ORGANOGENESIS: MOLECULAR MECHANISMS OF TUBULOGENESIS

Many vital human organs develop in the embryo fromsimple tubes that branch and ramify into complex, tree-like structures. In this way, the total cellular area avail-able to the body for metabolic processes, such as nutri-ent and gas exchange, is greatly increased, whereas thedistances over which substances have to travel arereduced. These features might have had an importantrole in the evolution of branched tubular systems asorganisms increased in size1. Although the final shapeand function of tube-based organs, such as the lung,mammary gland, kidney and blood vessels, differgreatly, studies at the cellular and molecular level arebeginning to reveal common features about the initialprocesses of tube formation and differentiation. Forexample, a fundamental feature of all tubular organs isthat they are made up of cells with apical–basal polarity— in other words, the cells have a clearly defined apicalmembrane facing the central lumen and a basal surfaceattached to a layer of extracellular matrix (FIG. 1). Theacquisition of this polarity is a defining event in tubulo-genesis, and it involves a series of dynamic and interde-pendent processes, including reorganization of thecytoskeleton, assembly of intercellular junctional com-plexes and the specification of membrane domains2–4.Understanding the different strategies for the formationof tubular organs is a challenge for both developmentalbiologists and cell biologists, for whom genetics hasbecome a powerful tool. This review highlights different

strategies for the generation and elaboration of tubes,and shows how insights into the underlying mecha-nisms have come from recent genetic analyses of modelsystems, such as Caenorhabditis elegans, Drosophila andzebrafish.

In vertebrates, tubular organs often contain manydifferent cell types that are localized to specific regionsand specialized for functions such as fluid secretion orgas exchange. The genesis of these organs might involveextensive cell proliferation and programmed cell death.There is also turnover of cells throughout life, or, in thecase of the mammary gland and hair follicle, in periodiccycles. These organs contain stem cells, which are oftenlocated with specialized mesenchymal cells that controlstem-cell behaviour through extracellular signals. Bycontrast, tubular systems in simpler organisms, such asDrosophila, usually consist of only a few cell types anddevelop without cell proliferation and apoptosis. Thisreview — and others in this issue5 — show how geneticanalysis can also link the generation of cell diversity withtube morphogenesis, and how some of these mecha-nisms have been conserved during evolution.

Strategies for making tubesStrategies for making tubes can be classified accordingto whether the participating cells already haveapical–basal polarity or whether such polarity isacquired in response to local cues (FIG. 2). Examples in

MOLECULAR MECHANISMS OFTUBULOGENESISBrigid L. M. Hogan*‡ and Peter A. Kolodziej‡

As organisms have evolved in size and complexity, tubular systems have developed to enable the efficient transport of substances into and out of tissues. These tubular systems are generatedusing strategies that are based on common elements of cell behaviour, including cell polarization,tube migration to target sites, cell-fate diversification and localization of specialized cells todifferent regions of the tube system. Using examples from both invertebrate and vertebratesystems, this review highlights progress in understanding these basic principles and brieflydiscusses the possible evolution of strategies to regulate the morphogenesis of tubular systems.

PRIMARY TRACHEAL SACS

The precursors to the Drosophilatrachea that form a sack-likestructure, before any branchingoccurs.

NATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 513

*Howard Hughes Medical Institute and‡Department of Cell andDevelopmental Biology,Vanderbilt Medical Center,Nashville, Tennessee 37232-2175, USA. Correspondenceto B.L.M.H.e-mail:[email protected]:10.1038/nrg840

R E V I E W S

O R G A N O G E N E S I S

© 2002 Nature Publishing Group

TIGHT JUNCTION

A connection betweenindividual cells in epitheliumthat forms a diffusion barrierbetween the two surfaces of anepithelium.

ADHERENS JUNCTION

A cell–cell and cell–extracellular-matrix adhesion complex that iscomposed of integrins andcadherins that are attached tocytoplasmic actin filaments.

DESMOSOME

A patch-like adhesiveintercellular junction that islinked to intermediate filaments— cytoskeletal components.They are found in vertebratetissues, especially in tissuesundergoing mechanical stress.

IMAGINAL DISC

An epithelial sheet that gives riseto the external adult structuresof insects, such as the wing, eyeand antennae.

514 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics

R E V I E W S

and shape of the tube. The placode of the Drosophilasalivary gland consists of ~100 cells only and initiallygives rise to a flask-shaped tube (see below). By contrast,the primordium of the vertebrate central nervous sys-tem, known as the neural plate, extends broadly medio-laterally along most of the body axis. The folding of thisplate into a tube is, therefore, a complex process involv-ing several intermediate stages, which, if interrupted bygenetic mutations or environmental factors, can giverise to human neural-tube defects, such as spina bifida6.

Another way of making tubes from epithelial sheetsis illustrated by the formation of veins in insect wings7.The veins are derived from prepatterned stripes of cellsin an epithelial monolayer, the wing IMAGINAL DISC. Thisfolds back on itself to form a bilayer in which the veincells in each half become aligned. Vein cells lack inte-grins that are expressed on intervein cells , and this dif-ference in cell adhesiveness probably contributes to theability of vein cells to form fluid-tight tubes (FIG. 2B).

the first category, schematized in FIG. 2Aa, include theDrosophila salivary glands and PRIMARY TRACHEAL SACS, andthe vertebrate lung, liver and neural tube. Initially, agroup of cuboidal cells within a polarized epithelialsheet forms a thickened placode. Cells within the pla-code are, at first, columnar and elongated along theirbasal–apical axis. They then become wedge-shaped asthe placode invaginates. This shape change mightinvolve constriction of the apical cytoskeleton, increasedadhesion between placode cells and/or alterations to theunderlying extracellular matrix. Independently, thenuclei move basally, possibly by a microtubule-depen-dent mechanism. Inward movement of the nascent tubeproduces a structure that has proximal and distal polar-ity surrounding a common lumen; this structure under-goes further elaboration as a result of changes in theshape and behaviour of the individual cells, whichmight be influenced by signals from underlying cells.The dimensions of the placode influence the initial size

Apical

Recognition

Mature epithelium

RhoCdc42Rac

a

b c

Basal

Primitive-junctionformation

c

Figure 1 | Acquisition of apical–basal polarity is essential for lumen formation. Events during the maturation of the interfacebetween two epithelial cells are shown, on the basis of data from vertebrates and invertebrates. a | Primitive contacts are formedthrough homophilic cadherin interactions (green rectangle). The F-actin (red) is not yet well polarized. Other cell-adhesion molecules,such as Jam, a TIGHT-JUNCTION component (yellow), might also be involved. b | Early contacts are consolidated. A proto-ADHERENS

JUNCTION (green rectangle) develops; it associates with the atypical protein kinase C (aPKC)—PAR-3—PAR-6 complex (purplecircles) and, via cadherin, with p120, β-catenin and α-catenin (blue ellipses). Interactions (arrows) between junction-associatedcomplexes and F-actin-associated proteins, such as Short Stop, and between α-catenin and apical surface determinants (orangeline) (for example, crumbs and Discs Lost, white ellipses) specify the apical surface and adherens junctions. Rho-family GTPases(Rho, Rac and Cdc42) probably mediate some of these interactions. Other proteins, such as the PDZ-domain-containing Scribble(orange triangles), localize laterally and stabilize the adherens junctions and confine apical surface determinants. At the basal side(mauve line), cells are attached to a basement membrane (broken line). c | The mature epithelium contains an apically polarized F-actin cytoskeletal network (red) and several types of intercellular junctions (adherens, green; tight, yellow; DESMOSOME, black/grey;gap, light blue cylinder), each of which is made up of many proteins.



