3rd).
Supplemental References
Bobinnec, Y., Marcaillou, C., Morin, X., and Debec, A. (2003). Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster. Cell Motil Cytoskeleton 54, 217-225. Casso, D., Ramirez-Weber, F., and Kornberg, T. B. (2000). GFP-tagged balancer chromosomes for Drosophila melanogaster. Mech Dev 91, 451-454. Halfon, M. S., Gisselbrecht, S., Lu, J., Estrada, B., Keshishian, H., and Michelson, A. M. (2002). New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes. Genesis 34, 135-138. Le, T., Liang, Z., Patel, H., Yu, M. H., Sivasubramaniam, G., Slovitt, M., Tanentzapf, G., Mohanty, N., Paul, S. M., Wu, V. M., and Beitel, G. J. (2006). A new family of Drosophila balancer chromosomes with a w- dfd-GMR yellow fluorescent protein marker. Genetics 174, 2255-2257. Lee, T., and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24, 251-254.
COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila
Satish Arcot Jayaram., Kirsten-Andre´ Senti., Katarı´na Tiklova´, Vasilios Tsarouhas, Johanna Hempha¨la¨, Christos Samakovlis* Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
Abstract
Background: Tube expansion defects like stenoses and atresias cause devastating human diseases. Luminal expansion during organogenesis begins to be elucidated in several systems but we still lack a mechanistic view of the process in many organs. The Drosophila tracheal respiratory system provides an amenable model to study tube size regulation. In the trachea, COPII anterograde transport of luminal proteins is required for extracellular matrix assembly and the concurrent tube expansion.
Principal Findings: We identified and analyzed Drosophila COPI retrograde transport mutants with narrow tracheal tubes. cCOP mutants fail to efficiently secrete luminal components and assemble the luminal chitinous matrix during tracheal tube expansion. Likewise, tube extension is defective in salivary glands, where it also coincides with a failure in the luminal deposition and assembly of a distinct, transient intraluminal matrix. Drosophila cCOP colocalizes with cis-Golgi markers and in cCOP mutant embryos the ER and Golgi structures are severely disrupted. Analysis of cCOP and Sar1 double mutants suggests that bidirectional ER-Golgi traffic maintains the ER and Golgi compartments and is required for secretion and assembly of luminal matrixes during tube expansion.
Conclusions/Significance: Our results demonstrate the function of COPI components in organ morphogenesis and highlight the common role of apical secretion and assembly of transient organotypic matrices in tube expansion. Intraluminal matrices have been detected in the notochord of ascidians and zebrafish COPI mutants show defects in notochord expansion. Thus, the programmed deposition and growth of distinct luminal molds may provide distending forces during tube expansion in diverse organs.
Citation: Jayaram SA, Senti K-A, Tiklova´ K, Tsarouhas V, Hempha¨la¨ J (2008) COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila. PLoS ONE 3(4): e1964. doi:10.1371/journal.pone.0001964 Editor: Francois Schweisguth, Ecole Normale Superieure, France Received January 31, 2008; Accepted March 1, 2008; Published April 9, 2008 Copyright: ß 2008 Jayaram et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Swedish Research Council (VR), Cancerfonden, SSF to CS. EMBO postdoctoral fellowship to KAS. The funding agencies had no role in any part of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work.
Introduction luminal liquid is replaced by gas to generate a functional respiratory network [6]. Tube diameter expansion occurs within The acquisition of optimal tube dimensions during development a defined time interval without changes in the number or shape of is critical for the function of organs like the lung, renal and the constituent cells [7]. During this interval the tracheal vascular systems. This is reflected by the high incidence of human epithelium deposits proteins and polysaccharides into the lumen pathologies associated with tube size defects. For example, cystic and assembles a massive chitinous extracellular matrix. Mutations overgrowth in the collecting ducts is a landmark of polycystic affecting chitin biosynthesis (kkv) and matrix assembly (knk) grow kidney disease, whereas stenotic tubes cause obstructions of blood disproportional cystic and over elongated tubes [8–10]. In contrast vessels and other organs [1,2]. We use the Drosophila respiratory to diametric expansion, tube elongation is continuous during network as a genetic model to understand the determinants of tube tracheal development. Its termination requires secreted chitin size expansion. The tracheal system derives from twenty deacetylases that presumably modify the structure of the ECM and metameric cell clusters that undergo their last cell division as they thereby provide a stop signal to the epithelium [11,12]. invaginate from the epidermis. Cell migration and a series of Collectively, tube size mutants in flies and worms suggest an coordinated tracheal cell rearrangements generate a tubular instructive role of the extracellular luminal matrices in tube growth network that sends branches to all developing tissues [3–5]. coordination and termination [13]. Branching is followed by three precise morphological events What triggers and drives tube expansion? Studies of lumen transforming the nascent tubes into mature airways. First, a formation in other systems indicate that apical polarization and secretory burst of luminal proteins drives the diametric expansion targeting of membrane and vesicles to the lumen are central of the tubes. Later the luminal proteins are cleared and finally, the processes in the initiation of tube morphogenesis [2,14]. In the
PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e1964 COPI in Tubulogenesis zebrafish gut, luminal fluid influx generates a distending force that in metamere 6 was reduced by 54% in cCOP mutants drives coalescence of narrow tubes into a single lumen [15]. The (3.660.5 mm, n = 10) compared to wild type (6.260.36 mm, developing trachea tubes and other organs deposit transient solid n = 9) (Figure 1G). By contrast, DT metamere 6 length was not matrices, which may provide a distending force expanding tube altered in the mutants (32.761.1 mm in wild type n = 4 and diameter. Mutants affecting COPII vesicle budding and coat 33.160.7 mm in cCOP mutant embryos n = 4). These measure- assembly show defects in the secretion of luminal proteins and ments indicate that the DT volume capacity of the cCOP embryos diametric tube growth. The failure in the assembly and expansion is reduced to one third compared to wild type. of the chitinous matrix may be the underlying cause of the narrow Wild type embryos initiate diametric tube expansion at stage 14 tube phenotypes in these mutants. Alternatively, the diametric and nearly complete it within 3.5 hours [6]. To determine when expansion defects could be due to reduced delivery of apical the cCOP phenotype is manifested, we imaged live wild type membrane or transmembrane regulators to the cell surface embryos and cCOP mutants expressing btl.GFP-CAAX. We [6,16,17]. found that cCOP mutant embryos already show a narrower DT Secreted proteins traffic through the Endoplasmic Reticulum diameter at stage 14 and later expand their tubular diameter with (ER), the Golgi apparatus and the exocytic post-Golgi structures to a slower rate (0.45 mm/h) compared to wild type (0.71 mm/h) their final destination. Anterograde and retrograde transport (Figure 1H). Thus, zygotic cCOP function is required before and between the ER and the Golgi depends on two distinct types of during the diameter expansion interval. coated vesicles: COPII vesicles bud from the ER and shuttle To investigate where and when cCOP is expressed, we nascent secreted proteins to the Golgi apparatus. Conversely, performed mRNA in situ hybridizations. Maternal cCOP tran- COPI coated vesicles retrieve escaped luminal ER enzymes and scripts are abundant in early embryos (Figure S1). Zygotic cCOP recycling cargo adaptor proteins (such as the p23/24 type of expression commences in the epidermis and salivary glands (SG) proteins) from the Golgi and shuttle them back to the ER [18]. from stage 11, initiates in the trachea at stage 13 and at early stage Within the Golgi apparatus, COPI vesicles have been proposed to 16 is also detected in the foregut and hindgut tubes (Figure S1). mediate anterograde transport of secreted cargo or retrograde Consistent with the epidermal expression of cCOP, we found that traffic of Golgi enzymes between different cisterns of the Golgi cCOP mutants fail to complete dorsal closure. This defect can be [19]. The coatomer complex of COPI vesicles is composed of two rescued by ubiquitous cCOP expression from a tubulin1a-promoter different layers. The b, d, c, f subunits form the tetrameric in the mutant background demonstrating that cCOP is required subcomplex of the inner layer. The b- and c- subunits bind to for epidermal morphogenesis (Figure S1). active Arf1-GTP, cCOP also binds to p23. The outer coat layer cCOP and other genes encoding components of the secretory subcomplex is trimeric and contains the a, b9 and e subunits [20]. apparatus are prominently expressed in developing SGs [26]. This Here we present the functional analysis of COPI function in prompted us to analyze SG size growth in cCOP mutants. We used tube organogenesis. We find that COPI vesicle transport is antibodies against the apical regulator Crumbs and cytoplasmic a- required to: a) secrete of multiple luminal proteins, b) assemble a Spectrin to highlight SG cellular outlines and Piopio to label the luminal matrix and c) expand the diameter of tubular organs. SG lumen in wild type and cCOP embryos [27]. Like in tracheal These defects are not confined to the chitin-lined tracheal tubes tubes, the salivary glands of cCOP mutants were thinner compared but also extend to the salivary glands. In tubular epithelia of cCOP to wild type (Figure 1I, J, L, M). The SG tube diameter is strikingly mutant embryos, the structure of both the ER and Golgi is reduced in cCOP mutants from 5.060.76 mm (n = 7) in the wild compromised indicating that efficient luminal secretion is highly type to 1.960.36 mm (n = 7) in cCOP (Figure 1O) (errors are SD). dependant on a functional secretory apparatus. The results This phenotype can be rescued by ubiquitous expression of tub1a- highlight the major role of membrane traffic in the expansion of cCOP in mutant embryos (Figure 1K, N, O). Also SG tube luminal extracellular matrices and apical membrane during elongation is impaired in cCOP null mutant embryos. The distance tubulogenesis. between the most distal cells of the salivary duct expressing btl.GFP-CAAX to the gland tip visualized by E-Cadherin (E-cad) Results staining, was reduced from 50.265.8 mm (n = 7) in wild type to 3664.3 mm (n = 11) in cCOP mutants at stage 16 (p,0.001). The cCOP is required for tube diameter expansion similarities of tracheal and salivary gland phenotypes in cCOP We identified cCOPP1 mutants by screening a collection of lethal mutants suggest a common cellular mechanism expanding tubular P-element insertions for tracheal tube size defects [21–25]. At early organs. stage 16, the dorsal trunk (DT) tubes of cCOPP1 mutant embryos were narrower compared to wild type. Using imprecise excision, cCOP is required for luminal secretion and assembly of we generated two independent molecular null alleles including the ECM components cCOPD114 lethal allele, which we analyzed for tube expansion A COPII-mediated secretory burst of luminal proteins at late (hereafter referred to as cCOP mutants) (Figure S1). To visualize stage 13 drives the diametric expansion of tracheal DT tubes [6]. the tracheal lumen, we stained embryos for a secreted chitin During this interval luminal chitin and chitin binding proteins binding protein, Gasp in embryos expressing GFP-CAAX in the assemble into an expanding matrix. To test whether luminal trachea (btl.GFP-CAAX) [6]. At early stage 16, the DT tubes of protein deposition is affected in cCOP mutants, we stained wild cCOP mutants were thinner compared to wild type (Figure 1A, B). type and mutant embryos expressing btl.GFP-CAAX for the This failure in tube expansion was also evident by comparison of luminal antigen 2A12 and the chitin binding proteins Gasp and yz-confocal sections (Figure 1D, E). Re-expression of cCOP in the Verm. While 2A12, Gasp and Verm predominantly localize to the trachea of mutant embryos rescued the phenotype, indicating that tracheal lumen in wild type stage 15 embryos, those markers are cCOP is required in the tracheal epithelium for diametric tube strongly retained inside tracheal cells in cCOP mutants (Figure 2A, growth (Figure 1C, F). To quantify tube diameter, we measured B, D, E, G, H). This retention can be rescued by tracheal specific the distance between the apical surfaces of cells lining the DT in btl.cCOP expression in cCOP mutants (Figure 2C, F, I). Thus, confocal sections of wild type and cCOP mutant embryos cCOP is required in the tracheal epithelium for luminal protein expressing btl.GFP-CAAX. At early stage 16, the DT diameter secretion and the completion of diametric tube expansion. Does
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Figure 1. cCOP is required for efficient lumen diameter expansion. (A–F, I–N) Confocal micrographs of early stage 16 wild type (A, D, I, L), cCOP mutant (B, E, J, M) and btl.cCOP; cCOP/cCOPP1 (C, F) or tub-cCOP; cCOP/cCOPP1 embryos (K, N). GFP stainings of btl.GFP-CAAX embryos reveal tracheal DT cells (A–C, in magenta and D–F, in white). Anti-Crumbs and anti-a-Spectrin stainings show SG cells (I–K, in magenta and L–N, in white). Embryos were stained for the luminal antigens Gasp (A–C) and Piopio (I–K) (green). All micrographs show confocal sections except for (D–F) and (L–N) which show yz-confocal sections of DT and SG. (G and O) Lumen diameter of DT metamere 6 using btl.GFP-CAAX (G) and diameter of SG measured for Crumbs stainings (O). cCOP mutant embryos show significantly (p,0.001) narrower trachea and SG tubes. Transgenic expression of cCOP specifically in the trachea (C, F) or under the tubulin1a promoter for SG (K, N) largely rescues the narrow lumen phenotype of cCOP mutant embryos. (H) Plot showing DT diameter expansion of live wild type and cCOP embryos expressing btl.GFP-CAAX. Embryos were recorded from late stage 13 to stage 15. Error bars are means6SD. Scale bars are 10 mm (A–C, I–K), 5 mm (D–F) and 10 mm (L–N). doi:10.1371/journal.pone.0001964.g001
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Figure 2. cCOP is required for efficient luminal protein secretion and matrix expansion in the trachea. (A–I) Confocal micrographs of DT of wild type (A, D, G, J), cCOP mutant (B, E, H, K), btl.cCOP; cCOP/cCOPP1 rescued embryos (C, F, I,). btl.GFP-CAAX embryos were stained for the tracheal luminal antigens 2A12 (A–C), Verm (D–F) and Gasp (G–I) (green), GFP (magenta) and with a chitin binding probe (CBP), white in (J,K). All micrographs are single confocal sections except (J–K), which represent confocal projections. The inserts in J and K show y-z projections. cCOP mutant embryos show strong intracellular retention of tracheal luminal markers. Transgenic re-expression of cCOP largely rescues the luminal secretion phenotype of cCOP mutant embryos. (K and M) The luminal ECM is defective in the trachea of cCOP mutants. Scale bars are 10 mm in A–K and 0.5 mm in L and M. doi:10.1371/journal.pone.0001964.g002 the defective deposition of chitin binding proteins into the tubes with a failure in the secretion and assembly of the intraluminal affect the assembly and expansion of the luminal matrix? Staining matrix? The molecular composition of the SG luminal matrix is with a fluorescent chitin binding probe and TEM analysis revealed unknown, but unlike the tracheal one it does not depend on chitin that the tracheal luminal matrix in cCOP embryos remained biosynthesis (data not shown). Recently, the SG lumen has been narrow and dense at early stage 16 embryos (Figure 2J, K, L, M). shown to contain glycans with a single N-acetyl-Galactosamine O- The developing SGs also form an intraluminal matrix. Embryos linked to Serine or Threonine [30]. To visualize the luminal lacking pasilla or a gene cluster encoding putative prolylhydrox- secretion of those O-glycans in the salivary gland, we stained fixed ylases show disrupted matrix structure and cystic and malformed embryos with the specific Tn antibody. Directly after SG glands [28,29]. Do the cCOP defects in gland expansion coincide invagination, minor amounts of Tn antigen were detected in the
PLoS ONE | www.plosone.org 4 April 2008 | Volume 3 | Issue 4 | e1964 COPI in Tubulogenesis lumen. The Tn antigen levels markedly increased and localized deformed and abnormally electron dense luminal matrix in the in intracellular puncta and in the SG lumen at stage 12 and 13. narrow SG lumen (Figure 3I, J). Additionally, the lack of However from stage 14 onwards, the intracellular and luminal abundant dark apical granules in cCOP mutants further supports levels decline until only very little remains along the apical lining the role of cCOP in luminal material deposition. Thus, cCOP of the SG a stage 15 (Figure 3A, B, C, D). A similar, dynamic function is required for the secretion of luminal antigens and localization of Tn antigen is also evident in the trachea (data not assembly of an extracellular matrix. shown). Thus, salivary gland expansion is accompanied by a sharp burst of luminal secretion of O-glycosylated proteins. Is cCOP required for this secretory burst? Importantly at stage 13, dCOP mutants phenocopy cCOP secretion and diameter cCOP mutant embryos showed strongly reduced intracellular phenotypes accumulation and luminal deposition of Tn antigens compared As the coatomer complex comprises seven subunits that form to wild type embryos. This phenotype was fully rescued by the the COPI vesicle coat, we asked if mutants in other coatomer tub1a-cCOP transgene (Figure 3E, F, H). Like cCOP, sar1 mutant components show defects in tube expansion. dCOP mutants retain embryos also show a reduced secretion in the SG (Figure 3G), the Verm and Gasp luminal proteins inside the tracheal cells at suggesting both COPI and COPII are required for efficient stage 15 and fail to fully expand DT tube diameter at early stage luminal deposition in the SG. To determine if the defects in O- 16 (Figure 4A, B, C, D). The diameter of the DT tube of glycan secretion are accompanied by defects in luminal metamere 6 was reduced by 28% in dCOP mutants compared to extracellular matrix assembly, we analysed the SGs of cCOP the wild type (Figure 4E, F, I). A similar phenotype is evident in mutant embryos by TEM. In striking contrast to wild type the SGs (Figure 4G, H). These results confirm that COPI vesicle embryos that show a uniform and space-filling luminal matrix at trafficking mediates luminal secretion and efficient diametric early stage 16, cCOP mutant embryos showed an atrophic, expansion of tracheal and salivary gland tubes.
