Genetic control of branching morphogenesis during Drosophila tracheal development

731

Genetic control of branching morphogenesis during Drosophila tracheal development Markus Affolter* and Ben-Zion Shilo† Branching morphogenesis is a widely used strategy to increase the surface area of a given organ. A number of tissues undergo branching morphogenesis during development, including the lung, kidney, vascular system and numerous glands. Until recently, very little has been known about the genetic principles underlying the branching process and about the molecules participating in organ specification and branch formation. The tracheal system of insects represents one of the bestcharacterised branched organs. The tracheal network provides air to most tissues and its development during embryogenesis has been studied intensively at the morphological and genetic level. More than 30 genes have been identified and ordered into sequential steps controlling branching morphogenesis. These studies have revealed a number of important principles that might be conserved in other systems. Addresses *Biozentrum, Abteilung Zellbiologie, Klingelbergstrasse 70, 4056 Basel, Switzerland †Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Current Opinion in Cell Biology 2000, 12:731–735 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations Bnl Branchless Btl Breathless Dfr–Vvl Drifter–Ventral veinless Dof Downstream of FGFR DSRF Drosophila serum response factor EGF epidermal growth factor FGF fibroblast growth factor FGFR fibroblast growth factor receptor Kni–Knrl Knirps–Knirps-related Sal spalt Tkv Thick veins Trh trachealess

Introduction The establishment of the larval tracheal system of Drosophila represents a paradigm for studying branching morphogenesis. The trachea consists of a large tubular network that provides air from the spiracular openings to virtually every cell in the organism. During embryonic development, tracheal cells are set aside in 10 clusters of approximately 80 cells on each side of the embryo. Upon invagination and the formation of a sac-like structure, each tracheal metamere sprouts into five to six finer branches, some of which ultimately fuse to form a tree-like structure throughout the embryo. Remarkably, cells do not divide during the branching process, which occurs exclusively by cell migration and cell shape changes. The entire process has been characterised and described in great detail [1] and

a large number of genes have been identified that play important roles in tracheal morphogenesis [2,3]. These include genes determining the tracheal fate in epidermal cells, genes determining branch identities, genes encoding a chemoattractive ligand (Branchless–fibroblast growth factor [Bnl–FGF]) and signal transducers of the latter, as well as genes involved in cell fusion and tracheal cell specialisations. Although many questions remain, the first coherent molecular picture of the formation of this branched organ starts to emerge (see Figure 1).

Assignment of tracheal fate to ectodermal cells Tracheal cells are set aside within a larger field of lateral ectodermal cells in 10 clusters of approximately 80–100 cells on each side of the embryo. Analogous with other organ systems, the determination of tracheal cells is brought about by the locally restricted expression of a combination of transcription factors [2], in this particular case, the products of the trachealess (trh) and the drifter–ventral veinless (dfr–vvl) loci. trh encodes a bHLH–PAS domain protein that forms a complex with Tango, a broadly expressed bHLH–PAS protein, whereas dfr–vvl encodes a POU domain transcriptional regulator. The territories of Trh and Dfr–Vvl expression are determined by the genes organising the anteroposterior and the dorsoventral axis of the embryo. Recent studies suggest that Trh and Dfr–Vvl might directly interact with each other, and in this manner cooperate to specify tracheal cell fates [4]; however, additional genes must be required to confer complete tracheal cell identity within the ectodermal field [5] and different combinations of factors might assist in the transcriptional activation of the battery of genes essential for proper tracheal development. One of the first functions of Trh and Dfr–Vvl is to activate a program leading to invagination of the tracheal placodes, thereby generating a luminal space that is then further expanded within the embryo via directed cell migration. Little is known about the genetic control of the invagination process but initial studies suggest that epidermal growth factor (EGF) receptor signalling might contribute to the concerted invagination [6]. EGF receptor activation is triggered by increased levels of Rhomboid (Rho) in the tracheal placode, resulting from induction of rho transcription, which requires both Trh and Dfr–Vvl [4]. Invagination certainly relies upon dramatic changes in the organisation of the actin cytoskeleton; indeed, actin redistribution prefigures the internalisation of the placode and is controlled by Trh [6]. A pivotal role of tracheal determinants is the induction of tracheal-specific expression of a number of components essential for cell signalling events that control the subsequent branching morphogenesis. Such components include