R E V I E W S

PLACODE

A disc-shaped group ofcolumnar epithelial cells thatdetaches from an epithelial sheetto give rise to an organ.

EXCRETORY CELL

A cell in the nematodeCaenorhabditis elegans thatallows waste excretion. Theintracellular lumen of anexcretory cell is known as theventral canal.

Mesodermcells

Tip cell

Primary branch

Secondary branch

DuctMicrolumen

Futuresecretory

region

*

2 3

Placode2 3 4

B

D

Ab1

Aa1

VeinIntervein

2 3C1

Bud

Figure 2 | Basic strategies for generating tubular systems. Aa | Invagination of cells from an epithelial sheet (for example, in Drosophila salivary gland andmouse lung) begins (1) with a polarized epithelial sheet in which cuboidal cells have an apical domain (orange), junctional complexes and a basolateral domainthat is adjacent to a continuous basal lamina (broken line). (2) Before invagination, cells in a contiguous group become columnar, forming a PLACODE thatinvaginates (3) to form a flask-shaped structure (4). The basal lamina beneath the epithelial cells might be discontinuous at the distal tip. Ab | Extension ofpolarized epithelial cells. (1) Primary branches of the Drosophila tracheal system can be two cells wide and elongate by cell convergence and extension. (2) The distal tips cells (blue) express the receptor tyrosine kinase, Breathless, and extend processes from their basolateral surfaces towards localized sources of fibroblast growth factor (Fgf ) ligand (Branchless, red dots), which is released by specific mesoderm cells (red circles). The special pattern of these targetsdetermines the stereotypic branching pattern of the primary tubes46. (3) Tip cells elongate to form unicellular secondary and terminal branches. The lumen of thefinest tracheoles is intracellular. B | Formation of tubes by apposition of two sheets of cells with different adhesive properties (veins in a Drosophila wing). Thebasolateral surfaces of the future vein cells (red) have lower adhesive properties than the corresponding surfaces of the intervein cells, which eventually die. C | Lumen formation within a protrusion of unpolarized cells (for example, in mammalian mammary and salivary glands, pancreas or hair follicle). (1) Duringdevelopment of the mammary gland36, a placode gives rise to a bud that is ~20 cells in diameter. (2) As the bud begins to branch, a small lumen forms. Cellsthat are adjacent to the lumen become polarized, as evidenced by microvilli projecting into the lumen and by the re-expression of junctional complexes. It seemsthat, in the mammary gland, peripheral cells undergo apopotosis. (3) As the rudiment continues to branch, multiple lumena are formed and subsequentlycoalesce, eventually becoming a continuous lumen (not shown). D | Formation of a channel or lumen within individual cells (such as Caenorhabditis elegansEXCRETORY CELLS and secondary branches of the Drosophila tracheal system). In the C. elegans excretory system, a single cell elaborates a fluid-filled channel orcentral canal that is closed at one end (asterisk) and might be open at the other. The apical subdomain of the cell membrane and its associated cytoskeleton(orange) faces the lumen, whereas the basolateral subdomain contacts a basement membrane and epidermal cells.



R E V I E W S

therefore, provides a paradigm for understanding morecomplex systems, including tubular organs of higherorganisms, such as the kidney9 (see accompanyingreview by Seppo Vainio and Yanfeng Lin on p533 in thisissue for more on kidney organogenesis). Mutations inmore than a dozen genes cause morphological abnor-malities in the C. elegans excretory cell, including cysticdilation of the CENTRAL CANAL. The phenotypes indicatethat these genes have roles in cell extension, in localizingmolecules beneath the lumenal membrane and in orga-nizing the apically polarized filamentous actin (F-actin)cytoskeleton. One of the genes, sma-1, encodes βH-spec-trin10, an F-actin-crosslinking protein that is associatedwith the apical membranes of diverse cells, includingthose of Drosophila trachea. Another gene, exc-5, is the C. elegans homologue of human FDG1, which ismutated in the X-linked skeletal disorder faciogenitaldysplasia11. exc-5/FDG1 encodes a guanine nucleotideexchange factor (GEF) that regulates Rho-family GTPaseactivity and affects the apical accumulation of F-actin inthe excretory cell12,13. Mutations in RhoA also affectlumen formation in the Drosophila trachea14 indicatingthat mechanisms of intra- and intercellular lumen for-mation might share some common controlling factors.

Tubulogenesis by invaginationAn elegant model for studying tubulogenesis from anepithelial sheet is the Drosophila salivary gland, whichdevelops into only two cell types, without proliferationor apoptosis15–17. The homeotic gene, Sex combs reduced(Scr), specifies the size and position of the salivary glandprimordium, which arises in the ventral ectoderm ofPARASEGMENT 2. Elsewhere in the ectoderm, dorsal–ventraland anterior–posterior patterning genes such as

A third basic strategy for tubulogenesis involves pre-cursors that initially do not have apical–basal polarity orspecialized intercellular connections. Instead, organssuch as the mammary gland, hair follicle and early pancreas bud (see accompanying review by HelenaEdlund on p524 in this issue for more on pancreasorganogenesis) (FIG. 2C) develop from primordia inwhich unpolarized cells are initially closely packedtogether into a three-dimensional cluster or bud thatdoes not have a lumen. In other cases — for example, thezebrafish gut (FIG. 3) or early blood vessels (FIG. 4) — theunpolarized cells are aligned into cord-like structuresthat do not initially have a lumen. The lumen is formedsubsequently when cells in the centre of the cluster or inthe cord acquire apical–basal polarity and form junc-tional complexes.

Finally, tubes can be made by and within individualcells (FIG. 2D). For example, cells in the secondarybranches of the Drosophila tracheal system formlumena either by making junctions with themselves(autocellular junctions) or by forming an intracellularlumen8. The fine terminal branches or trachioles thatramify into tissues in response to local hypoxia are cyto-plasmic extensions of single cells with a subcellularlumen of less than 1 µm in diameter. Another exampleis the highly elongated C. elegans excretory cell. It is~100–200 µm long, with a fluid-filled central lumen~1 µm in diameter, that is elaborated with numerousmicro-invaginations (see FIG. 2D and below)9.

Intracellular lumen formation: basic principlesThe membrane that surrounds an intracellular lumen isequivalent to the apical membrane of epithelial cells.Genetic analysis of lumen formation in single cells,

CENTRAL CANAL

A fluid-filled channel in thecentre of the single excretorycell.

PARASEGMENT

Segmentation genes patternparasegments, regions of theDrosophila embryo that containthe posterior part of ahemisegment and the anteriorpart of its neighbor. Mesodermalthickenings and ectodermalgrooves demarcate parasegmentborders.

Early junction formation Junctions cluster in rod centre Lumen forms

Poor clustering of junctions Ectopic lumen formation

A cell within the cord:

a

b

Figure 3 | Central lumen formation in a cord of cells. a | Initially, cells in a cord, such as in the zebrafish intestinal primordium, areweakly polarized. They assemble primitive adherens contacts (green) at the junctions between several cells42 (left). F-actin (red)associated with these contacts might help them cluster in the centre (centre panel). The lumen (blue) develops in the centre of thecord (right). The inset shows a close-up of an individual cell. Lumen formation might involve retracting the apical surface (orange) ofeach cell from the centre of the cord (large arrow) and secreting vesicular material (blue circles) into the resulting cavity (small arrow).b | The junction clustering and assembly is delayed in the zebrafish heart and soul (has) mutant (left); consequently, the junctions areoften in multiple clusters within the cord and more than one lumen forms (right).