Figure 3. cCOP is required for deposition of O-glycans and luminal ECM assembly in the SG. (A–H) Confocal projections show developmental stages of salivary gland (A–D) stained for Tn-antigen (green) and Crumbs in (magenta). (A9–D9) represent confocal sections of the (A– D) projections focused inside the lumen. (A, A9) show stage 11, (B, B9) stage 12, (C, C9) stage 13, (D, D9) stage 15 wild-type embryos. The salivary gland epithelium dynamically deposits Tn antigen into the lumen. While at stage 11 luminal levels of Tn antigen are low, they increase dramatically during stages 12 and 13 and localise to intracellular puncta and lumen. Later, luminal Tn antigen levels decline but a minor proportion remains along the apical epithelial membrane. (E–H) Confocal projections show stage 13 wild-type (E), cCOP/cCOPP1 (F), sar1DEP28 (G) and tub-cCOP; cCOP/cCOPP1 mutant embryos (H) stained for Tn antigen (white). cCOP and sar1 mutant embryos show reduced intracellular puncta and luminal deposition of Tn antigen. (I and J) TEM of salivary gland cross-sections from stage 16 wild-type and cCOP mutants. The intraluminal matrix and the electron-dense granules are severely reduced in cCOP mutants compared to wild type. Scale bars are 10 mm in A–H. and 2 mm in I and J. doi:10.1371/journal.pone.0001964.g003
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mutants (Figure 5P–S). Thus, loss of cCOP or sar1 does not affect epithelial integrity (Figure 5E, J, O). In summary, this suggests that apical secretion and luminal matrix assembly are common strategies for tubular organ expansion in Drosophila.
cCOP co-localizes with ER and Golgi markers We used S2 cells and an antiserum against the mouse cCOP [31] to address the subcellular localization of cCOP in Drosophila. The cCOP antibody showed a punctate cytoplasmic signal in S2 cells or cells treated with double stranded (ds) RNA against GFP. cCOP dsRNAi treatment abolished the punctate staining and showed a specific reduction of a 96 kDa protein in cCOP dsRNAi treated cells compared to GFP dsRNAi treated cells (Figure S2). To reveal the identity of the cCOP puncta, we co-stained S2 cells with either ER or Golgi markers. Confocal analysis showed a partial overlap of cCOP with the ER markers KDEL (the ER retention signal found in many ER proteins) and Calreticulin (Figure S2 and not shown). Co-staining for the cis-Golgi markers GM130 and Lava lamp showed a discrete co-localisation of cCOP with these structures. By contrast, the cCOP puncta did not overlap with the median (gp120) and trans Golgi marker (peanut agglutinin) staining (Figure S2). Thus cCOP localizes in the ER and cis-Golgi units in Drosophila cells, consistent with the localization of its mammalian and plant homologs [31,32].
ER and Golgi are disrupted in cCOP mutant embryos The prominent secretory defects in the cCOP mutants and the co-localization of cCOP with ER and cis-Golgi markers suggested that the basal secretory apparatus may be defective in cCOP embryos. We therefore stained wild type and mutant embryos for KDEL, the transmembrane adaptor p23/Baiser and Calreticulin, an abundant ER chaperone [33–35]. We observed a drastic reduction in the staining intensities of all markers in the trachea and SGs of the mutants compared to wild type embryos Figure 4. dCOP mutants show defects in secretion and luminal (Figures 6A–D, K, L and S3) suggesting a disruption of ER diameter expansion. (A–H) Confocal micrographs show the DT in integrity in cCOP embryos. The reduced intensity of Calreticulin wild type (A, B) and dCOP mutants embryos (C, D). Embryos were staining in cCOP mutants was also evident in epidermal cells stained with anti-Gasp (green) together with anti-Crumbs and anti-a- Spectrin to show tracheal cells (magenta). All micrographs are single (Figure S3), which also showed an abnormally high intracellular confocal sections except (E–H) which represent yz- confocal projections accumulation of Verm at stage 16 (data not shown). We used of DT of metamere 6 for stage 16 wild type (E), dCOP (F) and SG TEM to visualize the tracheal and SG ER in wild type and cCOP projections of wild type (G), dCOP (H). dCOP mutant embryos retained a mutants. In contrast to the uniformly organized tubular ER of considerable amount of Gasp inside tracheal cells at stage 15 (C). At the wild type, ER structures were severely bloated in the mutants early stage 16, dCOP embryos (D, F) show narrower DT lumen in particularly in the trachea (Figure 6I, J, O, P). These structural comparison to wild type (B, E). dCOP mutant embryos stained for Crb (H) show narrow SG lumen compared to wild type (G) at stage 16. (I) defects are accompanied by a mild activation of the unfolded Graph showing the lumen diameter of the DT at metamer 6 in wild type protein response evidenced by the presence of processed xbp1 and dCOP embryos at early stage 16 using Crb staining to visualize transcripts in cCOP mutants (Figure S3) [36]. The above data apical cell membrane. dCOP mutant embryos show a significant show that COPI vesicle transport is required for maintenance reduction in lumen diameter (p,0.001) when compared to wild type. of ER integrity in epithelial tissues. We analyzed the Golgi status 6 Error bars are means SD. Scale bars are 10 mm. by staining embryos for the Lava lamp and gp120 markers [37]. doi:10.1371/journal.pone.0001964.g004 As expected the intensity of Lava lamp and gp120 puncta were reduced in the trachea (Figure 6E–H) and salivary glands Epithelial organization is not affected in zygotic cCOP of mutant embryos (Figures 6M, N and S3). Thus, the mutants deficits in luminal protein deposition and tube expansion are Reduced apical secretion in cCOP mutants may be an indirect due to structural defects in the secretory apparatus of cCOP consequence of defects in epithelial polarization. We addressed mutants. this by staining for apical and junctional epithelial polarity markers, such as Crumbs, the adherence junction marker E- COPI and COPII vesicle trafficking drive tube expansion Cadherin and the septate junction marker Coracle in the trachea COPI coatomer subunit mutations cause tube expansion and and SGs. Except for a minor decrease in the staining intensity of cellular defects that closely resemble the phenotypes of COPII coat the markers, their localization and the revealed epithelial cell mutants [6]. However, only cCOP mutants show dorsal closure shapes were indistinguishable between mutants and wild type phenotypes suggesting a selective role of COPI vesicles. To embryos (Figure 5A–O). Further, we found that both adherens determine if mutants in COPI and COPII components show junction structure (Figure 6I, J), as well as the transepithelial additive phenotypes in the trachea, we analyzed zygotic single sar1 barrier function of the septate junctions were intact in cCOP or cCOP null mutant embryos and sar1 cCOP double mutants.
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Figure 5. cCOP mutant embryos show normal epithelial organization. (A–O) Confocal micrographs show DTs and SG of early stage 16 wild type (A, C, F, F9, H, H9, K, M), cCOP mutant embryos (B, D, G, G9, I, I9, L, N) and sar1 mutant embryos (E, J, J9, O). All micrographs are single confocal sections except (F9–J9) which represent projections. GFP stainings of btl.GFP-CAAX embryos label tracheal cells (magenta). The first two columns from the left represent tracheal DT and the next three columns show SG. Embryos were stained for the apical marker Crb (A–E), the adherens junction marker E-cad (F–J) and septate junction marker Coracle (K–O) (either green or white). No defects in the localization or organization of epithelial markers were detected in cCOP mutant embryos. (P–S) Wide field images of live wild type (P, P9), cCOP (Q, Q9), sar1P1 (R, R9), and Atpa (S) embryos injected with 10 kDa fluorescent dextran (P, Q, R, S). The DT is visualized by expression of GFP-CAAX (P9,Q9,R9). The transepithelial barrier function in the trachea is not affected in cCOP and sar1P1 mutant embryos. Scale bars are 10 mm. doi:10.1371/journal.pone.0001964.g005
Interestingly, sar1 cCOP double mutant embryos showed as narrow cCOP and sar1 mutants show qualitatively similar phenotypes in DT tubes, as sar1 or cCOP single mutants (Figure 7B, C, D). The tube expansion in the trachea and the SG. cCOP and sar1 gene lack of an additive phenotype in the double mutants suggests that products are deposited in the oocyte at sufficient levels to support anterograde COPII and retrograde COPI vesicles transport are early embryogenesis. Distinct levels in maternal contribution or equally important to maintain both ER and Golgi structures and zygotic expression of cCOP and Sar1 may contribute to the that the dynamic bidirectional ER-Golgi traffic is essential for the quantitative differences in tube expansion phenotypes. In sharp secretory activity of epithelial cells during tube expansion. contrast to the similarities of tubular defects, cCOP mutant embryos fail to complete dorsal closure while sar1 mutants close Discussion normally (data not shown). While we cannot exclude that the cCOP maternal product is less stable than Sar1, this phenotypic COPI and COPII mutations strictly affect diameter but not difference suggests that not all developmental processes require length expansion in the trachea. SG expansion is mainly defective both COPII and COPI function equally. This may be attributed in diameter and to lesser degree also in length. This differential to a specific COPI function in intra Golgi trafficking. effect may be explained by the dramatic expansion of SG in both The developing trachea and SG tubes initiate a dynamic diameter and length during the SG secretory burst. The tracheal secretory burst that deposits transient solid matrices. The tubes on the other hand, elongate continuously and the diametric expansion defects in mutants for COPI and COPII programmed boost of secretory activity precedes the short interval components may in part be due to reduced delivery of apical of diametric expansion. membrane or transmembrane regulators to the cell surface. The
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Figure 6. Defective ER and Golgi in cCOP embryos. (A–H, K–N) Confocal sections of stage 16 wild type (A, B, E, F, K, M) and cCOP mutant embryos (C, D, G, H, L, N) expressing btl.GFP-CAAX (magenta). Embryos were stained with anti-KDEL for ER in white (A, C, K, L), green in (B, D) and with anti-Lava lamp for Golgi in white (E, G, M, N), green in (F, H). cCOP mutant embryos show reduced ER staining intensity (C, D, L) and a marked decrease in the number of Golgi puncta both in trachea and SG cells (G, H, N). TEM sections of DT (I–J) and SG (O–P) of stage 16 wild type (I, O) and cCOP embryos (J, P). In wild type both tracheal and SG cells show a tubular organization of the rough ER, studded with ribosomes (white arrow in I, O). cCOP mutant cells show disrupted and bloated rough ER structure (white arrow in J, P). The SG intraluminal matrix is indicated by an asterix in (O) and (P). Scale bars are 10 mm (A–H, K–N), 0.5 mm in (I–J) and 1 mm in (O, P). doi:10.1371/journal.pone.0001964.g006
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proposed to generate the turgor for notochord expansion [42]. Zebrafish mutants in coatomer encoding genes fail to expand their notochord and larval tail. These phenotypes could be partly due to defects in laminin secretion and the assembly of the basal extracellular matrix [43] . The recent visualisation of vacuolar membrane fusion with the luminal cavity in ascidians and our analysis of COPI mutants further implicate luminal secretion in notochord expansion. Thus, the programmed deposition of swelling luminal matrices may be a common strategy of tube expansion.