732

Cell differentiation

Figure 1

a) Determination of tracheal cells

component Rho. These regulatory activities are part of the program that sets the stage for the branching process. Ec

Subdivision of the tracheal placode While all tracheal cells express the trachea determining genes trh and dfr–vvl, subpopulations of cells giving rise to distinct branches during morphogenesis express other, specific nuclear transcriptional regulators. Expression of the zinc finger proteins Knirps–Knirps-related (Kni–Knrl) is induced in ventral and dorsal tracheal cells as a response to Dpp signalling; the ligand Dpp is secreted from neighbouring ectodermal cells positioned ventral and dorsal to the placode ([7–10]; see Figure 1). Dpp signal reception and kni–knrl activation in tracheal cells is not only essential to determine the correct number of cells in dorsal and ventral branches, but is also critical in allowing cells to respond to the chemoattractant Bnl ([7]; see below) and to determine the size of the tube to be formed later during morphogenesis [11•]. The same functions appear to be provided by the spalt (sal) gene in the dorsal trunk [10,12]; sal induction requires the activation of the EGF receptor by high levels of rho in tracheal cells [8].

Tr Trh, Tango, Vvl/Dfr

Ec

b) Invagination of the placode EGFR (Rho)? c) Determination of branch identity Dpp signaling, Kni/Knrl; EGFR signalling, Sal

= Dpp

d) Reponse to chemoattractant (Bnl) Sfl, Sgl; Bnl/Btl signalling, Dof Response to other cues? Bridge cell factor, Adrift?

Determination and differentiation of distinct tracheal celltypes Bnl/Btl signalling, Pnt, DSrf/Bs, Esg, Sry, Hdc, N signalling Extension of terminal branches to hypoxic tissues

= Bnl

(e)

(f)

(g)

Bnl signalling

Although determinants for other branches remain to be identified, it appears that branching morphogenesis does not occur in the absence of the genetic program that leads to branch specification; indeed, branch outgrowth is virtually abolished when both DPP and EGF signalling are defective [8,9]. The identification of target genes regulated by these branch-specific transcription factors is thus crucial for the understanding of the functional interconnection between organ induction and branching morphogenesis per se. The identification of these genes in the near future might be greatly assisted by DNA array technologies.

Current Opinion in Cell Biology

Branching via directed cell migration Sequential steps in tracheal branching morphogenesis. Panels show the different steps in tracheal development. The gene products involved in these steps are listed. Schematic representations shown in the centre are limited to a single tracheal metamere. (a) The embryo on the right was hybridized with a trh RNA probe. trh is expressed in the 10 tracheal placodes and in the invaginating salivary gland (arrow). Ec-ectoderm; Tr-tracheal placode. (b) The first three placodes of an embryo are visualised using immunostaining against β-galactosidase expressed under the regulatory region of the trh gene. Tracheal cells in (c) and (d) are visualised using the same procedure. (c) dpp transcript distribution around the tracheal pits is shown in blue. (d) bnl transcript distribution around the tracheal pits is shown in blue. (e) Tracheal system of a stage 15 Drosophila embryo. Tracheal cell nuclei are shown in red, the tracheal lumen in green. (f) Fine extensions formed by a larval terminal cell visualised with Tau-GFP (arrow). (g) Fusion of a dorsal branch from the left side with a dorsal branch from the right side of the larva; The lumen visualised in this photograph shows the specialized structure formed at the junction of the two fusion cells. The nuclei of the two fusion cells are visualized with nuclear GFP and only faintly visible. Ec, ectoderm; Tr, tracheal placode; GFP, green fluorescent protein.

the FGF receptor (FGFR), Breathless (Btl), the FGF signalling component. Downstream of FGFR (Dof), the Dpp type I receptor Thick veins (Tkv) and the EGF signalling