R E V I E W S

many other stages of gland development. In fkhmutants, the nuclei of the primordium move basallybut the apical cell surfaces do not constrict, showingthat these two subcellular processes can be separatedmechanistically21. It is interesting to note that a verte-brate forkhead family member, Foxe1 (TTF2 inhuman) is required in the mouse embryo for theDELAMINATION and migration of the thyroid bud fromthe floor of the anterior foregut22. Mutations in thehuman TTF2 gene are associated with thyroid AGENESIS

and other congenital abnormalities23.Once the secretory portion of the salivary gland is

formed, it extends dorsally and then posteriorly, to liein close contact with the muscle cells of the body wall.This realignment requires sequential changes in cellshape, together with the activity of several genes,including zipper, raw and ribbon (rib)24. zipper, whichencodes a non-muscle myosin II heavy chain25; raw,which regulates Jun-kinase signalling26; and rib geneti-cally interact during epithelial morphogenesis24.Recent research has shown that rib encodes a 71-kDanuclear protein that has an amino-terminal BTB/POZ(Bric a brac, Tramtrack, Broadcomplex/Poxvirus, ZincFinger) protein–protein interaction domain and aconserved DNA-binding motif, both of which arefound in various transcription factors27,28. The func-tion of rib is not confined to salivary-gland morpho-genesis — it is also required for epithelial migration inother contexts, including the migration of the pri-mary branches in the Drosophila tracheal system. Asshown in FIG. 2A, these branches migrate towards spa-tially restricted sources of Fgf ligand (Branchless,Bnl), which is produced by clusters of nearby meso-dermal cells (for a review, see REF. 29). The tip cells ofthe tracheal tubes extend cytoplasmic processes fromtheir basal surface towards these targets; in ribmutants, the extensions are particularly prominent,indicating that they are still able to transduce the Bnlsignal through Breathless (Btl), a transmembranereceptor tyrosine kinase (RTK). However, the cellbodies and apical surface do not follow the directedbasal extensions and no net forward movementoccurs. It remains to be seen how rib functions to cou-ple the extension of the basal surface with the move-ment of the rest of the cell and why mutations in ribaffect some branches more than others28.

We are beginning to understand how simpleepithelial tubes undergo morphogenesis inDrosophila30, but less is known about the formation,elongation and branching of epithelial buds in theorgans of more complex organisms. In part, this isbecause in vivo mesodermal cells surround andobscure the tubulogenic epithelial cells. This problemcan be circumvented by the use of transgenic animalsin which the epithelial cells express reporters, such asβ-galactosidase or green fluorescent protein, and byculturing mesoderm-free tissues in three-dimensionalgel matrices30–33. This approach has been particularlyuseful for observing changes in the shape, cytoskeletalorganization and gene expression of mouse embryoniclung buds and lacrimal-gland epithelial cells budding

Decapentaplegic (Dpp), Abdominal-B and teashirt blockthe salivary-gland-inducing activity of Scr. About 100cells, located in a lateral domain of the primordium, giverise to the secretory portion of the gland, whereas ~30 cells, located more ventrally, generate the ducts thatconnect the glands to the mouth. Genetic analysis hasidentified dozens of genes that are expressed during sali-vary-gland development16,18. Some of these genes con-trol the acquisition of specialized functions or cell iden-tity — for example, components of the epidermalgrowth factor (EGF) signalling pathway. The activity ofothers, such as ribbon, described below, is associatedwith morphogenesis, not only of the salivary gland butalso of other tubular systems, and with processes such asDORSAL CLOSURE of the Drosophila embryonic epidermisthat involve extensive changes in cell shape19.

The development of the secretory portion of thesalivary gland begins with a stereotypical sequence ofevents — the generation of a cup-shaped epithelialplacode that is formed when cells change shape fromcuboidal to columnar. Invagination occurs in a dorsal-to-ventral wave in the placode cells as their nucleimigrate basally and their apical surfaces constrict. Thisprocess is regulated by Huckebein (Hkb), an Sp1-likeZn2+-finger family transcription factor that isexpressed in a pattern that predicts the order in whichthe placode cells invaginate17. The downstream signalsthat initiate and control the morphogenetic wave arenot yet known. However, they might include thesecreted protein Folded Gastrulation (Fog), which actsthrough the small GTPase RhoA18,20. Among the genesthat are known to regulate the invagination of the sali-vary-gland primordium is forkhead (fkh), whichencodes a winged helix transcription factor required at

Intersomitevessel

Somite

Dorsal aorta

Midline source of VEGF

Lateralmesoderm

Vein

a

b

c

d

e

Figure 4 | Vasculogenesis and angiogenesis in the vertebrate embryo. This model depicts several processes in primary blood-vessel formation in an idealized vertebrate embryo. a | ANGIOBLASTS are already specified as precursors of arteries (red) or veins (blue) in the lateralmesoderm. b | Artery precursors migrate towards the MIDLINE in response to vascular endothelialgrowth factor (VEGF) that is secreted by cells such as the HYPOCHORD in Xenopus59. c | En route,they might align into cords, forming a network or plexus. d | Angioblasts coalesce into largervessels, such as the DORSAL AORTA, but it is not known precisely when lumen formation is initiated.e | Intersomitic vessels are assembled sequentially from three types of endothelial cells, which aredistinguished by their morphologies (green, purple and blue)74.

DORSAL CLOSURE

An event in Drosophilaembryogenesis when theepidermis migrates as a sheettowards the dorsal midline. Thesides of the sheet meet at themidline, enclosing the embryo.

DELAMINATION

The detachment of cells from anepithelial sheet.

AGENESIS

Failure of a tissue to develop.

ANGIOBLAST

Precursor of endothelial cells inblood-vessel wall.

MIDLINE

The anterior–posterior linebisecting the dorsal side of thevertebrate embryo.

HYPOCHORD

A thin rod-like structure of cellsthat runs along the length of theXenopus embryo below thenotochord, and may be involvedin inducing dorsal aortaformation.

DORSAL AORTA

The main artery that runs to theembryonic heart.



R E V I E W S

time, and several small lumena are formed (FIG. 3). hasencodes a member of the protein kinase C family, theactivity of which is independent of calcium and diacyl-glycerol (an atypical PKCλ)42. Atypical PKC isoformsparticipate in an evolutionarily conserved complexwith the PAR-3–PAR-6 proteins, which are keyadherens-junction-associated proteins that establishepithelial polarity in many invertebrate and vertebratedevelopmental contexts (FIG. 1). The PAR-3–PAR-6complex also interacts with Cdc42 and Rac, Rho familyGTPases that regulate key pathways of F-actin assem-bly. Clustering the adherens junctions in the centre ofthe tube might, therefore, involve regulating their inter-actions with F-actin-containing structures.

Elongation of tubes by convergent extensionThe primary function of many tubular systems is totransport gases and nutrients to internal tissues and to remove wastes from them. As organisms increased insize during evolution, tubes had to become longer, moreextensively branched and able to alter their shape inresponse to the physiological needs of the target tissue.One of the strategies used for tube elongation is conver-gent extension, the process by which individual cellsintercalate between one another and extend along theproximal–distal axis of the tube. The process has animportant role in the elongation of the Drosophilahindgut44. In Xenopus and zebrafish, the geneStrabismus, which encodes a member of the non-canonical Wnt planar cell-polarity signalling pathway,has been implicated in the elongation of the body axisand neural tube44. The mammalian homologue ofStrabismus is a candidate for the semidominant looptail(Lp) mutation in mice, which is widely studied as amodel for neural-tube defects. In affected embryos, theneural tube does not close, probably because it fails toelongate along the body axis and, consequently, it is toobroad to fold up easily into a tube45.