Materials and Methods Drosophila Strains The P element alleles l(3)s057302 and KG06383 (cCOPP1 and cCOPP2 respectively; Figure S1) fail to complement each other. Insertion sites were determined by plasmid rescue and PCR. P- element excision of cCOPP2 generated a precise excision reverting the tracheal phenotype, along with two independent null lethal alleles: D128, a smaller deletion, taking out the 59UTR and a major part of the first exon and D114 (cCOP in text) which lacks the 59UTR, first exon and the majority of second exon (Figure S1). All the analysis was performed on D114. Rescue with btl.cCOP or tubulin1a-cCOP was performed in cCOP/cCOPP1 trans- heterozygous mutant background. Other mutants used were KG07426 (dCOP), l(3)05712 (sar1P1), sar1D28 (sar1 EP3575D28) [6], kkvDZ8 [8], AtpaS067611 [22], and NP5464 (p23/baiser).
Molecular biology ds-RNAi, RT-PCR. pUAST-cCOP and tubulin1a-cCOP were generated by sub-cloning the cDNA from RE37840 into pUAST and ptub1alpha-GAL80 [44]. Down regulation of cCOP by RNAi in S2 cells was performed as described in [45]. Primer pairs tailed with T7 RNA polymerase promoter were used to amplify a PCR fragment from the cDNA clone. Primers used in PCR amplification 59-TTAATACGACTCACTATAGGGAGACCAG Figure 7. COPI and COPII function in tube size control. (A–D) GAGGCTTTGAACAGCGACA-39 and 59-TTAATACGACTC Confocal sections of early stage 16 wild type (A), sar1D28 (B), cCOP (C) ACTATAGGGAGACGCACAATAGGGCTCTCCAAGATGA-39. and sar1D28 cCOP double mutant embryos (D). Cells of tracheal DT The 900 bp PCR product was then used as template for stained for anti-Crumbs and anti-a-Spectrin shown in magenta (A–D) dsRNA production with the MEGAscript RNAi kit (Ambion). and for the tracheal luminal antigen Gasp (A–D) (green). (E) Schematic For RT-PCR, total mRNA was isolated from stage 16 wild type illustration of a cross-section through an epithelial tube. The ER, Golgi and post-Golgi vesicles carry secreted proteins (green) into the lumen. and cCOP mutant embryos using oligo(dT)-coupled beads Both COPI and COPII vesicular transport between the ER and Golgi are (Dynabeads). Unfolded protein response was induced in S2 cells required for intraluminal matrix assembly and apical membrane by treatment with 10mM DTT. Reverse transcription was addition (red). Green arrows indicate the proposed pressure exerted performed with SuperScript-II (Invitrogen). Primers flanking the on the cells by the expanding matrix. Scale bars are 10 mm. splice-site of xbp-1 mRNA were used, 59-CGCCAGCG- doi:10.1371/journal.pone.0001964.g007 CAGGCGCTGAGG-39 and 59-CTGCTCCGCCAGCA- GACGCGC-39. actin mRNA was amplified using 59-GACCCA- strict correlation between the luminal matrix assembly defects and GATCATGTTCGAGACC-39 and 59-GCATTTGCGGTGAAC the tube expansion phenotypes strongly suggest that the swelling GATTCCGGG-39 on the same beads to provide a quantification luminal matrixes directly enlarge the tubes from the inside control. (Figure 7E). This is analogous to the distending pressure from the transient fluid influx into the lumen that expands the lung and Immunostaining, TEM and Western blotting coalesces the gut lumen in zebrafish [15,38]. ‘‘Inside out molding’’ Immunostainings, Western blots and TEM were performed as by swelling luminal matrixes may be a common mechanism in in [6,45] with the following additional antibodies: Rabbit anti- tubulogenesis extending to vertebrates. The notochord of many Mouse cCOP1 raised against C-terminal region [31] (The ascidian species expands by coalescing vacuoles into a single Drosophila cCOP protein is 50% identical to mouse cCOP1), continuous lumen stretching through the entire notochord [39], rabbit anti-Lava Lamp [37]. rabbit anti-Galleria mellonella calreti- while other vertebrate species rely on an immense intracellular culin [35]. Rabbit anti-p23/Baiser raised against cytoplasmic vacuolarisation [40]. The luminal/vacuolar turgor of the noto- peptide tail [33]. Fluorescein-Peanut agglutinin (PNA) (Sigma), chord is contained by a thick basement membrane ECM. This rabbit anti-dGM130 [46] (Abcam). The mAB anti-Tn clone B1.1 renders the expanding notochord into a stiff, rod-shaped organ (Biomeda) [30] recognizes the unmodified N-acetyl-Galactos- that elongates the tail and the entire animal [41]. The luminal amine-group O-linked to serines or threonines on protein secretion of water absorbing glycosaminoglycans has been substrates (Tn antigen). To examine the SJ barrier function in
PLoS ONE | www.plosone.org 9 April 2008 | Volume 3 | Issue 4 | e1964 COPI in Tubulogenesis tracheal cells, embryos were injected at late stage 16 with 10 kDa Found at: doi:10.1371/journal.pone.0001964.s002 (4.12 MB TIF) Rhodamine-Dextran (Molecular Probes). Figure S3 Defective ER and Golgi in cCOP embryos. (A–N) Confocal sections of wild type (A, B, E, F, I, M), cCOP mutant Supporting Information embryos (C, D, G, H, J, N), heterozygous for p23/baiser (K) and Figure S1 cCOP genomic locus and expression pattern. (A) The P23/baiser mutant embryos at early stage 16 (L). (A–H) Embryos cCOP locus and positions of P element insertions and deletions. (B– expressing btl.GFP-CAAX were stained for the ER marker E) Bright field images of wild type stage 1 (B), early stage 16 (C) Calreticulin for ER in white (A, C), green in (B, D) and the Golgi and cCOP mutant embryos stained for cCOP (E) with anti-sense markers gp120 for Golgi in white (E, G), or green (F, H). GFP RNA probes (grey). (D) shows an embryo stained with a ‘‘sense’’ localization in the DT is in magenta. (I–L) show SG staining for RNA probe. Expression of cCOP transcripts was strongly reduced p23/Baiser and (M, N) depict epidermis stained for Calreticulin. in zygotic cCOP mutant (E). (F–K) Confocal sections of 1-eve-1 cCOP mutants show reduced ER staining intensity (C, J, N) and a embryos showing zygotic expression of cCOP in the SG (F) and DT decreased number of Golgi units (G). (O) An agarose gel showing (H, I) with cCOP anti-sense probes. No staining was detected with RT-PCR products detecting splicing of XbpI mRNA in cCOP the sense probe in SG (G) and DT (J, K). Tracheal cells are mutant embryos. cCOP mutants show a mild Unfolded Protein visualized by anti-b-Gal staining (magenta in I, K). Zygotic Response (UPR). Scale bars are10 mm. expression of cCOP transcripts is observed in SG and trachea. (L– Found at: doi:10.1371/journal.pone.0001964.s003 (5.06 MB TIF) N) Confocal projections of wild type (L), cCOP mutant (M) and tub- P1 cCOP; cCOP/cCOP embryos (N) stained for Coracle to visualize Acknowledgments the dorsal epidermis. cCOP mutant embryos fail to close dorsally. Scale bars are 30 mm in (B–E) and 10 mm in (F–G, H–K, L–N). We would like to thank Peter Maroy, Rick Fehon, John Sisson, Randy Found at: doi:10.1371/journal.pone.0001964.s001 (4.14 MB TIF) Schekman, Felix Wieland, Liqun Luo, the Bloomington, Szeged and Kyoto stock centers, the Developmental Studies Hybridoma Bank (DSHB) at the Figure S2 cCOP co-localizes with ER and Golgi markers. (A–J) University of Iowa, and the Drosophila Genomics Resource Center for Confocal sections of S2 cells stained with anti-cCOP (green) and antibodies, reagents and fly strains. We are grateful to Kelly Ten Hagen for gp120 (A–D, I), or KDEL (F), or GM130 (G), or Lava lamp (H), or suggestions on the Tn antibodies and Stefan Luschnig for exchange of PNA (J) (red). S2 cells were either mock treated (A) or treated with unpublished information. We thank Inger Granell and Monika Bjo¨rk for dsRNA for GFP (B) or for cCOP for 3 days (C) or 6 days (D). Golgi their excellent technical assistence. and cCOP staining were reduced in cCOPi treated cells. (E) Western blot of S2 cell extracts show a marked reduction of Author Contributions ,97 kDa protein (equivalent to predicted molecular weight) in Conceived and designed the experiments: CS SJ KS. Performed the dscCOP treated cells, but not in untreated cells. Lamin was used as experiments: SJ KS KT VT JH. Analyzed the data: CS SJ KS KT VT. loading control. cCOP shows partial overlap with KDEL and Contributed reagents/materials/analysis tools: JH. Wrote the paper: CS SJ clear co-localization with cis-Golgi markers. Scale bars are 10 mm. KS.
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LETTERS
Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila
Katarína Tiklová1, Kirsten-André Senti1,2, Shenqiu Wang1, Astrid Gräslund3 and Christos Samakovlis1,4
Iron is an essential element in many biological processes. In MTf is a GPI (glycosyl phosphatidylinositol)-anchored, iron-binding vertebrates, serum transferrin is the major supplier of iron to protein found in several mammalian epithelial tissues8. Human MTf tissues, but the function of additional transferrin-like proteins is apically localized in polarized cells9, and its levels are increased in remains poorly understood. Melanotransferrin (MTf) is a melanoma patients. Consequently, MTf has been suggested to modulate phylogenetically conserved, iron-binding epithelial protein. melanoma-cell migration and metastasis10. However, its function in vivo Elevated MTf levels have been implicated in melanoma remains elusive. Mice that lack or overexpress MTf have no detectable pathogenesis. Here, we present a functional analysis of MTf in abnormalities8,1112, presumably because of compensatory mechanisms Drosophila melanogaster. Similarly to its human homologue, involving the other proteins of the transferrin-like family. Drosophila Drosophila MTf is a lipid-modified, iron-binding protein attached MTf (CG10620), similarly to its vertebrate homologues, contains a sig- to epithelial cell membranes, and is a component of the nal peptide, two putative iron-binding domains and a carboxy-terminal septate junctions that form the paracellular permeability barrier GPI modification sequence (Fig. 1a). In embryos, MTf is expressed in epithelial tissues. We demonstrate that septate junction in all ectodermal epithelial tissues13. We generated MTf null mutants assembly during epithelial maturation relies on endocytosis (Supplementary Information, Fig. S1a) and focused our analysis on the and apicolateral recycling of iron-bound MTf. Mouse MTf trachea. The dorsal trunk airways were tortuous in MTf-mutant embryos, complements the defects of Drosophila MTf mutants. Drosophila compared with wild-type (Fig. 1b, c and Supplementary Information, provides the first genetic model for the functional dissection of Fig. S1b). To investigate whether insect and mammalian MTf are phylo- MTf in epithelial junction assembly and morphogenesis. genetically conserved, we expressed Drosophila or mouse MTf cDNA in the trachea of MTf mutants. Both the Drosophila and mouse MTf rescued Epithelial sheets provide diffusion barriers that generate distinct chemi- the tracheal phenotypes in 100% (n = 9) and 60% (n = 10) of the mutant cal compartments within tissues and organisms. Specialized epithelial- embryos, respectively (Supplementary Information, Fig. S1c, d). Thus, cell junctions—tight junctions in vertebrates1 and septate junctions in mouse MTf can largely substitute for Drosophila MTf in trachea suggest- insects2—prevent passive diffusion between cells and segregate the api- ing that they share essential molecular functions. cal and basal environments. Despite structural differences, septate- and The airway phenotype of MTf-mutant embryos resembles the defects tight-junctions share essential components, such as claudins, and might of mutants lacking septate junction components. Septate junctions are be derived from a common ancestral junction3. Genetic and molecular required for luminal accumulation of the putative chitin deacetylases, ver- analysis of Drosophila septate junctions has identified 15 components, miform and serpentine, and for chitin matrix modification14. These modifi- including adhesion molecules and scaffolding proteins4. Among these, cations are thought to limit branch elongation. MTf-mutant embryos failed Coracle, Neurexin IV and Na+, K+- ATPase α form a functional sub- to secrete vermiform into the growing tracheal tubes and showed diffuse group with the FERM-protein, Yurt (Yurt–Cora group)5, to maintain luminal chitin and tube overelongation (Supplementary Information, epithelial polarity. Subsequently, septate junction protein complexes Fig. S1e-h). We further assessed septate junction permeability in MTf assemble into junctions to establish the paracellular epithelial barrier6 mutants by dye-injection experiments15. In MTf mutants, but not in wild- and control apical membrane growth during tubular organ expansion7. type embryos, 10 K fluorescent dextran leaked rapidly into the airways The maturation mechanism of septate junction complexes into func- (Supplementary Information, Fig. S1i, j). These phenotypes indicate that tional junctions remains unknown. septate junction functions are compromised in MTf-mutant embryos.
1Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden. 2Current address: IMBA, Dr. Bohr-Gasse 3, 1030 Vienna, Austria. 3Department of Biochemistry and Biophysics, Stockholm University, S-10691 Stockholm, Sweden. 4Correspondence should be addressed to C.S. (e-mail: [email protected])
Received 04 May 2010; accepted 02 September 2010; published online 10 October 2010; DOI: 10.1038/ncb2111
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a MTf DDYY DNYH h WT i WT SS Fe-binding (28%) Fe-binding (35%) GPI
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Figure 1 MTf is a septate junction component. (a) Schematic representation confocal microscopy image of embryonic tracheal cells immunostained of the MTf protein domains. The iron-binding domains of Drosophila MTf to indicate localization of MTf. (i) Merged confocal microscopy images of show 28% and 35% similarity to the mouse MTf domains. SS; signal peptide. embryonic tracheal cells immunostained for MTf and Nrx. (j, k) Confocal D, Y, H and N indicate potential iron-binding residues. (b, c) Wild-type microscopy images of ATPα (j) and cora (k) mutant embryos immunostained (b) and MTf mutant (c) embryos at stage 16, stained with 2A12 antibody to indicate localization of MTf. Scale bars, 10 μm. (l) Cell extracts from to visualize trachea. Scale bar, 25 μm. (d, e) Confocal microscopy section wild-type embryos at stage 14–16 were immunoprecipitated with anti-MTf of a wild-type (d) and an MTf embryo (e) immunostained for Nrx. Scale (left) or anti-Nrx (right). Co-immunoprecipitated proteins were resolved and bar, 10 μm. (f, g) Transmission electron micrographs of wild-type (f) and identified by western blotting using the indicated antibodies. Pre-immune MTf mutant (g) embryos at stage 16. Brackets indicate paracellular septa. sera were used as negative controls. Input; 5% of extract that was used for Arrows indicate adherens junctions. Scale bar, 0.2 μm. (h) Representative immunoprecipitation.
To investigate septate junction integrity, wild-type and MTf-mutant junction components were not detected in the anti-MTf immunopre- stage-16 embryos were stained for the septate junction proteins, cipitates, and anti-DE-cadherin antibodies did not co-precipitate MTf, Neurexin IV (Nrx16; Fig. 1d, e) and Coracle (Cora17; Supplementary indicating that MTf interacts specifically with septate junction proteins Information, Fig. S1k, l). In contrast to wild-type, where these mark- (Supplementary Information, Fig. S1v). Thus, MTf is a septate junction ers were localized apically on the lateral membrane, Nrx and Cora component required for junction integrity. were spread basolaterally in the trachea of MTf mutants. Transmission Next, we assessed how septate junction proteins assemble into func- electron microscopy (TEM) analysis of the mutants indicated a strong tional junctions. We monitored MTf and Nrg localization in tracheal reduction in paracellular septa, compared with the ladder-like septate and hindgut cells of wild-type embryos during early- and late-stages of junctions of wild-type embryos (Fig. 1f, g). No defects were detected in epithelial morphogenesis. At stage 13, before septate junction forma- the adherens junctions of MTf mutants (Supplementary Information, tion, MTf and Nrg localized evenly along the lateral and basal epithelial Fig. S1n). Therefore, MTf is selectively required to establish or maintain cell membranes. A fraction of the proteins were also detected in intra- septate junctions. cellular puncta (Fig. 2a, c and Supplementary Information, Fig. S2a). We hypothesized that MTf might be a septate junction component. Interestingly, co-immunoprecipitation experiments using stage 10–13 Immunostaining of embryos with antibodies against MTf (Supplementary embryonic extracts demonstrated that a substantial portion of MTf is Information, Fig. S1o, p), Nrx (Fig. 1h, i and Supplementary Information, already forming a complex with Cont and Nrx at early stages of septate Fig. S1r, s) and Discs large (Dlg)18 (Supplementary Information, Fig. S1t, junction formation (Supplementary Information, Fig. S2c). The wide- u) demonstrated that MTf co-localizes with these septate junction mark- spread distribution of these complexes along basolateral membranes ers both in the trachea and in the hindgut. To determine whether MTf at stage 13 markedly contrasts their restricted apicolateral localization localization depends on septate junction components, MTf localization at stage 16 (Fig. 2b, d and Supplementary Information, Fig. S2b). TEM was analysed in cora and ATPα mutants19,20 (Fig. 1j, k). Although the analysis and epithelial permeability assays from previous studies suggest staining intensity was not affected, a pronounced basolateral spread of that mature septate junctions are established during the interval between MTf was detected in the mutants. Thus, MTf localization requires septate stage 13 and 16 (ref. 2). We hypothesized that septate junction assem- junction integrity, and vice versa, suggesting that MTf is an integral septate bly may involve endocytosis of septate junction components from the junction component. To directly test if MTf associates with septate junc- basolateral surface. We therefore assessed the potential co-localization of tion proteins, co-immunoprecipitation was performed from embryonic septate junction proteins with endosomal markers at different stages. We extracts. The MTf antiserum co-immunoprecipitated Nrx, Contactin observed a substantial overlap of MTf staining with the GFP–2×FYVE (Cont)21 and neuroglian (Nrg)6. In a complementary experiment Nrx and the GFP–Rab5 markers in early endosomes and multivesicular bod- antibodies precipitated MTf and Cont (Fig. 1l). Thus, MTf forms com- ies at stage 13 (Fig. 2e, f). To extend these findings and avoid potential plexes with Nrx, Cont and Nrg during late embryogenesis. Adherens reporter-derived artifacts, endogenous Rab5 (ref. 22), Rab11 (ref. 23) and
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a WT b WT i 1 23456789 Anti- Calreticulin Anti- Syntaxin16 MTf MTf Anti- c WT d WT Syntaxin Anti- Rab5 Anti- MTf MTf MTf e btl>GFP–2×-FYVE f btl>GFP–Rab5
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Anti-Nrg st.10-13
Anti-Nrg st.14-17 MTf Rab5 MTf Rab11 69B>GFP–Rab5 69B>GFP–Rab11 69B>GFP–GPI
Figure 2 MTf co-localizes with early and recycling endosomes. (a-d) anti-MTf and anti-Rab11 (h). Scale bar a–h, 10 μm. Images on the Confocal microscopy section of trachea (a, b) and hindgut (c, d) tissues right of e–h show localization of individual proteins (top, middle) and immunostained with anti-MTf. MTf is localized along the basolateral a merge of these images (bottom) from a region of the image on left. membrane and in intracellular puncta at stage 13 in tracheal (a) and (i) Immunoblots of fractions (1; top–9; bottom) from an equilibrium hindgut (c) cells. At stage 16, MTf accumulates predominantly at density gradient of wild-type membrane extracts. (j) Membrane extracts the apicolateral membrane in the trachea (b) and hindgut (d) cells. from wild-type embryos at stage 10–13 and stage 14–17 expressing (e, f) Confocal microscopy section of tracheal cells from a stage 13 GFP–Rab5, GFP–Rab11 and GFP–GPI were immunoprecipitated with embryo, stained with anti-MTf and expressing GFP–2×FYVE (e) or anti-GFP. Co-immunoprecipitated proteins were resolved and identified GFP–Rab5 (f). (g, h) Confocal microscopy of hindgut sections from by western blotting using the indicated antibodies. Input; 25% of stage 13 embryos, immunostained with anti-MTf and anti-Rab5(g) and extract that was used for immunoprecipitation.
MTf were immunostained in the hindgut epithelium. MTf co-localized barely detectable in the GFP–Rab5 and GFP–Rab11 immunoprecipitates with Rab5 puncta at stage 13 indicating that MTf is internalized by Rab5- from later embryonic stages, supporting the hypothesis that these pro- mediated endocytosis (Fig. 2g and Supplementary Information, Fig. teins transiently associate with early and recycling endosomes (Fig.2j S2d–f). Interestingly, partial co-localization of MTf with Rab11 at stage and Supplementary Information, Fig. S3m). The immunofluorescence, 13 was also detected, suggesting that MTf is recycled to the apicolateral fractionation and immunoprecipitation experiments indicate that MTf membrane region by Rab11 (Fig. 2h). At stage 16 however, when septate and septate junction components enter and traffic through early and junctions are fully functional, we did not detect MTf puncta or co-locali- recycling endosomes during septate junction maturation. zation of MTf with endocytic markers (Supplementary Information, Fig. To address the potential role of endocytosis in septate junction matura- S2g–j). Next, we investigated whether other septate junction proteins like tion, we expressed dominant-negative forms of Rab5 (rab5DN) and Rab11 Cora, gliotactin and sinu are also redistributed during septate junction (rab11DN) in ectodermal epithelia and analysed MTf localization. In the maturation. All three septate junction proteins were transiently detected hindgut of wild-type embryos, MTf was first detected distributed evenly in intracellular puncta at stage 13, but not at stage16 (Supplementary along basolateral cell membranes (Fig. 3a), and then accumulated at the api- Information, Fig. S3a-l’), consistent with the low turnover of septate junc- colateral region (Fig. 3b). In contrast, all of the embryos expressing rab5DN tion proteins at stage 16 (ref. 24). To corroborate the endosomal MTf (n = 10), and 40% of those expressing rab11DN (n = 12), retained the broad localization, we fractionated wild-type membrane extracts by equilibrium MTf distribution at stage 16, resembling wild-type embryos at stage 13 density-gradient centrifugation and monitored MTf levels in the frac- (Fig. 3c, d). Also, in clathrin heavy chain (chc1) and dynamin (shits) mutants, tions. A substantial part of MTf was present in the early endosomal mem- shifted to the restrictive temperature at stage 11, MTf and Cora failed to brane fraction marked by Rab5 (Fig. 2i). In a complementary experiment, accumulate to the apicolateral region of hindgut cells at stage 16 (Fig. 3e, we expressed GFP–Rab5, GFP–Rab11 or GFP–GPI using 69B–GAL4, f and Supplementary Information, Fig. S4g) and showed the characteris- and used anti-GFP antibodies for immunoprecipitations. MTf and Nrg tic spread-localization of earlier stages (Supplementary Information, Fig. were specifically detected in the complexes from both the GFP–Rab5 S4a–f). Embryos expressing rab5DN or rab11DN, and chc1 and shits mutants, and GFP–Rab11 embryonic extracts at stage 10–13. However, they were also demonstrated MTf mislocalization, and overelongated dorsal trunks
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a WT b WT To elucidate the molecular role of MTf in septate junction assembly, we analysed its conserved sequence motifs. First, we tested if Drosophila MTf is a GPI-linked membrane protein, similarly to its human homologue. We probed membrane and cytosolic fractions of embryonic lysates for MTf, the transmembrane protein Syntaxin 1A (Syx1A)29, and the GPI- linked protein, Knickkopf (Knk)30. MTf was predominantly detected in the membrane fraction together with Syx1A and Knk (Fig. 4b). MTf MTf Incubation with phoshatidylinositol-phospholipase Cγ (PI-PLCγ) lib- DN DN c 69B>rab5 d 69B>rab11 erated MTf and Knk, but not Syx1A, into the supernatant, indicating that MTf associates with the plasma membrane through a GPI-modification motif (Fig. 4b). To test significance of the GPI-modification motif, we deleted it from MTf (generating a secreted MTfΔGPI protein), or exchanged it for the mouse CD8 transmembrane domain (MTf-TM). We overexpressed MTf, or the MTfΔGPI or MTf-TM constructs in the trachea of MTf mutants. Soluble MTfΔGPI rescued Nrx mislocaliza- MTf MTf tion and tube overelongation, presumably by binding to membrane- e chc1 f shits associated septate junction proteins. (Fig. 4c–e). Notably, MTf-TM did not rescue these MTf phenotypes, although it was expressed at similar levels (Fig. 4f). This suggests that the MTf GPI-modification may be cleaved or have another function. Next, we investigated if Drosophila MTf binds iron. Iron-binding by serum transferrin is mediated by Tyr 188 of the amino-terminal 31 32 MTf MTf lobe and the Tyr 426 of the carboxy-terminal lobe . To test the iron- binding capacity of Drosophila MTf, we mutated the corresponding tyrosines, Tyr 231 or Tyr 533, to phenylalanine33, and also deleted the Figure 3 Endocytosis is required for MTf localization. (a, b) Confocal microscopy of hindgut sections from stage 13 (a) and stage 16 (b) wild- GPI-modification sequence to generate soluble forms of His-tagged type embryos immunostained with anti-MTf. (c, d) Confocal microscopy wild-type and mutated MTf in S2 cells (Fig. 4a). All constructs were of hindgut sections, immunostained with anti-MTf, from stage 16 expressed and secreted at similar levels, suggesting that the substitutions embryos expressing dominant-negative rab5 (c) and dominant-negative did not alter protein secretion or stability. Secreted MTf proteins were rab11 constructs (d). (e, f) Confocal microscopy of hindgut sections, immunostained with anti-MTf, from stage 16 chc1- and shits-mutant affinity purified along with mouse MTf and vermiform as positive and embryos. Scale bar, 10 μm. negative controls, respectively. Atomic absorption spectroscopy detected similar levels of bound iron to the wild-type Drosophila and mouse MTf (Supplementary Information, Fig. S4). The paracellular barrier integrity proteins (Supplementary Information, Fig. S6a). The Y231F substitu- of chc1 mutants was compromised, as dye leaked into the trachea in 36% of tion in the N-terminal lobe of MTf reduced iron binding, whereas the the analysed mutants (n = 11). This genetic analysis indicates that endocy- Y533F mutation completely abolished it. To further investigate MTf tosis is required for functional septate junction assembly. Endocytosis also iron binding, the electron paramagnetic resonance (EPR) spectra of the has a prominent role in adherens junction remodelling and stabilization. MTf proteins were compared (Fig. 4i). The Drosophila and mouse MTf Previous studies demonstrated that DE-cadherin accumulates intracel- spectra were strikingly similar. These EPR profiles are comparable to lularly in pupal tissues of shits mutants at the restrictive temperature25,26. those of other Tf proteins, and originate from a high-spin Fe(III)-ion34. In contrast, the localization and the levels of Crb and DE-cadherin were Vermiform and MTFY533F, at comparable protein concentrations, did not not affected in our experiments (Supplementary Information, Fig. S4). have a detectable EPR signal (Fig. 4i), supporting the hypothesis that the This reveals a direct role for endocytosis in functional septate junction MTf C-terminal lobe is the site of Fe(III)-binding specificity. Next, we assembly, independent of its earlier function in DE-cadherin recycling investigated if Fe(III)-binding is required for MTf function in epithelial and adherens junction reinforcement27. The rab11DN phenotypes suggest junction assembly. Tracheal overexpression of the Y231F MTf protein, that septate junction components are targeted from the basolateral to the which only partly reduces iron-binding, fully rescued the tube over- apicolateral membrane region through recycling endosomes. The similari- growth and the Nrx mislocalization defects in MTf mutants (Fig. 4g). ties of the septate junction defects in MTf-mutant embryos and in mutants This construct also partially rescued the lethality of MTf mutant larvae compromising endocytosis prompted us to test the endocytic capacity of when expressed by the ectodermal driver 69B–GAL4 (data not shown). MTf epithelial cells. The intensity and the localization of Rab5 and Rab11 Conversely, expression of the iron-deficient Y533F construct failed to immunostaining were not affected in embryos with the MTf mutation rescue the MTf mutant phenotypes (Fig. 4h). This indicates that iron- (Supplementary Information, Fig. S5a–d). Additionally, the endocytic binding is crucial for MTf function and suggests that iron-bound MTf clearance of the tracheal luminal protein Gasp28 was unaffected in MTf has a direct role in septate junction assembly. Additionally, it may reflect mutants, compared with wild-type (Supplementary Information, Fig. a role for MTf in iron homeostasis. To test this, we monitored ferritin S5e–h). Thus, the endocytic machinery is intact in MTf mutants, sug- levels, which provide a sensitive indicator of aberrant cellular iron con- gesting that the junction assembly mechanism relies on endocytosis of centration35. Fer1 and Fer2 levels remained unaffected in MTf mutants MTf and other septate junction proteins. indicating that MTf does not have a major role in cellular iron uptake
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Figure 4 MTf is a GPI-anchored iron-binding protein. (a) Schematic and in MTf mutants expressing MTf (d), MTf∆GPI (e) MTf-TM (f), MTfY231F representation of the MTf protein. Arrows indicate the residues at which (g) and MTfY533F (h) under the control of the btl–Gal4 driver. Cells were substitution mutations were made (Y231F and Y533F), and the GPI domain, immunostained with antibodies specific to MTf and Nrx; images on the left which was deleted in the MTf∆GPI mutant. (b) Proteins in membrane and indicate localization of Nrx and images on the right indicate co-localization of supernatant fractions of lysed wild-type embryos treated with PI-PLCγ, as Nrx and MTf. Scale bar, 10 μm. (i) EPR spectra of MTf, MTf Y533F, mouse MTf indicated, were resolved and identified by western blotting. The GPI-anchored (mMTf) and vermiform (Verm). A g-factor signal of 4.3 indicates a high-spin protein Knk and the transmembrane protein Syx1A were used as controls. Fe(III) atom is bound to the protein. The signal was absent in the preparations (c–h) Confocal microscopy images of the trachea in an MTf mutant (c), containing the mutated MTf or the unrelated protein vermiform.