A very important role in generating the branched morphology of the developing tracheal system is attributed to the FGF-like molecule encoded by the bnl locus [13] and the FGFR molecule encoded by the btl locus [14]. The FGF ligand Bnl is expressed in groups of ectodermal cells around the invaginated placode that prefigure the formation of tracheal branches. The Bnl ligand acts as a chemoattractant upon the nearest tracheal cells that express the Btl FGF receptor in addition to the appropriate branch-specific determinants. Under the control of Bnl, branches emerge from the tracheal sac towards Bnl-secreting ectodermal cells via directed cell migration. Recent studies have identified several components important for Bnl–induced cell migration. Mutations in sugarless and sulfateless, two enzymes involved in the synthesis of heparan sulfate glycosaminoglycans, result in phenotypes similar to those observed in the absence of ligand or receptor [15]. Clearly, heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand–receptor oligomerization and might contribute to chemotaxis.

Genetic control of branching morphogenesis during Drosophila tracheal development Affolter and Shilo

Surprisingly, genetic studies have also lead to the identification of a molecule that appears to act specifically in the FGF signalling pathway. The dof gene [16••] (also called heartbroken [17] or stumps [18]) encodes a novel cytoplasmic protein containing putative ankyrin repeats and a coiled coil segment and is present exclusively in cells that express FGF receptors [16••]. Dof is needed in tracheal cells for the FGF-mediated activation of the MAP kinase cascade and for directed cell migration. How Bnl signalling elicits a chemotactic response in tracheal cells and what the role of Dof might be in this process remains to be elucidated. Bnl signalling might control cellular motility via small G proteins [19] and actin polymerisation and/or via selective adhesion to specific substrates. As discussed above, a prerequisite for cells to respond to the chemoattractive role of distinct Bnl spots is the expression of the appropriate branch-specific markers. How this dual signalling requirement is manifested at the molecular level remains to be investigated. Wolf and Schuh [20••] have recently obtained evidence for the involvement of additional non-ectodermal cells in branching morphogenesis. It has been known for some time that the dorsal trunk, the main structure of the tracheal system, can also form when Btl is uniformly activated in all tracheal cells [21] and additional guidance cues have been proposed to be involved in dorsal trunk formation [13]. Wolf and Schuh identified a single mesodermal cell expressing the Hunchback (Hb) transcriptional regulator at the posterior lateral margin of each tracheal metamere during the onset of migration [20••]. After one cell division, the more dorsally located Hb-expressing cell connects to the posterior and anterior dorsal trunk outbuds, linking two adjacent tracheal metameres by acting as a ‘bridge cell’. In the absence of this cell (i.e. in hb mutant embryos), initial primary branch formation is normal, but subsequently dorsal trunk branches either stall or extend in the wrong directions whereas the other branches continue to develop normally. These defects can not be overcome via the ectopic expression of Bnl. Though the precise function of the bridge cell remains to be elucidated, this study identifies a non-ectodermal cell population that assists in the ordered outgrowth of tracheal branches. These cells might secrete short-range guidance factors, or serve as a migration matrix or as attachment sites for filopodia/lamellipodia. It is worth mentioning that visceral mesodermal cells are also crucial for the extended outgrowth of the visceral branches; in this case, however, the secretion of Bnl by these mesodermal cells appears to be the critical cue. Additional cells, such as neurons and glia cells, might also provide guidance cues for tracheal cells during later pathfinding events. The ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial cells, switching substrates five times before reaching its ultimate target in the CNS [22]. In adrift (aft) mutants, pathfinding along some of these substrates is abolished.

733

Since aft encodes a nuclear protein present in migrating tracheal cells, it has been suggested that it might regulate expression of tracheal genes required for pathfinding. These studies demonstrate that although initial branch outgrowth is controlled to a large extent by Bnl, pathfinding at later stages appears to involve distinct cell populations and might be regulated by other guidance cues.