Tip cells and directed epithelial-cell movementCells at the tips of tubes also have important roles in tubeelongation and morphogenesis. For example, the cells atthe tips of extending tracheal branches in the Drosophilaembryo respond to migration cues and pull the rest of thebranch along46. One of the factors promoting this migra-tion is Bnl, which is made by mesenchymal cells andupregulated in response to the physiological stimulus ofoxygen deprivation47. Cells at the tips of the fly salivarygland might also have a pivotal role in migration, as evi-denced by their specific expression of the guidance mole-cule Semaphorin II (REF. 17). Less is known about themigratory behaviour of cells at the distal tips of epithelialbuds in mammalian systems. In the embryonic mouselung, a continuous basal lamina surrounds most of theepithelium except at the tips, where there are discontinu-ities in the basement membrane48,49. These might help tipcells to sense gradients of ligands, such as the Fgf familymember Fgf10, which, like Bnl in the Drosophila trachealsystem, is expressed by distal mesenchymal cells32. Thesensing might occur through guidance and adhesionreceptors and RTKs on the basal surface of the epithelial

and/or migrating towards an external source of Fgf.This behaviour mimics the in vivo outgrowth of thelung and lacrimal-gland epithelium towards localizedsources of Fgf that emanate from mesodermalcells31,32,34,35.

Reorganizing cell junctions and cell polarityAs described above, tubular organs, such as the mammary gland, hair follicle, SUBMANDIBULAR GLAND andpancreas, arise from compact clusters of relatively unpo-larized cells36–39 that lack specialized junctional com-plexes, such as adherens junctions and DESMOSOMES

(FIG. 1). Two crucial aspects of tube formation that occurin these organs are the de novo establishment ofapical–basal polarity in the precursors and the elabora-tion of intercellular junctional complexes that link thenewly polarized cells around the nascent central lumen.FIG. 2C summarizes some of the key steps that areinvolved in the initial formation of the mammaryglands of the mouse. The ectodermal cells condense toform a primordium that initially projects above the surface of the epidermis before subsiding into the mes-enchymal mammary fat pad40. These changes appar-ently occur without ectodermal proliferation. Lef1(lymphoid enhancer binding factor-1), a component ofthe Wnt signalling pathway that is required for mam-mary-gland development, is the earliest gene known tobe expressed in the primordium; the list of players thatfollow is increasing rapidly and includes Msx1, Msx2(homeobox Msh-like-1) and Pthrp (parathyroid hor-mone related peptide) (for a review, see REF. 41). Beforebirth, the ectodermal buds elongate into solid cords, andtube formation begins when small intercellular spacesor microlumena appear. These gradually fuse into a sin-gle central lumen. Electron-microscope studies almost20 years ago revealed the absence of specialized junc-tions between tightly packed cells in early buds, andlater, the formation of morphologically distinct apicalmembranes and junctional complexes as the inner cellsorganize around the microlumena37. Consistent withthese ultrastructural changes, immunohistochemicalstudies have shown that junctional components areexpressed only at low levels in central cells early in theprocess, and their expression levels are restored aslumena form39. Finally, the ultrastructural studies revealthat cells that are excluded from the newly polarizedepithelium become PYCNOTIC and die.

Other insights into the process of tube formationwithin a solid cord of cells have come from thezebrafish gut (FIG. 3). As embryogenesis progresses,junctional complexes form locally between gut endo-derm cells, initially near any intersection of three cellsurfaces within the rod of cells, but then movinginward to form a single, central cluster (FIG. 3). So, thecells become wedge-shaped with tiny central apicalmembranes. As the apical membrane domains enlarge,a single, central lumen forms. The zebrafish heart-and-soul (has) mutant has defects in many epithelial organs,such as the heart, eye and gut. In the developing gut, theformation of the apical junctions is delayed and ineffi-cient so that they do not reach the centre at the same

SUBMANDIBULAR GLAND

Salivary gland locatedunderneath the jaw.

DESMOSOME

Specialized cell–cell junctionbetween epithelial cells that isconnected to intermediatefilaments, one of the cytoskeletalelement types.

PYCNOTIC

The appearance of a nucleus in acell that is undergoing apoptosisor programmed cell death as itshrinks and the chromosomescondense.



R E V I E W S

angiogenesis serves to extend vascular networks towards atarget, to form ANASTOMOSES between parts of the networkand, as discussed below, to assemble longer connectingbranches such as the intersomitic vessels.

Studies in the Xenopus embryo show that angioblastsmigrate from the lateral mesoderm over several hundredmicrons to the midline to form the dorsal aorta. Theyseem to do so in response to VEGF-A (vascular endothe-lial growth factor A), which is released by a group of mid-line cells that is known as the hypochord, which sits justunderneath the notochord59.Angioblasts express VEGF-R2 (Flk-1), a VEGF-A receptor57,60. The VEGF-A geneencodes several isoforms, some of which bind to heparinand the extracellular matrix, and some of which are solu-ble. VEGF-A isoforms also have different potencies interms of promoting cell survival and proliferation61.Diffusibility and activity differences among the VEGFisoforms might help to establish concentration gradientsalong the routes of angioblast migration — in this case,with the highest levels in the midline (FIG. 4).

Analysis of mouse embryos that have mutations ingenes that encode members of the VEGF family (at leastfour, including Vegfa) and the VEGF receptors (theRTKs Vegfr1, Vegfr2, Vegfr3 and the co-receptor neu-ropilin-1) has confirmed the importance of these mole-cules in blood- and lymphatic-vessel formation andindicated specific roles for these different receptors62–67.In the future, conditional and isoform-specific mousemutants will provide a more sophisticated analysis ofhow three-dimensional arrays of vessels are assembledin a tissue-specific manner. It will also be crucial tounderstand how other important modulators of angio-genesis that are presented in a cell-type-specific manner,such as the ANGIOPOIETINS68, interact with the VEGFs toregulate endothelial-cell behaviour and the formationand destruction of intercellular junctions.Vegfa loosensendothelial contacts, and angiopoietin-1 seems to stabi-lize them. How the activation of their correspondingRTKs leads to these opposing effects on cell–cell junc-tions needs to be further investigated.

Assembling physiologically distinct blood vessels.There are many morphological and physiological dif-ferences between arteries and veins. Given these differences, it is important to know at what stage ofdevelopment these blood vessels arise. Analysis of thezebrafish gridlock mutation indicates that angioblastsare already specified as arterial and venous precursorsubpopulations in the lateral mesoderm before theysort during migration and assemble into the mainblood vessels (and, therefore, well before the circula-tion begins). gridlock encodes a transcription factorthat is a target of Notch signalling, implicating thispathway in the specification of arterial and venousendothelial cells69. An important consequence ofNotch signalling might be the differential expressionof members of the ephrin B ligands and EphB RTKfamilies on these two different endothelial-cell popu-lations70–73. They might function as bi-directional sig-nalling molecules that confine arterial and venouscells to appropriate tubular systems.

cells. This mechanism would be particularly effective ifthe tip cells extend dynamic cytoplasmic processes invivo, as is the case in Drosophila tracheal cells.

Integrins are an important class of receptors forextracellular matrix molecules that are required for tubeextension in many systems, including the C. elegansexcretory cell and the developing mammalian lung andkidney ureter bud50–52. However, it is not knownwhether integrin or other signalling pathways are differ-entially activated in tip cells to promote branch migra-tion, and the roles of integrins in tube migration com-pared with cell survival and proliferation are difficult todisentangle experimentally. Other studies indicate thatsignalling systems that were first described in axon guid-ance, including SEMAPHORINS and the homologue of theSlit receptor, Roundabout (Dutt1/Robo1), might alsodirect epithelial branching in mammalian organs, suchas the lung; these systems could also act primarily on thetip cells53,54. Overall, it seems that tip cells integrate guid-ance cues from the environment through several differ-ent signalling systems to direct branch outgrowth and migration.