(Supplementary Information, Fig. S6b–e). This is consistent with the puncta and also along the lateral membranes of the receiving cells, functional analysis of mouse and human MTf 11. In converse experi- suggesting that it is endocytosed (Fig. 5g, h). MTFY533FΔGPI was not ments, no septate junction abnormalities were found in fer1 and fer2 detected in adjacent cells, despite its expression at comparable levels in mutants suggesting that iron homeostasis does not directly affect septate cells expressing en–GAL4, indicating that MTf internalization requires junction assembly (Supplementary Information, Fig. S6f, g). Thus, the Fe(III)-binding. Next, we investigated if MTf endocytosis induces sep- genetic analysis suggests that MTf iron binding and endocytosis are tate junction assembly in the recipient cells. We immunostained MTf specifically required for septate junction maturation. mutant epidermal cells, expressing GFP and MTf constructs, with anti- Next, we investigated if endocytocis of Fe(III)-bound MTf is suf- bodies specific to Nrx and MTf. The wild-type protein induced Nrx ficient to induce functional septate junction assembly. We expressed apicolateral accumulation only in the en–GAL4 expressing cells (Fig. 5a, wild-type MTf, MTfΔGPI and MTfY533FΔGPI in epidermal stripes of b). As expected, the iron-binding deficient construct, MTFY533FΔGPI, MTf mutants using en–GAL4. The MTf constructs were detected in en– did not rescue the MTf mutant (Fig. 5e, f). Conversely, MTfΔGPI from GAL4 expressing cells, which were visualized by parallel expression of the en–GAL4 cells fully rescued Nrx mislocalization in these cells, as UAS–GFP–CAAX (CAAX, membrane-targeting motif, where X is any well as in the adjacent cells (Fig. 5c, d). In these embryos there was also residue; Fig. 5a, c, e). The wild-type protein was undetectable in sur- rescue of the convoluted tracheal phenotype, indicating that exogenous rounding cells lacking transgenic MTf expression. Strikingly, MTfΔGPI MTf from the epidermis was endocytosed, which restored tracheal sep- (Fig. 5c, d) also accumulated in cells not expressing en–GAL4. Thus, tate junction assembly and function. These results demonstrate that epidermal cells that lack MTf bind and internalize exogenous, secreted endocytosis of Fe(III)-bound MTf is sufficient to induce functional MTfΔGPI. The exogenous MTfΔGPI partly localized in Rab11-positive septate junction assembly in MTf-mutant cells.
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a en>GFP–CAAX,MTf; MTf
MTf Nrx GFP MTf Nrx GFP b
MTf Nrx GFP MTf Nrx GFP
c en>GFP–CAAX,MTfGPI; MTf
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MTf Nrx GFP MTfNrxGFP e en>GFP–CAAX,MTf Y533FGPI; MTf
MTf Nrx GFP MTf Nrx GFP f
MTf Nrx GFP MTf Nrx GFP g en>GFP–CAAX,MTfGPI; MTf
MTf Rab11 GFP MTf Rab 11GFP h
MTf Rab11 GFP MTf Rab11 i Apical
Basal Endocytosis Mature SJs st.13 st.15 SJ components MTf Iron Early endosome Recycling endosome
Figure 5 Uptake of MTf induces septate junction assembly in MTf mutant en–GAL4 cells are expressing GFP. (h) Confocal microscopy x–z images of cells. (a–f) Confocal microscopy x–y images of embryonic ectodermal cells cells shown in g. Co-localization of MTf and Rab11 is visualized in white from en>GFP–CAAX, MTf; MTf (a, b), en>GFP–CAAX, MTf ΔGPI; MTf (right). GFP–expressing cells are marked by a blue bracket (right). In all (c, d) and en>GFP–CAAX, MTf Y533FΔGPI; MTf (e, f) embryos. Cells were x–y sections the apical cell surface is up. Scale bar, 10 μm. (i) Model immunostained with antibodies specific to MTf and Nrx, as indicated. of septate junction maturation during embryogenesis. Septate junction In all of the imaged embryos, engrailed cells were expressing GFP, as components at stage 13 are deposited along the whole lateral and at the indicated. a, c, and e show the epidermis from above, whereas b, d and f basal membranes. Fe(III)–MTf induces endocytosis and redistribution show cross sections. Image on the right is a merge of the images on the of septate junction complexes from the basolateral to the apicolateral left. (g) Confocal microscopy x–y images of en>GFP–CAAX, MTf ΔGPI; membrane. After septate junction assembly by stage15, Fe(III)–MTf may MTf embryos immunostained with antibodies specific to MTf and Rab11. also promote junction stability.
We have identified MTf as a component of Drosophila septate junctions. vesicles remains unknown but their localized delivery at the apicolateral We suggest that Fe(III)-binding may enhance its affinity for transmem- region may increase the local concentration of adhesion molecules on brane septate junction components and induce endocytosis and clearance neighbouring cell surfaces and promote septate junction formation. Once of septate junction protein complexes from the basolateral surface of epi- at the apicolateral membrane region, MTf may bind circulatory iron and thelial cells at stage 13. On internalisation, the complexes may dissociate also stabilize the junctions (Fig. 5i). Our data suggest that MTf may also in endosomes, freeing MTf and septate junction proteins for recycling function in paracellular junction assembly in vertebrates and provide clues to the apicolateral membrane. The targeting mechanism of the recycling for the elucidation of its postulated role in melanoma tumorigenesis.
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METHODS 13. Tomancak, P. et al. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3, research0088–0088.14 (2002). Methods and any associated references are available in the online version 14. Wang, S. et al. Septate-junction-dependent luminal deposition of chitin deacetylases of the paper at http://www.nature.com/naturecellbiology/ restricts tube elongation in the Drosophila trachea. Curr. Biol. 16, 180–185 (2006). 15. Lamb, R. S., Ward, R. E., Schweizer, L. & Fehon, R. G. Drosophila coracle, a member of Note: Supplementary Information is available on the Nature Cell Biology website the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells. Mol. Biol. Cell. 9, 3505–3519 (1998). ACKNOWLEDGMENTS 16. Baumgartner, S. et al. A Drosophila neurexin is required for septate junction and blood- We are indebted to S. Baumgartner, M. González-Gaitán, R. Fehon, G. Beitel, nerve barrier formation and function. Cell 87, 1059–1068 (1996). M. Bhat, F. Missirlis, H. Bellen, M. Hortsch, D. Ready, V. Auld, the Bloomington 17. Fehon, R. G., Dawson, I. A. & Artavanis-Tsakonas, S. A Drosophila homologue of mem- Drosophila Stock Center, the Drosophila Genomics Resource Center (DGRC; brane-skeleton protein 4.1 is associated with septate junctions and is encoded by the Indiana), and the Developmental Studies Hybridoma Bank (DSHB; Iowa) for coracle gene. Development 120, 545–557 (1994). Drosophila strains, clones and antibodies. We thank M. Sahlin for initial help with 18. Woods, D. F. & Bryant, P. J. The discs-large tumor suppressor gene of Drosophila iron detection, T. Astlind for the EPR measurements and M. Björk and I. Granell encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451–464 for expert technical assistance. We are grateful to members of the Samakovlis lab (1991). 19. Hemphala, J., Uv, A., Cantera, R., Bray, S. & Samakovlis, C. Grainy head controls api- for suggestions. K.S. was supported by an EMBO postdoctoral fellowship. The work cal membrane growth and tube elongation in response to Branchless/FGF signalling. was supported by grants from the G. Gustafsson Foundation, Vetenskapsrådet and Development 130, 249–258 (2003). Cancerfonden to C.S. 20. Paul, S. M., Ternet, M., Salvaterra, P. M. & Beitel, G. J. The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal AUTHOR CONTRIBUTIONS system. Development 130, 4963–4974 (2003). C.S., K.S. and K.T. designed the experiments. K.T. performed the experiments. S.W. 21. Faivre-Sarrailh, C. et al. Drosophila contactin, a homolog of vertebrate contactin, is identified the tracheal overelongation phenotype of the MTf P-element mutant. required for septate junction organization and paracellular barrier function. Development A.G. analysed the EPR data. C.S., K.T. and K.S. wrote the paper. 131, 4931–4942 (2004). 22. Wucherpfennig, T., Wilsch-Brauninger, M. & Gonzalez-Gaitan, M. Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. COMPETING FINANCIAL INTERESTS J. Cell Biol. 161, 609–624 (2003). The authors declare that they have no competing financial interests. 23. Emery, G. et al. Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 122, 763–773 (2005). Published online at http://www.nature.com/naturecellbiology 24. Laval, M., Bel, C. & Faivre-Sarrailh, C. The lateral mobility of cell adhesion molecules Reprints and permissions information is available online at http://npg.nature.com/ is highly restricted at septate junctions in Drosophila. BMC Cell Biol. 9, 38 (2008). reprintsandpermissions/ 25. Leibfried, A., Fricke, R., Morgan, M. J., Bogdan, S. & Bellaiche, Y. Drosophila Cip4 and WASp define a branch of the Cdc42–Par6–aPKC pathway regulating E-cadherin 1. Farquhar, M. G. & Palade, G. E. Junctional complexes in various epithelia. J. Cell Biol. endocytosis. Curr. Biol. 18, 1639–1648 (2008). 17, 375–412 (1963). 26. Georgiou, M., Marinari, E., Burden, J. & Baum, B. Cdc42, Par6 and aPKC regulate 2. Tepass, U. & Hartenstein, V. The development of cellular junctions in the Drosophila Arp2/3-mediated endocytosis to control local adherens junction stability. Curr. Biol. 18, embryo. Dev. Biol. 161, 563–596 (1994). 1631–1638 (2008). 3. Krupinski, T. & Beitel, G. J. Unexpected roles of the Na-K-ATPase and other ion trans- 27. Shaye, D. D., Casanova, J. & Llimargas, M. Modulation of intracellular trafficking porters in cell junctions and tubulogenesis. Physiology 24, 192–201 (2009). regulates cell intercalation in the Drosophila trachea. Nat. Cell Biol. 10, 964–970 4. Banerjee, S., Sousa, A. D. & Bhat, M. A. Organization and function of septate junctions: (2008). an evolutionary perspective. Cell Biochem. Biophys. 46, 65–77 (2006). 28. Tsarouhas, V. et al. Sequential pulses of apical epithelial secretion and endocytosis drive 5. Laprise, P. et al. Yurt, Coracle, Neurexin IV and the Na+, K+-ATPase form a novel group airway maturation in Drosophila. Dev. Cell 13, 214–225 (2007). of epithelial polarity proteins. Nature 459, 1141–1145 (2009). 29. Cerezo, J. R., Jimenez, F. & Moya, F. Characterization and gene cloning of Drosophila 6. Genova, J. L. & Fehon, R. G. Neuroglian, Gliotactin, and the Na+/K+ ATPase are essential syntaxin 1 (Dsynt1): the fruit fly homologue of rat syntaxin 1. Brain Res. Mol. Brain Res. for septate junction function in Drosophila. J. Cell Biol. 161, 979–989 (2003). 29, 245–252 (1995). 7. Laprise, P. et al. Epithelial polarity proteins regulate Drosophila tracheal tube size in 30. Moussian, B. et al. Drosophila Knickkopf and Retroactive are needed for epithelial tube parallel to the luminal matrix pathway. Curr. Biol. 20, 55–61 (2009). growth and cuticle differentiation through their specific requirement for chitin filament 8. Suryo Rahmanto, Y., Dunn, L. L. & Richardson, D. R. The melanoma tumor antigen, organization. Development 133, 163–171 (2006). melanotransferrin (p97): a 25-year hallmark—from iron metabolism to tumorigenesis. 31. He, Q. Y. et al. Inequivalence of the two tyrosine ligands in the N-lobe of human serum Oncogene 26, 6113–6124 (2007). transferrin. Biochemistry 36, 14853–14860 (1997). 9. Alemany, R. et al. Glycosyl phosphatidylinositol membrane anchoring of melanotransfer- 32. Mason, A. B. et al. Mutational analysis of C-lobe ligands of human serum transferrin: rin (p97): apical compartmentalization in intestinal epithelial cells. J. Cell Sci. 104, insights into the mechanism of iron release. Biochemistry 44, 8013–8021 (2005). 1155–1162 (1993). 33. Lambert, L. A., Perri, H., Halbrooks, P. J. & Mason, A. B. Evolution of the transferrin 10. Demeule, M. et al. Regulation of plasminogen activation: a role for melanotransferrin family: conservation of residues associated with iron and anion binding. Comp. Biochem. (p97) in cell migration. Blood 102, 1723–1731 (2003). Physiol. B, Biochem. Mol. Biol. 142, 129–141 (2005). 11. Sekyere, E. O., Dunn, L. L., Rahmanto, Y. S. & Richardson, D. R. Role of melanotrans- 34. Farnaud, S. et al. Biochemical and spectroscopic studies of human melanotransferrin ferrin in iron metabolism: studies using targeted gene disruption in vivo. Blood 107, (MTf): electron-paramagnetic resonance evidence for a difference between the iron- 2599–2601 (2006). binding site of MTf and other transferrins. Int. J. Biochem. Cell Biol. 40, 2739–2745 12. Rahmanto, Y. S. & Richardson, D. R. Generation and characterization of transgenic (2008). mice hyper-expressing melanoma tumour antigen p97 (Melanotransferrin): no overt 35. Hentze, M. W., Muckenthaler, M. U. & Andrews, N. C. Balancing acts: molecular control alteration in phenotype. Biochim. Biophys. Acta 1793, 1210–1217 (2009). of mammalian iron metabolism. Cell 117, 285–297 (2004).