Determination and differentiation of distinct tracheal cell types During the migration phase, cells within each tracheal branch are determined to distinct fates, which ultimately allow the formation of a continuous network of epithelial cells transporting oxygen to target tissues. Each branch forms a well-defined number of cell types at precise positions [1]. These cell types include fusion cells (which allow the interconnection of adjacent tracheal metameres), terminal cells (which allow oxygen exchange) and branch cells (which allow oxygen transport from the outside to target tissues). The formation of terminal and fusion cells requires Bnl and complex cell–cell interactions involving Notch signalling [23–27]. Reciprocal interactions assure that the correct stoichiometry of fusion and terminal cells are generated. Interactions between tracheal cells and neighbouring, non-tracheal cells appear to be involved in the correct positioning of fusion cells [25]. While the branch-specific selection of terminal and fusion cells is an important mechanism, these two cell types are most intriguing for their highly specialised biological function. Terminal cells differentiate under the control of the MADS box transcription factor DSRF–blistered [28] and form tree-like structures of long, bifurcate and hollow terminal extensions towards target tissues. Fusion cells are characterised by the expression of the zinc finger protein Escargot and form tight connections with tracheal cells from adjacent metameres via highly orchestrated cell shape changes assisted by high levels of E cadherin [29,30]. The molecular events underlying the formation of these two exquisite cell types are largely unknown and their elucidation remains a major challenge in developmental cell biology.

Branch size determination While terminal cells and fusion cells carry out precise cellular functions, branch cells appear to fulfil a more structural role; however, it is likely that the proper size of the epithelial tubes building the tracheal system is critical for the function of the tracheal system. Studies aiming at a better understanding of how tube size is controlled in the embryonic and the larval tracheal system have recently been initiated [11•]. A genetic analysis of tube size determination revealed that the tube size program can be divided into two major phases: an early phase in which tube size is specified and tracheal cells assemble into small tubular structures and an expansion phase in which the diameter and length of the tubes grow to their mature sizes. In the early phase, branch identity genes (i.e. kni–knrl and possibly sal) specify tube

734

Cell differentiation

size. Interestingly, genes that function exclusively in expansion of tubes and/or maintenance of expanded tubes (and have no function in cell migration) were also identified [11•]. Although the molecular analysis of these genes remains to be done, one could anticipate that tube expansion genes encode components or regulators of the actin cytoskeleton, molecules that mediate cell communication, regulators of vectorial secretion or molecules accumulating in the extracellular matrix or the exoskeleton. Molecular characterisation of the expansion genes will help to distinguish among these hypotheses.

Acknowledgements We thank Mark Krasnow and Reinhard Schuh for communication of results prior to publication, and Chris Bazinet and members of the Affolter and the Shilo laboratories for discussion. Our own studies were supported by a Human Frontiers of Science Program grant and grants from the Schweizerischer Nationalfond and the Kantons Basel-Stadt and Basel-Land.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Samakovlis C, Hacohen N, Manning G, Sutherland DC, Guillemin K, Krasnow MA: Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 1996, 122:1395-1407.

2.

Zelzer E, Shilo BZ: Cell fate choices in Drosophila tracheal morphogenesis. Bioessays 2000, 22:219-226.

3.

Metzger RJ, Krasnow MA: Genetic control of branching morphogenesis. Science 1999, 284:1635-1638.

4.

Zelzer E, Shilo BZ: Interaction between the bHLH–PAS protein Trachealess and the POU-domain protein Drifter, specifies tracheal cell fates. Mech Dev 2000, 91:163-173.

5.

Boube M, Llimargas M, Casanova J: Cross-regulatory interactions among tracheal genes support a co-operative model for the induction of tracheal fates in the Drosophila embryo. Mech Dev 2000, 91:271-278.

6.

Llimargas M, Casanova J: EGF signalling regulates cell invagination as well as cell migration during formation of tracheal system in Drosophila. Dev Genes Evol 1999, 209:174-179.

7.

Vincent S, Ruberte E, Grieder N, Chen CK, Haerry T, Schuh R, Affolter M: DPP controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. Development 1997, 124:2741-2750.