Finally, unlike the Drosophila salivary gland and tra-cheal system, tubulogenesis of mammalian organs usu-ally involves extensive cell proliferation, especially at thetips of buds once they have extended32,35,48. Moreover, itseems that cells at the distal tip of growing organs areoften undifferentiated and multipotential. The differen-tial expression of genes in the distal buds of mammalianbranching organs, such as the embryonic lung (for areview, see REF. 55), might, therefore, reflect the rapidproliferation of distal buds and their need to remainmultipotent, and not just the need for bud extension,guidance and branching.

Vasculogenesis — switching between statesDuring the development of the vertebrate vascular andlymphatic systems, endothelial cells form tubes by switch-ing between migratory states — in which they lack api-cal–basal polarity — and non-motile states — in whichthey form intercellular junctions and have anapical–lumenal surface (FIG. 4). The primary blood vesselsof the embryo — the dorsal aortae and the CARDINAL VEINS

— assemble from individual endothelial precursors(angioblasts) that arise in the lateral (peripheral) meso-derm, by a process called vasculogenesis56.Although thedetails of vasculogenesis vary between species, the dorsalaortae are formed from angioblasts that migrate towardsthe midline, whereas the veins assemble from precursorsthat remain in the more lateral positions. It seems thatcells do not assemble into tubes directly, but first aggre-gate side by side into a network of cords that are a fewcells thick that then coalesce into the larger vessels57,58.Precisely when and how the endothelial cells first generateadherens junctions and a central vessel lumen is not yetknown. This is partly because, unlike in developingepithelia, there are no markers for the early apical vesselsurface. Once assembled into vessels, endothelial cells cansprout from within or around the vessel and form abranch, probably through a cord-like intermediate. Thisprocess is known as angiogenesis56. Early in development,

SEMAPHORINS

A family of secreted ortransmembrane molecules thatall contain at least onesemaphorin repeat in theirextracellular domain, whichrepel migrating cells and growthcones in the nervous system andother tissues.

CARDINAL VEINS

The main veins carrying bloodout of the embryonic heart.

ANASTOMOSES

Plural of anastomosis, the site atwhich two blind-ended tubesjoin together to form a singletube.

ANGIOPOIETINS

Secreted factors that bind to theTie2 receptor and that modulatevascular permeability andremodelling.



R E V I E W S

the aorta before their morphogenesis or only looselyassociated with it, is not yet known. In the zebrafishmutant out of bounds (obd), ISV precursors migrateprematurely, originate from the wrong positions alongthe aorta and follow aberrant pathways74. It seems thatobd acts through the somite cells, which might providea cellular substrate to guide the migration of the ISVprecursors, but the precise mechanism remains to beelucidated. This mechanism might involve ephrin B2signalling. In mouse embryos that have an engineeredtruncation of the cytoplasmic domain of ephrin B2, theintersomitic vessels migrate abnormally and sproutinto the somites75.

Vascular remodelling The zebrafish study of ISV formation indicates that animportant feature of vascular remodelling is the fusionbetween two vessels to form a common lumen (anasto-mosis), but how this is achieved is not known. Cluesmight come from the anastomosis of tracheal cells inthe Drosophila embryo. During this process, specializedcells at the tips of the fusing branches recognize eachother by forming an E-cadherin (Shotgun) contactalong their apposing basolateral surfaces76,77 (FIG. 5).F-actin assembles at the contact, and then forms a track

We do not yet know how many different subpopu-lations of precursors there are, and to what extent dif-ferent vessels are a mosaic of non-equivalentendothelial cells. The zebrafish embryo has advan-tages for addressing these problems, given that individual precursors can be followed in vivo by time-lapse micrography. This technique has revealed thatthree different types of precursor are involved inbuilding intersegmental veins (ISV), the vessels thatpass between the SOMITES to link the dorsal aorta to theDORSAL LONGITUDINAL ANASTOMOTIC VESSEL (DLAV)74. Thefirst type migrates from the dorsal aorta and forms asingle-cell T-shaped junction with the DLAV. Thesecond type also travels from the dorsal aorta andforms a straight, single-cell connection with the firsttype. The third type forms another single-cell T-shaped junction, with the tail connected to the sec-ond precursor type and the head to the dorsal aorta(FIG. 4). Lumen formation occurs separately in eachpart of this nascent plumbing, and then anastomosescreate a continuous vessel that links the dorsal aortato the DLAV.

This process is highly stereotypical, and probablyinvolves interactions between endothelial cells and thesomite. Whether the ISV precursors are initially part of

HEMISEGMENTAL BOUNDARY

A boundary between twohemisegments, the portions of asegment between the ventral anddorsal midlines, along theanterior/posterior axis.

PLAKIN

A family of large (250–600 kDa)proteins that contain a centralrod domain, and sites forbinding F-actin, microtubules,and/or intermediate filaments.

SOMITE

Mesodermal balls of cellsadjacent to the neural tube thatwill differentiate into the muscle,vertebrae and dermis.

DORSAL LONGITUDINAL

ANASTOMOTIC VESSEL

(DLAV). The vein that runsparallel to the dorsal aorta in thezebrafish, and is connected tothe dorsal aorta by theintersegmental veins.

LL L

L

LL

L

L

Recognition andcontact assembly

Track assembly,contact stabilization andexpansion into both cells

Apical-surface extensionalong track

Intracellullar lumen forms andfuses with flanking lumens

a b

cd

Figure 5 | Lumen formation during anastomosis between two tubes. a | During anastomosis in the Drosophila trachea, fusioncells (purple) at the tips of tracheal branches form a new cadherin contact (green rectangle) at the HEMISEGMENTAL BOUNDARY. Thecontact typically initiates in the anterior fusion cell77, and is associated with filamentous actin (F-actin) and the PLAKIN Short Stop, anF-actin/microtubule-binding protein (red). The apical surface (orange) of the fusion cell faces the lumen (L). b | The contact broadens(green) to include both fusion cells, and the track extends across each cell to the existing apical surface. c | Apical surface markers(orange) and membrane are deposited along the track. d | A complex rearrangement ensues, in which the fusion cell bodies aredisplaced dorsally, and the central cadherin contact forms a ring that encircles the two continuous intracellular, doughnut shapedlumens formed in the fusion cells. These lumens extend through the two fusion cells to connect the two original lumens.



R E V I E W S

migration, as well as the subsequent transcriptionalprogramme that might be crucial for repolarizing thecells to form a lumen in the branch and anastomosiswith the parent lumen.

Future directions in tubulogenesisIn the future, a better understanding of tubulogenesiswill require advances in several different areas. Geneticand biochemical analyses of vertebrate and invertebratemodel systems will continue to reveal more about themechanisms that underlie fundamental processes, suchas lumen formation, tube migration, control of tubediameter and tube remodelling. Cell-culture systemsthat closely recapitulate key developmental processeswill also be very useful for discovering new tubulogenicfactors and visualizing intermediates in tubulogenesis,particularly as more molecular markers for the diversecell types that comprise tubes and their precursorsbecome available. Developing markers for the apical andbasolateral surfaces of cell membranes will be particu-larly important for determining more precisely whenlumen formation occurs, both in vitro and in vivo. Liveimaging of tubes as they assemble and migrate will indi-cate the intermediate steps that occur as cells respond tomigration cues or become polarized, and how theseprocesses are connected to particular signalling events.Ultrastructural studies of the cytoskeleton and vesicletrafficking in developing tubes, combined with mutantanalysis, will also help us to understand the key ques-tions that are associated with cell polarization, the gene-sis of intercellular connections, the control of tubediameter and lumen formation87, and the specificationand specialization of membrane domains. For example,some endothelial cells form unusual apical-surface spe-cializations, such as FENESTRATIONS, and understandinghow the development of these features is controlled willprovide insights into how cells in tubes alter their apicalsurfaces to perform specific physiological functions88.