NATURE CELL BIOLOGY VOLUME 12 | NUMBER 11 | NOVEMBER 2010 1077 © 2010 Macmillan Publishers Limited. All rights reserved METHODS DOI: 10.1038/ncb2111
METHODS 10 mM Tris at pH 7.5, 5 mM EDTA, 0.25 M sucrose and protease inhibitor cocktail Drosophila strains. The KG-type P element, KG01571, is inserted into the 5ʹ (Roche). KCl was added to a final concentration of 100 mM and the extract was UTR (untranslated region) of the MTf gene. Imprecise and precise excisions centrifuged at 3000g for 10 min. The supernatant was layered on top of discontinu- of the P element yielded several viable and lethal strains (non-complementing ous 2.5–30% OptiPrep gradient (Sigma). After centrifugation at 37,000g for 1 h, the lethality of Df(3L)5492). One of the lethal strains, excision #234, contains a nine factions were collected and analysed by western blotting. 626 bp deletion that removes the MTf 5ʹ UTR and 552-bp coding sequence, and The measurement of iron in His-tagged MTf, MTf Y231F, MTf Y533F , mMTf (all was used for the analysis. The deletion is flanked by two following sequences: of which had the GPI motif deleted) and vermiform was performed by atomic 5ʹ-CAAAACAAACTCATTAA-3ʹ and 5ʹ-GAACCGGCCAACCTAACACG-3ʹ. absorption spectroscopy (AAS) (London & Scandinavian Metallurgical). The pro- w 1118 was used as the wild-type strain. Other alleles used include Fer1HCH teins were expressed in Drosophila Schneider cells and purified from the medium and Fer2LCH36. For co-localization experiments we used the UAS transgenes using Ni-NTA agarose (Qiagen). GFP–2×FYVE, GFP–Rab5 and GFP–Rab11 (ref. 22). Other constructs used were UAS–Rab5DN (ref. 37) and UAS–Rab11DN (ref. 38). chc1 and shits mutants EPR spectroscopy. EPR spectra were recorded on an Elexsys CW X-band spec- are described in Flybase (http://flybase.org/). Crosses for ectopic expression using trometer (Bruker) with an ESR900 Helium Cryostat (Oxford Instruments) at btl–GAL4, en–GAL4 and 69B–GAL4 drivers were performed at 25 °C, except for 12.0 K ± 0.2 K, 0.63 mW microwave power, approximately 9.63 GHz microwave the shits mutant where embryos were raised at 22 °C for 8 h and than shifted to frequency and 0.5 mT modulation amplitude. A dual mode ER 4116DM micro- 31 °C for 5 h. In all experiments CyO, TM3 and FM7c balancer strains carrying wave cavity in perpendicular mode was used. All spectra were recorded under GFP or lacZ transgenes were used as necessary to identify embryos with the identical conditions. The estimated concentration of the measured proteins was: desired genotypes. MTf, 10 ng μl–1, MTf Y533F, 10 ng μl–1, mMTf, 20 ng μl–1 and vermiform, 25 ng μl–1.
Immunostaining, TEM and dextran injection. Immunohistochemistry, TEM Immunostaining and western-blotting antibodies. The following antibodies and 10 K dextran (Molecular Probes) injection was performed as previously were used at the indicated dilutions: guinea pig anti-MTf (1:500 for immunos- described14,15. Images were acquired with a Zeiss Meta confocal microscope and taining, 1:1,000 for western blotting); mouse anti-tracheal luminal 2A12 (1:5; Tecnai G 2 Spirit BioTWIN electron microscope. Images were processed with DSHB), guinea pig anti-Cor17 (1:5,000), rabbit anti-Nrx16 (1:1,000), mouse anti- Adobe Photoshop. Dlg (1:10; DSHB), mouse anti-Nrg 1B7 (1:500; ref. 41), guinea pig anti-Cont21 (1:1,000), rabbit anti-sinuous42 (1:300), mouse anti-gliotactin43 (1:200), rat anti- Molecular Biology. MTf antisera were generated against a recombinant protein DE-cadherin (1:50; DSHB), mouse anti-armadillo (1:50; DSHB), mouse anti-GFP corresponding to amino acid residues 32–183 of MTf fused to the 6×His-tag JL8 (1:1,000; Clontech), rabbit anti-GFP (1:500; Molecular Probes), guinea pig from pET28a. It was purified on Ni–NTA resin and used to immunize guinea anti-vermiform (1:500), guinea pig anti-gasp28 (1:1,000), rabbit anti-Galleria mel- pigs (Eurogentec). lonella calreticulin44 (1:500), mouse anti-syntaxin (1:100, 8C3; DSHB), rabbit anti- UAS–MTf and UAS–mMTf transgenes were generated by subcloning the syntaxin 16 (1:500; abcam), rabbit anti-knk30 (1:500), rabbit anti-Rab522 (1:50), inserts of LD22449 for MTf and BC040347 for mMTf into pUAST. MTf ΔGPI rabbit anti-Rab1123 (1:50), rabbit anti-Fer1HCH45 (1:400), rabbit anti-Fer2LCH45 and MTf-TM constructs were made by PCR amplification and subcloning into (1:400), rabbit anti-β-gal (1:500; Cappel), fluorescein-conjugated ChtB (1:500; pUAST: MTf ΔGPI lacks amino-acid residues 798–820 and MTf-TM carries New England BioLabs). the TM domain of mouse CD8a protein39 fused to MTf at amino-acid residue 798. The GPI modification site was predicted by http://mendel.imp.ac.at/gpi/ 36. Spradling, A. C. et al. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153, gpi_server.html (ref. 40). 135–177 (1999). QuickChange Site-Directed Mutagenesis Kit (Stratagene) was used to produce 37. Entchev, E. V., Schwabedissen, A. & Gonzalez-Gaitan, M. Gradient formation of the mutated forms of UAS– MTf. In the N-terminal lobe a Y231F substitution was TGF-β homolog Dpp. Cell 103, 981–991 (2000). 38. Li, B. X., Satoh, A. K. & Ready, D. F. Myosin V, Rab11, and dRip11 direct apical secre- made, and in the C-terminal lobe a Y533F substitution. tion and cellular morphogenesis in developing Drosophila photoreceptors. J. Cell Biol. 177, 659–669 (2007). Immunoprecipitation, membrane preparation and western Blotting. 39. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene Immunoprecipitation experiments were performed as described21. function in neuronal morphogenesis. Neuron 22, 451–461 (1999). Immunoprecipitates were analysed by immnunoblotting in a standard western 40. Eisenhaber, B., Bork, P. & Eisenhaber, F. Prediction of potential GPI-modification sites in proprotein sequences. J. Mol. Biol. 292, 741–758 (1999). blotting procedure. 41. Bieber, A. J. et al. Drosophila neuroglian: a member of the immunoglobulin super- Wild-type embryos were homogenized in a solution containing 10 mM Tris family with extensive homology to the vertebrate neural adhesion molecule L1. Cell at pH 8.0, 1 mM EDTA (ethylenediaminetetraacetic acid) and protease inhibitor 59, 447–460 (1989). cocktail (Roche). The homogenate was centrifuged for 10 min at 1,500g. The 42. Wu, V. M., Schulte, J., Hirschi, A., Tepass, U. & Beitel, G. J. Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control. pellet was resuspended in the same buffer and centrifuged. The supernatant was J. Cell Biol. 164, 313–323 (2004). centrifuged for 10 min at 4,000g. The supernatant was laid on a 0.25 M sucrose 43. Venema, D. R., Zeev-Ben-Mordehai, T. & Auld, V. J. Transient apical polarization of cushion and centrifuged at 70,000g for 1 h. The pellet was dissolved in PI-PLCγ Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila. buffer and treated with PI-PLCγ (PROzyme) for 2 h at 37 °C and centrifuged for Dev. Biol. 275, 301–314 (2004). 44. Kuraishi, T. et al. Identification of calreticulin as a marker for phagocytosis of apoptotic 30 min at 30,000g. Pellets and supernatants were analysed by western blot. cells in Drosophila. Exp. Cell Res. 313, 500–510 (2007). The fractionation of membranes on equilibrium density gradient was per- 45. Missirlis, F. et al. Characterization of mitochondrial ferritin in Drosophila. Proc. Natl formed with extracts of wild-type embryos homogenized in a solution containing Acad. Sci. USA 103, 5893–5898 (2006).
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DOI: 10.1038/ncb2111
Figure S1 MTf mutant embryos have non-functional SJs (a) The MTf locus stage 15 wild-type embryos reveals an intact paracellular barrier as the dye fails including the P element (KG01571) insertion site and the MTf mutant to penetrate into the lumen of the trachea (i). In MTf mutant embryos the dye deletion is indicated by the black bar. (b) Histogram showing that dorsal penetrates the lumen of the trachea indicating the defects of the paracellular trunk length is significantly different in MTf mutant compared to wild-type barrier (j). Scale bar: 25 μm. (k, l) A confocal microscopy section of wild-type embryos (p<0.0001, n=7). Dorsal trunk length is expressed as a ratio of dorsal and MTf mutant stained for the SJ component Cora. In MTf mutant, Cora is trunk length (metamer 1-8) / embryo length. (c, d) Expression of UAS-MTf mislocalized and spread basolaterally. (m, n) A confocal microscopy projection and UAS-mMTf in MTf mutants with the tracheal driver btl-GAL4 rescues of wild-type and MTf mutant trachea stained for DE-cad. DE-cad is properly the Verm secretion and tube over-elongation phenotypes. (e, f) Wild-type and localized in MTf mutant. (o, p) btl>GFP-CAAX; MTf mutant embryos stained MTf mutants expressing GFP-CAAX in the trachea stained for Verm and GFP. with MTf antiserum. GFP immunostaining visualized tracheal cells. (r, s) Verm is secreted to the tracheal lumen in wild-type embryos, but remains Immunostaining of embryonic hindgut cells reveals colocalization of MTf and intracellular in MTf embryos at stage 15. (g, h) Wild-type and MTf mutant Nrx in confocal sections. (t, u) Immunostaining of embryonic tracheal cells trachea labeled with a FITC-conjugated chitin binding probe (ChtB). Inserts shows colocalization of SJ components Dlg and MTf. Scale bar: 10 μm. (v) MTf display y-z projections. The filamentous cable is detected in the wild-type and Nrx are coimmunoprecipitated by MTf antibodies from extracts of wild-type embryos (g). Its structure is diffuse and expanded in the mutants (h). Scale embryos, but not DE-cad and Arm. DE-cad and Arm are precipitated by DE-cad bar: 10 μm. (i, j) Injection of rhodamine-labeled dextran into the body cavity of antibodies. 5% of extract that was used for IP was loaded in the input lane.