8.

Wappner P, Gabay L, Shilo BZ: Interaction between the EGF receptor and DPP pathways establish distinct cell fates in the tracheal placode. Development 1997, 124: 4707-4716.

9.

Llimargas M, Casanova J: ventral veinless, a POU domain transcription factor, regulates different transduction pathways required for branching in the Drosophila tracheal system. Development 1997, 124:3273-3281.

Physiology of oxygen transport Development of the stereotyped branching pattern of the tracheal tree relies upon the reiterated use of the Bnl signal, first to induce the outgrowth of primary branches, then to induce secondary branches (not discussed in detail; see [1,3]) and subsequently to determine fusion and terminal cell fates. During these processes, different Bnl signalling responses are likely to be generated under the control of the stereotyped developmental program initiated by the trachea determining genes and followed by the expression of branch-specific transcriptional regulators. The FGF-responsive enhancer of blistered–DSRF has been isolated [31] and the study of this and other cis-regulatory elements should give insight into the molecular coupling of these processes. Interestingly, Bnl also serves as a signal to convey information from oxygen-starved cells to terminal tracheal cells, which respond to this signal during larval development with the increased ramification of the fine terminal branches [32••]. Thus, a single growth factor, Bnl, is reiteratively used to pattern each level of airway branching, and the change in branch patterning during larval development results from a switch from different developmental to physiological control of its expression.

Conclusions Studying tracheal development has provided a framework for the understanding of branching morphogenesis in a developing organism. In brief, the genetic program involves a number of successive steps: one, determination of groups of tracheal cells (placodes); two, invagination of the placode to form a sac-like structure (pits); three, determination of specific branch identities; four, branch outgrowth via Bnl-mediated directed cell migration; five, determination and differentiation of distinct cell types, generating a functional branched organ; and six, extension of terminal branches towards hypoxic tissues. Many of these aspects of tracheal development are likely to be relevant for the formation of branched epithelial structures in vertebrates. A striking analogy between these different systems is the reiterated activation of tyrosine kinase signalling (Bnl in trachea, FGF2/10 in lung and mammary gland development, VEGF [vascular endothelial growth factor] in vasculogenesis) [3,33]. Future work will unravel whether other key molecules are shared between different branching processes and to what extend the underlying principles are similar.

10. Chen CK, Kühnlein RP, Eulenberg KG, Vincent S, Affolter M, Schuh R: The transcription factors KNIRPS and KNIRPS RELATED control cell migration and branch morphogenesis during Drosophila tracheal development. Development 1998, 125:4959-4968. 11. Beitel GJ, Krasnow M: Genetic control of epithelial tube size in • the Drosophila tracheal system. Development 2000, 127:3271-3282. The authors investigate the genetic control of tube size in the embryonic and larval tracheal system. They find that tube size is controlled by coordinately regulating the apical (luminal) surface of tracheal cells and not by the number and shape of tracheal cells in each branch. Genetic analysis shows that tube sizes are specified early by branch identity genes, and the subsequent enlargement of branches to their mature sizes involves a new set of genes, the tube expansion genes. 12. Kühnlein RP, Schuh R: Dual function of the region-specific homeotic gene spalt during Drosophila tracheal system development. Development 1996, 122:2215-2223. 13. Sutherland D, Samakovlis C, Krasnow M: branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 1996, 87:1091-1101. 14. Klämbt C, Glazer L, Shilo BZ: breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and specific midline glial cells. Genes Dev 1992, 6:1668-1678. 15. Lin X, Buff EM, Perrimon N, Michelson AM: Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development 1999, 126:3715-3723.

Genetic control of branching morphogenesis during Drosophila tracheal development Affolter and Shilo

16. Vincent S, Wilson R, Coelho C, Affolter M, Leptin M: The Drosophila •• protein Dof is specifically required for FGF signalling. Mol Cell 1998, 2:515-525. The authors identify a novel component of the FGF signalling pathway in Drosophila, Downstream of FGFR (Dof). Dof is a novel cytoplasmic protein with putative ankyrin repeats and acts downstream of the activated FGF receptors and upstream of Ras. Surprisingly, Dof is present only in those cells in which FGF receptors are expressed and is not required for signalling via other tyrosine kinases. 17.