Coordinated development of tubes and surrounding tissue.One topic that requires particular attention in thefuture is the way in which the epithelial cells of tubularorgans influence the growth and differentiation of thetissues that surround them, and vice versa. These inter-actions are particularly important in the complex,highly vascularized organs of vertebrates. It is possiblethat epithelial–mesenchymal interactions were elabo-rated during evolution to optimize the performance oftubular organs in response to increasingly sophisti-cated physiological and metabolic needs1. For example,in organs as diverse as the intestine, mammary glandand lung, a layer of smooth muscle cells surrounds theepithelial tubes. The muscle layer promotes the move-ment of nutrients or secretions along tubes or, in thecase of the lung, changes the diameter of the airwaytubes in response to physiological stimuli. Duringembryogenesis, the epithelial tubes of the lung pro-duce factors such as Sonic hedgehog (Shh) and Pdgfa(platelet-derived growth factor-α) that promote thegrowth and differentiation of smooth muscle from theundifferentiated mesenchyme89,90. The epithelial cells

that radiates outwards and eventually traverses thefusion cells to connect with their existing apical surfaces14. Track assembly and consolidation of the E-cadherin contact requires interactions between F-actin, microtubules and Short Stop, a giant plakin-likeprotein that binds to F-actin and to microtubules that isalso associated with the F-actin track. Apical surfacemarkers and membrane are then deposited along thetrack, and a lumen forms. The F-actin/Short Stop trackmight have several roles in this assembly process. Forexample, it might be a substrate for non-musclemyosin-based movements of membrane, inwards fromthe existing apical surfaces. In addition, it might targetintracellular vesicles that contain lumenal material tothe assembling lumen. In endothelial cells, vacuoles areproposed to provide lumenal material78. Examiningendothelial cells in developing vessels for the presence ofcytoskeletal structures that direct apical surface forma-tion and prefigure the position of the lumen is likely toreveal important aspects of how known angiogenicagents work, and lead to the identification of new ones.

Role of receptor tyrosine kinases in tubulogenesisReceptor tyrosine kinases have key roles in orchestratingthe different behaviours of tubulogenic cells. Tissuesthat influence how these cells form tubes in vivo expressRTK ligands — for example, VEGFs, FGFs, EGFs andglial-cell-line-derived neurotrophic factor. RTK activa-tion initiates receptor-specific, temporally separatedbehavioural responses that combine to elicit tubulogen-esis. Regulating these distinct responses involves phos-phorylating different sets of tyrosines in the cytoplasmicdomain of these receptors, and the consequent recruit-ment of specific signalling molecules33,79.

This principle is well illustrated by an in vitro modelin which c-met, the RTK for hepatocyte growth factor,drives the assembly of MDCK cells (a cell line that isderived from canine kidney epithelial cells) into tubesin three-dimensional, extracellular matrix gels80. Oneset of phosphorylation sites in c-met directs epithelialbudding and depolarization by activating phos-phatidyl-inositol-3-kinase (PI3K), phospholipase Cγ(PLCγ) and Rac81,82 (enzymes that regulate cell migra-tion and the organization of the cytoskeleton). Racactivity is also required for the assembly of laminin, akey component of the basement membrane that isinvolved in establishing the apical–basal polarity ofthese cells81. A second set of associated signalling mole-cules stimulates Ras–MAPK (mitogen-activated pro-tein kinase) to trigger further cell proliferation83;MAPK activation can also regulate tight-junctionassembly84 and integrin-dependent cell adhesion85.Finally, tube formation, which involves repolarization,re-forming of junctions and lumen formation, requiresthe transient binding of Stat3 (signal transducer andactivator of transcription 3) to its receptor, and Stat3translocation to the nucleus and regulation of its tran-scriptional targets86. So, c-met, and probably othertubulogenic RTKs, initiate both early changes in apicalpolarity, cytoskeletal organization and cell contacts thatare important for allowing tube cells to begin branch

FENESTRATION

A round window, 63–68 nm indiameter, that cuts throughsubsets of endothelial cells. Theyare covered by a membraneousdiaphragm that contains afibrillar meshwork.



R E V I E W S

endothelial cells of blood vessels of vertebrate embryosare required for the growth and differentiation oftubular organs, including the liver and pancreas92,93

(see also the accompanying review by Kenneth Zareton p499 of this issue). These factors might be requiredto coordinate tissue vascularization with proliferationand differentiation.

In conclusion, tubulogenesis is a fascinating topicthat brings together scientists working in many differentdisciplines. Its elucidation will probably reveal new, evo-lutionarily conserved biological processes, and prove tobe of great medical importance.

also make VEGF, a factor that stimulates the develop-ment of the vascular network so as to keep pace withthe ramifying epithelial network. Similar interactionsbetween tubular systems and associated cells are cru-cial in other organisms. Indeed, analysis of the expres-sion of VEGF and VEGF-receptor family membersduring Drosophila development has led to new ideasabout the evolution of blood vessels from a primitiveblood cell type that initially migrated freely within thebody and was attracted to organ systems by factors likeVEGF91. Reciprocally, there is growing evidence thatas-yet-unidentified factors that are made by the

1. West, G. B., Brown, J. H. & Enquist, B. J. The fourthdimension of life: fractal geometry and allometric scaling oforganisms. Science 284, 1677–1679 (1999).The authors use mathematical models to explore theimportance of branching tubular systems forincreasing the metabolic efficiency of organisms asthey increased in size during evolution.

2. Knust, E. Control of epithelial cell shape and polarity. Curr.Opin. Genet. Dev. 10, 471–475 (2000).

3. Ohno, S. Intercellular junctions and cellular polarity: thePAR–aPKC complex, a conserved core cassette playingfundamental roles in cell polarity. Curr. Opin. Cell Biol. 13,641–648 (2001).

4. Bilder, D. PDZ proteins and polarity: functions from the fly.Trends Genet. 17, 511–519 (2001).

5. Vainio, S. & Lin, Y. Coordinating early kidney development:lessons from gene targeting. Nature Rev. Genet. 3, 533–543(2002).

6. Colas, J.-F. & Schoenwolf, G. C. Towards a cellular andmolecular understanding of neurulation. Dev. Dyn. 221,117–145 (2001).A comprehensive review of the cellular and molecularmechanisms underlying neurulation, with emphasison the chick embryo, and the genetic andenvironmental factors that are associated with neural-tube defects in mammals.

7. Bier, E. Drawing lines in the Drosophila wing: initiation ofwing vein development. Curr. Opin. Genet. Dev. 10,393–398 (2000).

8. Manning, G. & Krasnow, M. A. in The Development ofDrosophila melanogaster Vol. 1 (eds Bate, M. & Martinez-Arias, A.) 609–686 (Cold Spring Harbor Laboratory Press,Plainview, New York, 1993).

9. Buechner, M., Hall, D. H., Bhatt, H. & Hedgecock, E. M.Cystic canal mutants in Caenorhabditis elegans aredefective in the apical membrane domain of the renal(excretory) cell. Dev. Biol. 214, 227–241 (1999).