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Figure S2 SJ protein complexes transiently colocalize with endosomal markers. serum was used as the negative controls. 5% of extract that was used for IP (a, b) A confocal section of hindgut shows Nrg localization along the basolateral was loaded in the input lane. (d , e, f) Overlap between MTf and Rab5 positive membrane and in intracellular puncta at stage 13 (a) and apicolateral intracellular vesicles at stage 13. (g, h) Minimal colocalization between MTf accumulation at stage 16 (b). (c) Nrx and Cont coimmunoprecipitate from and expessed GFP-2x-FYVE (g) and MTf and expressed GFP-Rab5 (h) in the extracts of stage 10-13 wild-type embryos with MTf. Cont precipitates Nrx tracheal cells at stage 16. (i, j) Minimal colocalization between MTf and Rab5 from stage 10-13 wild-type embryo extracts using Nrx antibody. Pre-immune (i) and MTf and Rab11 (j) in the hindgut cells at stage 16. Scale bar: 10 μm.
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Figure S3 SJs components partially colocalize with early and recycling l’). (g-i’) 69B-GAL4 embryos expressing GFP-Rab5 stained for GFP (g’, h’, endosomes at stage 13. Confocal microscopy sections of stage 13 hindgut i’) and SJs components: Cora (g), Glio (h), Sinu (i). (j-l’) 69B-GAL4 embryos (a-f’). (a-c’) 69B-GAL4 embryos expressing GFP-Rab5 stained for GFP (a’, b’, expressing GFP-Rab11 stained for GFP (j’, k’, l’) and SJs components: Cora c’) and SJs components: Cora (a), Glio (b), Sinu (c). (d-f’) 69B-GAL4 embryos (j), Glio (k), Sinu (l). Scale bar: 10 μm. (m) Western blots from stage 10-13 expressing GFP-Rab11 stained for GFP (d’, e’, f’) and SJs components: Cora and stage 14-17 wild-type embryos expressing GFP-Rab5, GFP-Rab11 and (d), Glio (e), Sinu (f). Confocal microscopy sections of stage 16 hindgut (g- GFP-GPI with 69B-GAL4 driver showing the expression of the constructs.
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Figure S4 The apical marker Crb and the AJ marker DE-cad are not altered in Wild-type embryos, (b’-b’’’’) 69B-GAL4 embryos expressing dominant-negative endocytic mutants. Confocal microscopy sections of stage 13 hindgut (a-e). rab5, (c’-c’’’’) 69B-GAL4 embryos expressing dominant-negative rab11, (d’- Wild-type embryos (a), embryos expressing dominant-negative rab5 (b) and d’’’’) chc1 mutant embryos and (e’-e’’’’) shits mutant embryos stained for the dominant-negative rab11 (c) in hindgut cells show MTf along the basolateral apical marker Crb (green), AJs component DE-cad (red) and SJs component membrane at stage 13, as visualized by MTf immunostaining. (d, e) chc1 MTf (blue). Crb and DE-cad localization is not altered in the mutant embryos, and shits mutant embryos display localization of MTf along the basolateral but MTf is mislocalized and the trachea displays a convoluted phenotype. (f, membrane at stage 13. Confocal microscopy sections of stage 16 hindgut (a’- g) chc1 mutant embryos show mislocalization of Cora along the basolateral a’’’, b’-b’’’, c’-c’’’, d’-d’’’, e’-e’’’) and trachea (a’’’’, b’’’’, c’’’’, d’’’’, e’’’’). (a’-a’’’’) membrane at stage 16 compare to the wild-type embryos. Scale bar: 10 μm.
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Figure S5 Endocytosis is not affected in MTf mutant. (a-d) MTf mutant mutant embryos expressing btl >GFP-CAAX at stage 16 and stage 17. embryos show similar levels and localization of Rab5 (b) and Rab11 Embryos are stained with anti-Gasp and anti-GFP. At stage 16, Gasp is (d) compared to wild-type (a, c) in the hindgut at stage 16. Dashed intra-luminal and at stage 17 after the luminal protein clearance, it is lines mark the apical and basal side of the cells. Basal is the upper detected in the cells of wild-type (e, f) and MTf mutant embryos (g, h). line. (e-h) Confocal sections of the trachea of wild-type and MTf Scale bar: 10 μm.
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Figure S6 MTf binds iron but Ferritin mutants do not show SJs defects section of wild-type and MTf mutant trachea at stage 16 stained for Fer1 (b, in the trachea. (a) Atomic absorption spectroscopy measurements of iron c) and Fer2 (d, e). The levels of Fer1 and Fer2 are similar in wild-type and in purified soluble MTf proteins. Decreased levels of iron are detected in MTf mutant. (f, g) Immunostainings of fer1 and fer2 mutant embryos show constructs containing mutated iron-binding residues. Mouse MTf was used MTf localized in SJs at stage 16. Tracheal tube length is not affected. Scale as positive control and Verm protein as a negative control. (b-e) A confocal bar: 10 μm.
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Figure S7 Full scans and panels of key Western data
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Control of tube diameter expansion by secreted chitin-
binding proteins
Katarína Tiklová and Christos Samakovlis1
Department of Developmental Biology, Wenner-Gren Institute, Stockholm
University, S-10691 Stockholm, Sweden
1Corresponding author: [email protected]
1 Abstract
The size and shape of epithelial tubes determine the transporting capacities of tubular organs. Here, we analyze two genes involved in airway tube size regulation in
Drosophila. Obst-A and gasp encode secreted proteins with chitin binding domains that are conserved among insect species. mRNA in situ hybridizations show that both genes are strongly expressed during airway tube expansion. Gasp protein is secreted into the airway tubes and colocalizes with a chitin binding probe and other chitin binding proteins. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are required for larval elongation and airway tube dilation. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and Obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied with a corresponding reduction of tube diameter in the mutants.
Conversely, overexpression of Obst-A or Gasp in the airways expands the tube circumference. Our results indicate that the level of distinct matrix binding proteins in the tubes determines the extent of diametric growth. We propose that Obst-A and
Gasp organize the assembly of the luminal matrix and thereby provide distending forces that stretch the apical cell membranes to expand tube diameter accordingly.
2 Introduction
An important late step in the morphogenesis of branched tubular organs is the final
acquisition of branch sizes. The fixed length and diameter of tubes dictate the flow
rates of the transported fluids and are therefore major determinants of optimal organ
function. The major tracheal airways, of the Drosophila respiratory network are made
by a single epithelial cell layer and provide a relatively simple system for the genetic
dissection of tube size control.
Genetic analysis in the Drosophila airways has revealed several of the cellular
mechanisms in epithelial tube size regulation (Ghabrial, Luschnig et al. 2003; Uv,
Cantera et al. 2003; Wu and Beitel 2004; Swanson and Beitel 2006; Casanova 2007;
Andrew and Ewald 2010; Schottenfeld, Song et al. 2010). The length and diameter of
the different tracheal branches are controlled independently, branch length increases
continuously but tube diameter expansion occurs step-wise. Furthermore, neither cell
size or cell number are primary determinants of tube expansion in the embryonic
trachea(Beitel and Krasnow 2000). Analysis of mutants with selective size defects
indicates that genes encoding proteins with very diverse molecular functions, control
airway tube size in Drosophila embryos.
One group of mutants shows predominantly overelongated tubes but some of the
members also display local dilations and constrictions. The mutations in this group
affect genes encoding proteins involved in chitin biogenesis or polymer assembly.
These genes include cystic/mummy (mmy), kkv, knk, retroactive (rtv), vermiform
(verm) and serpentine (serp). mmy encodes the Drosophila homolog of UDP-N- acetylglucosamine diphosphorylase. This enzyme is required for the production of
UDP-N-acetylglucosamine, a substrate for chitin synthesis (Araujo, Aslam et al.
2005; Devine, Lubarsky et al. 2005; Tonning, Helms et al. 2006). kkv encodes a chitin
3 synthase that links UDP-N-acetylglucosamine residues into β1,4 glucan chains
(Ostrowski, Dierick et al. 2002; Tonning, Hemphala et al. 2005). knk and rtv encode novel proteins implicated in chitin filament organization (Ostrowski, Dierick et al.
2002; Moussian, Tang et al. 2006). verm and serp encode two related putative luminal chitin deacetylases with a chitin binding domain and are required for the structural modification of the apical ECM (Luschnig, Batz et al. 2006; Wang, Jayaram et al.
2006). An intraluminal chitin cable is detected at stage 13, just before the expansion of the tracheal tubes. The luminal chitin matrix forms a tightly packed cylindrical structure that grows during tube expansion at stages 14 and 15 and disappears at stage
16 (Devine, Lubarsky et al. 2005; Tonning, Hemphala et al. 2005). The analysis of the mutant phenotypes in the “chitin group” of genes suggests that the apical chitin matrix provides either a physical scaffold that defines the shape of the underlying epithelial cells or it locally signals back to the epithelial cells to adjust their shape in a coordinate manner. Interestingly, kkv mutants show an irregular subapical cytoskeleton (Tonning, Hemphala et al. 2005), suggesting that the chitin cable provides cues for the coordinated organization of the apical epithelial cell surfaces along the length of the tubes.
A second group of tube size regulators consists of genes encoding proteins associated with the paracellular septate junctions (SJs). This group of mutants shows overelongated tubes with a defective paracellular barrier. One possible mechanism of
SJ-mediated control of tracheal tube size is through the regulation of the activity of the apical polarity proteins such as Crumbs (Crb) and atypical protein kinase C
(aPKC) (Laprise, Paul et al. 2010). Another way to explain the functions of the SJ- associated proteins in tube size control derives from their potential involvement in the assembly of the luminal chitin matrix (Wu and Beitel 2004). SJs have been directly
4 implicated in the luminal deposition of the chitin deacetylases Verm and Serp, which
suggests that the function of SJ components is to facilitate the targeting of luminal
proteins and to the apical cell surface (Wang, Jayaram et al. 2006). A third group of
mutants shows selective defects in tube diameter expansion. They are characterized
by narrow airways and they all affect genes encoding COPII and COPI vesicle
components (Tsarouhas, Senti et al. 2007; Grieder, Caussinus et al. 2008; Jayaram,
Senti et al. 2008; Forster, Armbruster et al. 2010; Norum, Tang et al. 2010).
Here, we describe two new genes, obst-A and gasp required for larval growth and diametric tube expansion. They encode secreted luminal proteins with chitin binding domains. Obst-A and Gasp are required for the assembly of the intraluminal chitin filament and taenidial integrity. Overexpression of either of the proteins in wild type animals can cause tube dilation. Our analysis argues that Obst-A and Gasp modify the
luminal matrix to provide an expanding mold that determines airway tube expansion.
5 Results and Discussion
Obst-A and Gasp are expressed and secreted into the airways during tube expansion
To identify new genes involved in tube morphogenesis, we surveyed the BDGP gene expression database for genes, which are expressed in the trachea (Tomancak, Beaton et al. 2002). Several members of the Obstructor gene family were strongly expressed in the airways during the tube expansion period. This gene family is conserved among insects and can be subdivided into two subgroups, based on protein domain arrangement (Behr and Hoch 2005). We focused our analysis on Obstructor subgroup
1 because it contains genes with a pronounced tracheal expression pattern. The members of this subgroup (Obst-A, B, C, D) are weakly expressed during early embryogenesis but their expression increases dramatically during late embryogenesis.
This increase in gene expression coincides with cuticle formation in ectodermal tissues and tube expansion in the airways. Obst-A (Fig. 1a) and Obst-B are expressed in the trachea and epidermis; Obst-C (Gasp) (Fig. 1b) shows expression in the tracheal system, foregut and salivary glands. Obst-D and Obst-E are predominantly detected in the midgut and epidermis. The expression of the genes in subgroup 2 is not elevated during embryogenesis, arguing against a role for them in tube morphogenesis. To examine the phylogenetic relationships among the Obst family members, we constructed a phylogenetic tree based on the protein sequences (Fig.
1c). Obst-A and Gasp are the most closely related genes expressed in the trachea. The predicted proteins contain a signal peptide at the amino terminus, followed by three chitin-binding domains type 2 (CBD2) (Fig. 1d). CBD2 is an extracellular domain that contains six conserved cysteines that probably form three disulphide bridges.
These domains are connected with linker regions of similar length in both proteins.
6 Alignment of the protein sequences revealed 37% identity. The features of the two proteins and the timing of their expression in the trachea indicate that they may function in tube expansion.
To investigate the function of Obst-A and Gasp proteins during tracheal development we characterized strong loss of function mutants for both genes. We first generated a null obst-A mutant by the FLP-FRT technique (Parks, Cook et al. 2004). mRNA in situ hybridizations of mutant and wild type embryos confirmed that obst-A was deleted since we could not detect any obst-A mRNA expression in the mutants (Fig.
S1b). For gasp, we used a transposon-induced mutation, which carries a transposon
66bp upstream of the predicted translation initiation site of the gasp gene. We analyzed these mutants using a polyclonal antibody against recombinant Gasp expressed in bacteria (Tsarouhas, Senti et al. 2007). We could not detect any Gasp protein in the mutant embryos suggesting that they can be considered as null (Fig.