Michelson AM, Gisselbrecht S, Buff E, Skeath JB: Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila. Development 1998, 125:4379-4389.

18. Imam F, Sutherland D, Huang W, Krasnow MA: stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migrations of tracheal and mesodermal cells. Genetics 1999, 152:307-318. 19. Hall A: Rho GTPases and the actin cytoskeleton. Science 2000, 279:509-514. 20. Wolf C, Schuh R: Single mesodermal cells guide outgrowth of •• ectodermal tubular structures in Drosophila. Genes Dev 2000, 14:2140-2145. Wolf and Schuh describe the discovery of a mesodermal cell, the ‘bridge cell’, that links the leading cells of outgrowing dorsal trunk branches of adjacent tracheal metamers. The authors show that this bridge cell serves as an essential guidance post that provides cues acting in concert with Branchless–FGF signalling to mediate directed branch outgrowth and/or position-specific branch fusion. This study suggests that similar bridge cells might exist for other tracheal branches and incites the search for them. 21. Reichman-Fried M, Dickson B, Hafen, E, Shilo B-Z: Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration. Genes Dev 1994, 8:428-439.

735

25. Steneberg P, Hemphälä J, Samakovlis C: Dpp and Notch specify the fusion cell fate in the dorsal branches of the Drosophila trachea. Mech Dev 1999, 87:153-163. 26. Hacohen N, Kramer S, Sutherland D, Hiromi Y, Krasnow MA: sprouty encodes a novel antagonist of FGF signalling that patterns apical branching of the Drosophila airways. Cell 1998, 92:253-263. 27.

Steneberg P, Englund C, Kronhamn J, Weaver TA, Samakovlis C: Translational readthrough in the hdc mRNA generates a novel branching inhibitor in the Drosophila trachea. Genes Dev 1998, 12:956-967.

28. Guillemin K, Groppe J, Dücker K, Treisman R, Hafen E, Affolter M, Krasnow M: The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. Development 1996, 122:1353-1362. 29. Samakovlis C, Manning G, Steneberg P, Hacohen N, Cantera R, Krasnow M: Genetic control of epithelial tube fusion during Drosophila tracheal development. Development 1996, 122:3531-3536. 30. Tanaka-Matakatsu M, Uemura T, Oda H, Takeichi M, Hayashi S: Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot. Development 1996, 122:3697-3705. 31. Nussbaumer U, Halder G, Groppe J, Affolter M, Montagne J: Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development. Mech Dev 2000, 96:27-36.

23. Llimargas M: The Notch pathway helps to pattern the tips of the Drosophila tracheal branches by selecting cell fates. Development 1999, 126:2355-2364.

32. Jarecki J, Johnson E, Krasnow MA: Oxygen regulation of airway •• branching in Drosophila is mediated by Branchless FGF. Cell 1999, 99:211-220. The authors show that ramification of the fine terminal branches of the larval tracheal system is variable and regulated by oxygen levels. This process is controlled by a local Bnl–FGF signal produced by oxygen-starved cells. During larval life, oxygen deprivation stimulates expression of Bnl–FGF, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. These observations combined with previous studies demonstrate that a single growth factor is reiteratively used to pattern different levels of airway branching.

24. Ikeya T, Hayashi S: Interplay of Notch and FGF signalling restricts cell fate and MAPK activation in the Drosophila trachea. Development 1999, 126:4455-4463.

33. Hogan BL, Yingling JM: Epithelial/mesenchymal interactions and branching morphogenesis of the lung. Curr Opin Genet Dev 1998, 8:481-486.

22. Englund C, Uv AE, Cantera R, Mathies LD, Krasnow MA, Samakovlis C: adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS. Development 1999, 126:1505-1514.