10. McKeown, C., Praitis, V. & Austin, J. sma-1 encodes a βH-spectrin homolog required for Caenorhabditis elegansmorphogenesis. Development 125, 2087–2098 (1998).

11. Pasteris, N. G. et al. Isolation and characterization of thefaciogenital dysplasia (Aarskog-Scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor. Cell 79, 669–678 (1994).

12. Suzuki, N. et al. A putative GDP–GTP exchange factor isrequired for development of the excretory cell inCaenorhabditis elegans. EMBO Rep. 2, 530–535 (2001a).This paper and reference 13 implicate the Rho familyof GTPases in controlling lumenal diameter andsmoothness, by identifying a GDP–GTP exchangefactor (GEF) as a key regulator of these importanttubular properties. Rho GTPases might regulate theapical F-actin network, although this is not shownexplicitly in this paper.

13. Gao, J., Estrada, L., Cho, S., Ellis, R. E. & Gorski, J. L. TheCaenorhabditis elegans homolog of FGD1, the humanCdc42 GEF gene responsible for faciogenital dysplasia, iscritical for excretory cell morphogenesis. Hum. Mol. Genet.10, 3049–3062 (2001).The authors show that the C. elegans GEF that affectslumenal development of the excretory cell is thehomologue of a human GEF that is involved infaciogenital dysplasia. Lumenal development in C. elegans, therefore, provides a model for this humangenetic disease.

14. Lee, S. & Kolodziej, P. A. The plakin Short Stop and theRhoA GTPase are required for E-cadherin-dependent apicalsurface remodeling during tracheal tube fusion.Development 129, 1509–1520 (2002).

The authors investigate the mechanism of de novolumen formation. They describe the assembly of an F-actin-rich track that is associated with new E-cadherin contacts between tracheal branches. This track directs lumen formation in branch tip cellsto connect tracheal branches. RhoA probablyfunctions upstream of Shot to regulate Shotlocalization to the track and track assembly.

15. Andrew, D. J., Henderson, K. D. & Seshaiah, P. Salivarygland development in Drosophila melanogaster. Mech. Dev.92, 5–17 (2000).

16. Bradley, P. L., Haberman, A. S. & Andrew, D. J. Organformation in Drosophila: specification and morphogenesis ofthe salivary gland. Bioessays 23, 901–911 (2001).

17. Myat, M. M. & Andrew, D. J. Organ shape in the Drosophilasalivary gland is controlled by regulated, sequentialinternalization of the primordia. Development 127, 679–691(2000).

18. Lammel, U. & Saumweber, H. X-linked loci of Drosophilamelanogaster causing defects in the morphology of theembryonic salivary glands. Dev. Genes Evol. 210, 525–535(2000).

19. Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L.& Montague, R. A. Multiple forces contribute to cell sheetmorphogenesis for dorsal closure in Drosophila. J. Cell Biol.149, 471–490 (2000).

20. Costa, M., Wilson, E. T. & Wieschaus, E. Putative cell signalencoded by the folded gastrulation gene coordinates cellshape changes during Drosophila forkhead gastrulation. Cell76, 1075–1089 (1994).

21. Myat, M. M. & Andrew, D. J. Fork head prevents apoptosisand promotes cell shape change during formation of theDrosophila salivary glands. Development 127, 4217–4226(2000).

22. De Felice, M. et al. A mouse model for hereditary thyroiddysgenesis and cleft palate. Nature Genet. 19, 395–398(1998).

23. Clifton-Bligh, R. J. et al. Mutation of the gene encodinghuman TTF-2 associated with thyroid agenesis, cleft palateand choanal atresia. Nature Genet. 19, 399–401 (1998).

24. Blake, K. J., Myette, G. & Jack, J. ribbon, raw, and zipperhave distinct functions in reshaping the Drosophilacytoskeleton. Dev. Genes Evol. 209, 555–559 (1999).

25. Young, P. E., Richman, A. M., Ketchum, A. S. & Kiehart, D. P. Morphogenesis in Drosophila requiresnonmuscle myosin heavy chain function. Genes Dev. 7,29–41 (1993).

26. Byars, C. L., Bates, K. L. & Letsou, A. The dorsal–opengroup gene raw is required for restricted DJNK signalingduring closure. Development 126, 4913–4923 (1999).

27. Bradley, P. L. & Andrew, D. J. ribbon encodes a novelBTB/POZ protein required for directed cell migration inDrosophila melanogaster. Development 128, 3001–3015.(2001).This paper and reference 29 describe a novel nuclearprotein, Ribbon, that is required for trachealbranching. The authors propose that Ribbonregulates the transcription of genes that are involvedin steps in cell migration that occur after processextension.

28. Shim, K., Blake, K. J., Jack, J. & Krasnow, M. A. TheDrosophila ribbon gene encodes a nuclear BTB domainprotein that promotes epithelial migration andmorphogenesis. Development 128, 4923–4933 (2001).The authors compare the tracheal phenotypes ofribbon mutants to those of mutants in signallingpathways, and also describe the effects of Ribbon onother tubular epithelia, most notably the salivarygland. Given the phenotypic similarities with wingless

(wg) mutants, the authors propose that ribbon mightfunction in the wg pathway or parallel to it.

29. Metzger, R. J. & Krasnow, M. A. Genetic control of branchingmorphogenesis. Science 284, 1635–1639 (1999).

30. Srinivas, S. et al. Expression of green fluorescent protein inthe ureteric bud of transgenic mice: a new tool for the analysisof ureteric bud morphogenesis. Dev. Genet. 24, 241–251(1999).

31. Makarenkova, H. P. et al. FGF10 is an inducer and Pax6 acompetence factor for lacrimal gland development.Development 127, 2563–2572 (2000).

32. Weaver, M., Dunn, N. R. & Hogan, B. L. Bmp4 and Fgf10 playopposing roles during lung bud morphogenesis.Development 127, 2695–2704 (2000).

33. Fisher, C. E., Michael, L., Barnett, M. W. & Davies, J. A. ErkMAP kinase regulates branching morphogenesis in thedeveloping mouse kidney. Development 128, 4329–4338(2001).

34. Miura, T. & Shiota, K. Time-lapse observation of branchingmorphogenesis of the lung bud epithelium in mesenchyme-free culture and its relationship with the localization of actinfilaments. Int. J. Dev. Biol. 44, 899–902 (2000).

35. Nogawa, H., Morita, K. & Cardoso, W. V. Bud formationprecedes the appearance of differential cell proliferation duringbranching morphogenesis of mouse lung epithelium in vitro.Dev. Dynamics 213, 228–235 (1998).

36. Hogg, N. A. S., Harrison, C. J. & Tickle, C. Lumen formation inthe developing mouse mammary gland. J. Embryol. Exp.Morphol. 73, 39–57 (1983).

37. Hieda, Y., Iwai, K., Morita, T. & Nakanishi, Y. Mouse embryonicsubmandibular gland epithelium loses its tissue integrityduring early branching morphogenesis. Dev. Dyn. 207,395–403 (1996).

38. Nanba, D., Hieda, Y. & Nakanishi, Y. Remodeling ofdesmosomal and hemidesmosomal adhesion systems duringearly morphogenesis of mouse pelage hair follicles. J. Invest. Dermatol. 114, 171–177 (2000).

39. Nanba, D., Nakanishi, Y. & Hieda, Y. Changes in adhesiveproperties of epithelial cells during early morphogenesis of themammary gland. Dev. Growth Differ. 43, 535–544 (2001).

40. Mailleux, A. A. et al. Role of FGF10/FGFR2b signaling duringmammary gland development in the mouse embryo.Development 129, 53–60 (2002).Mouse mammary glands develop from five pairs ofplacodes. Surprisingly, one of the five placodesdevelops independently of FGF10–FGFR2b, theligand–receptor combination that directs branchingmorphogenesis of the other four.