S1d). During these experiments we also noticed that the Gasp protein and the unknown antigen recognized by the 2A12 antibody show a similar pattern of protein localization in the trachea. Initially during embryonic stages 13 and 14, both Gasp and the 2A12 antigen predominantly localized in the cytoplasm. At stage 15 both of them began to be deposited into the lumen and by stage 16 we only detected a luminal signal for both antigens. Surprisingly, when we stained gasp mutant embryos for both
Gasp and the 2A12 antigen we did not detect any signal from either of these antibodies (Fig. 2b, c). Parallel stainings for other luminal markers in the mutants still gave the expected expression patterns. These results suggested that the antigen for
2A12 is Gasp or that the 2A12 antigen is encoded by another gene, whose expression is severely reduced in Gasp mutants. To test this, we expressed Gasp protein using the en-GAL4 driver in the dorsal part of the hindgut. We stained those embryos for Gasp
7 (Fig. 2d), 2A12 (Fig. 2e) and Verm, a luminal chitin deacetylase (Fig. 2f). Exogenous expressed Gasp was detected both by Gasp and 2A12 antibodies. We could not detect any signal with the Verm antibody. This argues that the 2A12 monoclonal antibody recognizes specifically an epitope on the Gasp protein. Ongoing experiments address whether the 2A12 antibody recognizes recombinant Gasp on Western blots. These results identify the antigen recognized by the most widely used airway marker in
Drosophila. The discovery of Gasp as the 2A12 antigen opens the possibility to investigate the signals determining the different routes for the apical deposition of
Verm and 2A12 (Wang, Jayaram et al. 2006; Massarwa, Schejter et al. 2009).
Obst-A and Gasp control larval size
Mutations in several of the genes involved in chitin synthesis and assembly affect larval body size (tweedleD) (Guan, Middlebrooks et al. 2006) and cuticle differentiation (mummy, retroactive, knickkopf) (Moussian, Seifarth et al. 2006;
Moussian, Tang et al. 2006; Tonning, Helms et al. 2006). Therefore, we analyzed the size and cuticle structure of obst-A and gasp single and double mutants at the first instar larval stage (Fig. 3).
Both obst-A and gasp single mutant larvae are shorter compared to heterozygote siblings. The body length of obst-A larvae was reduced by 8% and the length of gasp mutants was 14% shorter compared to the wild type. obstA; gasp double mutants showed a more pronounced reduction of 25% compared to the wild-type larval length
(Fig. 3a-e). The width of the mutant larvae did not show any major defects both in single and double mutant combination (Fig. 3f). The increased phenotype severity in the double mutants suggests that Obst-A and Gasp have overlapping additive roles in regulating larval length. We compared cuticle preparations of first instar larvae of the
8 three mutant genotypes to wild-type preparations to assess the potential roles of role of Obst-A and gasp in epidermal differentiation (Fig. 3g-j). Both obst-A and gasp single mutants showed normal cuticular structures. We did not detect any obvious patterning defects or characteristic defects for dorsal closure or head involution phenotypes. In contrast the obst-A; gasp double mutant showed a bloated cuticle. The cuticle defect in the double mutants suggests a function for Obst-A and Gasp in chitin chitin fibril assembly and cuticle integrity.
Obst-A and Gasp are required for apical matrix formation in the airways
To examine the potential roles of Obst-A and Gasp in tube morphogenesis we analyzed the trachea of the mutants. The airways of single obst-A and gasp mutants were indistinguishable from wild type and became gas filled at the end of embryogenesis (Fig. 3a-c). However, the double mutant showed a fully penetrant gas- filling defect (Fig. 3d). We also analyzed the epithelial apical cell membrane and adherens junction integrity by staining of single and double mutants for apical markers, like Crumbs and AJs proteins, like DE-cadherin (Fig. S2). Both single and double mutants did not show defects in cell epithelial organization.
To assess the function of ObstA and Gasp in the assembly of the transient luminal chitin cable, we visualized its deposition using a fluorescent chitin-binding probe
(ChtB) (Fig. 4a-d). Chitin accumulates inside the dorsal trunk tubes in wild-type embryos at stage 14. The size of the cable increases as the tracheal lumen expands in diameter and reaches its final size at stage 16 (Tonning, Hemphala et al. 2005).
Both obst-A and gasp single mutants showed a decreased and irregular staining intensity of the chitin cable compared to wild type embryos (Fig. 4b, c). In obst-A; gasp double mutants the staining of the intraluminal matrix was even more diffuse
9 compared to the single mutants or to wild type embryos (Fig. 4d). To further
investigate if other chitinous matrix modifying proteins are affected, we used antisera
against Verm (Wang, Jayaram et al. 2006). Verm staining was reduced both in single
and double mutants (Fig. 4e-h). The reduction of ChtB and Verm point to function of
Obst-A and Gasp in luminal chitin assembly. It will be interesting to determine
whether the reduction in the staining intensities of chitin polymers and Verm in the
mutants are due to decrease in the expression of genes involved in polymer synthesis
and assembly or due to a direct reduction of matrix stability and integrity. The
presence of three chitin binding domains in Gasp and Obst-A suggest that the proteins
bind to the luminal chin polymers and aid their assembly into a homogeneous and
stable chitin filament.
To further test the potential structural role of Obst-A and Gasp in chitin assembly, we
analyzed the chitin in the taenidia, the tracheal cuticular ridges that line the airway tubes (Fig. 5). Transmission electron microscopy (TEM) reveals three layers of cuticle in wild-type embryos: the envelope layer, the proteinaceous epicuticle and the chitin-rich procuticle (Fig. 5a). TEM analysis of obst-A; gasp double mutants indicated an irregular shape and size of the taenidial folds (Fig. 5b). The outermost layers of the stratified cuticle, the envelope, and the dark epicuticle appeared normal in the mutants. The chitin-rich space between the epicuticle and the epidermal surface, was however, disorganized and granular compared to the uniform procuticle layer of the wild-type embryos.
Thus, the reduced staining intensity of the luminal chitin cable and the defects in the procuticle layer and the irregular taenidial folds in obst-A; gasp mutant embryos suggest a direct function of Obst-A and Gasp in chitin fibril organization and maintenance.
10
Obst-A and Gasp selectively regulate tracheal tube diameter
To assess the impact of Obst-A and Gasp function in tube size control we measured tracheal tube diameter of single and double mutant embryos. We stained obst-A and gasp single mutants for Crumbs and α-Spectrin. Crumbs is localized in the apical membrane (Tepass, Theres et al. 1990) and α-Spectrin (Pesacreta, Byers et al. 1989) is distributed along the lateral domains of epithelial cells. The combination of these two markers visualizes the apical and lateral cell surfaces and facilitates the measurement of tube diameter. Because the airway tubes are tapered we measured dorsal trunk (DT) diameter in three consecutive tracheal metameres to obtain a representative view (n=6) (Fig. 6a). Both mutants showed a small reduction in airway diameter, suggesting a role for Gasp and Obst-A in tube expansion. obst-A mutants showed an average 5% reduction in tube diameter whereas in gasp mutants diameter was reduced by 10% compared to wild type embryos. In obst-A; gasp double mutants the DT diameter reduction was by 15% in comparison to wild type. This suggests that
Obst-A and Gasp secretion into the lumen aids the assembly of the chitin cable, which in turn provides a distending force that stretches the apical surfaces of the epithelial cells. To address if overexpression of Obst-A or Gasp in the trachea may be sufficient to induce an increase in tube diameter we expressed Obst-A or Gasp or an unrelated secreted protein (ANF-GFP) with the btl-GAL4 driver in wild type embryos. We labeled the trachea with antibodies against Crumbs and α-Spectrin and performed the diameter measurements of the DT tubes as described above. Obst-A overexpression caused a ~7% increase and Gasp overexpression generated a ~13% enlargement in the diameter of the DT tubes. ANF-GFP expression did not have any effect on tube size
11 (Fig. 6b). These results suggest that the levels of the chitin binding proteins in the tracheal tubes can directly influence tube diameter expansion.
What is the role of Obst and Gasp in tube diameter expansion? There are few possibilities. One is that the luminal chitin has to be organized to a certain structure, which swells and generates and intra-luminal force that expands the apical surface of the underlying epithelial cells. Expanded chitin maybe sensed by unknown receptors on the apical membrane, which cause changes in cell shape leading to tube expansion.
In another model, luminal chitin-binding proteins or chitin modifying enzymes may generate and send chemical signals to the cells to modify their apical cytoskeleton and allow tracheal expansion.
12 Figure legends
Figure 1
Obstructor family
(a, b) Whole-mount in situ hybridization of wild-type embryos with obst-A (a) and gasp (b) probes. obst-A and gasp mRNA are detected in tracheal cells from stage 13 onward. Note the gaps in obst-A expression correspond to the position of the fusion cells. Scale bar: 25 µm (c) A phylogenetic tree of Obstructor proteins based on
ClustalW method. (d) Schematic representation of the Obst-A and Gasp protein domains. N-terminal signal sequence is followed by three chitin-binding domains type
2 (CBD2).
Figure 2
An unknown antigen recognized by 2A12 antibody is Gasp protein
(a) Wild-type embryo stained with antibodies against the luminal antigen 2A12. (b, c) gasp mutant embryo stained with 2A12 (b) and Gasp (c) antibodies. 2A12 and Gasp staining is not detected in gasp mutant. Scale bar: 25 µm (d-f) Confocal sections of embryonic hindgut of en>gasp embryos labeled with Gasp (d), 2A12 (e) and Verm (f) antibodies. The luminal staining is more pronounced with the Gasp antiserum. Gasp protein expressed in the dorsal part of the hingut is recognized by Gasp and 2A12 antibodies, but not by Verm antibody. Scale bar: 10 µm
Figure 3
Gas filling and cuticle defects in obst-A; gasp mutant
(a-d) Bright field photomicrographs of wild type (a), obst-A (b), gasp (c) and obst-A; gasp (d) first instar larvae. Refracted light makes the gas filled trachea clearly visible
13 in the wild type and single mutant larvae and shows that the trachea is not gas filled in the double mutant. Scale bar: 100 µm (e, f) Body size measurement of wild type, obst-A, gasp and obst-A; gasp first instar larvae. Body length (e) and body width (f)
(n=10). P value is p<0.0001 in all cases comparing wild type animals with mutants.
(g-j) Dark field of cuticle preparations of wild type (g), obst-A (h), gasp (i) and obst-
A; gasp (j) mutant embryos. obst-A; gasp double mutant shows dilated cuticle. Scale bar: 25 µm
Figure 4 obst-A and gasp mutants have defects in the luminal chitin
(a-d) Confocal microscopy projections of the trachea labeled with a FITC-conjugated chitin-binding probe (ChtB). The filamentous chitin is detected in the wild type embryos at stage 16 (a). The chitin intensity is weaker in the obst-A (b), gasp (c) and obst-A; gasp (d) mutant embryos. (e-h) The tracheal sections stained for Verm. Verm is secreted to the tracheal lumen in wild type embryos at stage 16 (e). The amount of secreted Verm is reduced in both obst-A (f) and gasp (g) single mutant embryos and obst-A; gasp (h) double mutant embryos. Scale bar: 10 µm
Figure 5
Ultrastructure of the tracheal lumen of obst-A; gasp mutant
(a-c) Transmission electron micrographs of wild type and obst-A; gasp dorsal trunk at the end of embryogenesis. The wild type embryos (a) show uniformly distributed chitin rich procuticle. In obst-A; gasp double mutant (b, c) an irregular shape and size of the taenidial folds and the procuticle with amorphous composition can be seen. pro
= procuticle. Scale bar: 0.5 µm
14
Figure 6
Lumen diameter expansion requires Obst-A and Gasp
(a, b) Tracheal diameter measurements of wild type, obst-A, gasp and obst-A; gasp embryos (a) and embryos overexpressing Obst-A and Gasp with tracheal specific driver (b) at stage 16.1. The diameter measurements are in three metamers: 4, 5 and 6.
Number of embryos used for measurement of each genotype n=6. y axis represents the diameter in micrometers. P value is p<0.05 in all cases comparing wild type embryos with mutant or overexpressed embryos.
Supplementary figure legends
Figure S1 obst-A and gasp mutants are the null mutants
(a, b) Whole-mount in situ hybridization of obst-A heterozygous (a) and obst-A mutant (b) embryos with obst-A probe and GFP antibody. GFP stained the balanced chromosome to separate the mutant and heterozygote embryos. The obst-A mRNA is not detected in obst-A mutant embryos. (c, d) Wild type (c) and gasp mutant (d) embryos stained with antibodies against the protein Gasp. Gasp staining is not detected in gasp mutant embryos. Scale bar: 25 µm
Figure S2
Apical membrane and AJs are not affected in the obst-A and gasp mutants
Confocal microscopy sections of the tracheal dorsal trunk labeled with an apical marker Crumbs (Crb) (a-d) and adherent junctions marker DE-cad (e-h). Crb is localized apically both in wild type (a) and obst-A (b), gasp (c), obst-A; gasp (d)
15 mutant embryos. DE-cad localization is the same in wild type (e) and obst-A (f), gasp
(g), obst-A; gasp (h) mutant embryos. Scale bar: 10 µm
Materials and Methods
Drosophila strains
The obst-A null mutant was generated by flip FRT technique using two piggyBac
elements (f02348, f03687). w1118 was used as the wild type strain. gasp (d02290)
mutant is described in Flybase. Crosses for ectopic expression using btl-GAL4, en-
GAL4 and 69B-GAL4 drivers were performed at 25 °C. In all experiments CyO, TM3
and FM7c balancer strains carrying GFP or lacZ transgenes were used as necessary to
identify embryos with the desired genotypes.
Immunostaining, TEM
Immunohistochemistry and TEM were performed as described (Lamb, Ward et al.
1998; Wang, Jayaram et al. 2006). The following antibodies were used: mouse anti-
tracheal luminal 2A12 (DSHB), rat anti-DE-Cad (DSHB), mouse anti-Crb (DSHB),
mouse anti-αSpectrin (DSHB), rabbit anti-GFP (Molecular Probes), gp anti-Verm
(Wang, Jayaram et al. 2006), gp anti-Gasp (Tsarouhas, Senti et al. 2007), rabbit anti-
β-gal (Cappel), fluorescein-conjugated ChtB (New England BioLabs). Images were acquired with a Zeiss Meta confocal and Tecnai G2 Spirit BioTWIN electron microscope. Images were post-processed with Adobe Photoshop.
Molecular Biology
UAS-Obst-A transgene was generated by subcloning the insert of LD43683 into pUAST.
16
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