41. Hennighausen, L. & Robinson, G. Signaling pathways inmammary gland development. Dev. Cell 1, 467–475 (2001).

42. Horne-Badovinac, S. et al. Positional cloning of heart and soulreveals multiple roles for PKC lambda in zebrafishorganogenesis. Curr. Biol. 11, 1492–1502 (2001). The first characterization of a member of thePAR3–PAR6 complex in vertebrates. The resultsindicate that, in a rod of cells destined to form a tube,atypical PKC coordinates the movement of F-actin-richstructures towards the rod centre. These structuresprefigure lumen formation.

43. Lengyel, J. A. & Iwaki, D. D. It takes guts: the Drosophilahindgut as a model system for organogenesis. Dev. Biol. 243,1–19 (2002).A scholarly review of Drosophila hindgut developmentand the insights it provides into the evolutionarilyconserved genetic pathways regulating gutdifferentiation and morphogenesis.

44. Park, M. & Moon, R. T. The planar cell-polarity gene stbmregulates cell behavior and cell fate in vertebrate embryos.Nature Cell Biol. 4, 20–25 (2001).



R E V I E W S

45. Kibar, Z. et al. Ltap, a mammalian homolog of DrosophilaStrabismus/Van Gogh, is altered in the mouse neural tubemutant Loop-tail. Nature Genet. 28, 251–255 (2001).Reports the positional cloning of a classic mousemutant that is widely used as a model for commonneural-tube defects in humans.

46. Sutherland, D., Samakovlis, C. & Krasnow, M. Branchlessencodes a Drosophila FGF homolog that controls trachealcell migration and the pattern of branching. Cell 87,1091–1102 (1996).

47. Jarecki, J., Johnson, E. & Krasnow, M. A. Oxygen regulationof airway branching in Drosophila is mediated by branchlessFGF. Cell 99, 211–220 (1999).

48. Mollard, R. & Dziadek, M. A correlation between epithelialproliferation rates, basement membrane componentlocalization patterns, and morphogenetic potential in theembryonic mouse lung. Am. J. Respir. Cell Mol. Biol. 19,71–82 (1998).

49. Bluemink, J. G., Van Maurik, P. & Lawson, K. A. Intimate cellcontacts at the epithelial/mesenchymal interface inembryonic mouse lung. J. Ultrastruct. Res. 55, 257–270(1976).

50. Zent, R. et al. Involvement of laminin binding integrins andlaminin-5 in branching morphogenesis of the ureteric budduring kidney development. Dev. Biol. 238, 289–302 (2001).Laminin and its integrin receptors are implicated inureteric-bud formation in vitro.

51. Mercurio, A. M., Rabinovitz, I. & Shaw, L. M. The α6β4integrin and epithelial cell migration. Curr. Opin. Cell Biol. 13,541–545 (2001).

52. Trusolino, L., Bertotti, A. & Comoglio, P. M. A signalingadapter function for α6β4 integrin in the control of HGF-dependent invasive growth. Cell 107, 643–654 (2001). The authors show that the α6β4 integrin forms acomplex with the HGF receptor, the Met receptortyrosine kinase, that is required for Met signalling.

53. Ito, T. et al. Repulsive axon guidance molecule Sema3Ainhibits branching morphogenesis of fetal mouse lung. Mech.Dev. 97, 35–45 (2000).

54. Xian, J. et al. Inadequate lung development and bronchialhyperplasia in mice with a targeted deletion in theDutt1/Robo1 gene. Proc. Natl Acad. Sci. USA 98,15062–15066 (2001). This paper implicates Robo1, a receptor formammalian Slits, in controlling epithelial organizationor cell number during lung development. Slits arechemorepellants for axonal growth cones andlymphocytes, but have not yet been implicated in lungdevelopment.

55. Warburton, D. et al. The molecular basis of lungmorphogenesis. Mech. Dev. 92, 55–81 (2000).

56. Risau, W. Mechanisms of angiogenesis. Nature 386,671–674 (1997).

57. Drake, C. J. & Fleming, P. A. Vasculogenesis in the day 6.5 to9.5 mouse embryo. Blood 95, 1671–1679 (2000).

58. Drake, C., Brandt, S., Trusk, T. & Little, C. TAL1/SCL isexpressed in endothelial progenitor cells/angioblasts anddefines a dorsal-to-ventral gradient of vasculogenesis. Dev.Biol. 192, 17–30 (1997).

59. Cleaver, O. & Krieg, P. A. VEGF mediates angioblastmigration during development of the dorsal aorta in Xenopus.Development 125, 3905–3914 (1998).

60. Yamaguchi, T. P., Dumont, D. J., Conlon, R. A., Breitman, M. L. & Rossant, J. flk-1, an flt-related receptortyrosine kinase is an early marker for endothelial cellprecursors. Development 118, 489–498 (1993).

61. Ferrara, N. Molecular and biological properties of vascularendothelial growth factor. J. Mol. Med. 77, 527–543 (1999).

62. Carmeliet, P. et al. Abnormal blood vessel development andlethality in embryos lacking a single VEGF allele. Nature 380,435–439 (1996).

63. Carmeliet, P. et al. Impaired myocardial angiogenesis andischemic cardiomyopathy in mice lacking the vascularendothelial growth factor isoforms VEGF164 and VEGF188.Nature Med. 5, 495–502 (1999).

64. Fong, G. H., Rossant, J., Gertsenstein, M. & Breitman, M. L.Role of the Flt-1 receptor tyrosine kinase in regulating theassembly of vascular endothelium. Nature 376, 66–70(1995).

65. Shalaby, F. et al. Failure of blood-island formation andvasculogenesis in Flk-1-deficient mice. Nature 376, 62–66(1995).

66. Dumont, D. J. et al. Cardiovascular failure in mouse embryosdeficient in VEGF receptor-3. Science 282, 946–949 (1998).

67. Kawasaki, T. et al. A requirement for neuropilin-1 inembryonic vessel formation. Development 126, 4895–4902(1999).

68. Yancopoulos, G. D. et al. Vascular-specific growth factorsand blood vessel formation. Nature 407, 242–248 (2000).

69. Zhong, T. P., Childs, S., Leu, J. P. & Fishman, M. C. Gridlocksignalling pathway fashions the first embryonic artery.Nature 414, 216–220 (2001).The authors show that Notch signalling controls veinversus artery identity.

70. Helbling, P. M., Saulnier, D. M. & Brandli, A. W. The receptortyrosine kinase EphB4 and ephrin-B ligands restrictangiogenic growth of embryonic veins in Xenopus laevis.Development 127, 269–278 (2000).

71. Shin, D. et al. Expression of EphrinB2 identifies a stablegenetic difference between arterial and venous vascularsmooth muscle as well as endothelial cells, and markssubsets of microvessels at sites of adult neovascularization.Dev. Biol. 230, 139–150 (2001).The authors found that the arterial marker Ephrin B2 is expressed on capillaries, which indicatesthat capillaries actually have arterial or venousidentity, rather than belonging to a third class ofvessel. They also found that Ephrin B2 is expressed inadult arterial smooth muscle, indicating a possiblerole for Ephrin B2 in the physiology of arterial smoothmuscle.

72. Gale, N. W. et al. Ephrin-B2 selectively marks arterial vesselsand neovascularization sites in the adult, with expression inboth endothelial and smooth-muscle cells. Dev. Biol. 230,151–160 (2001).

73. Takahashi, T. et al. Temporally compartmentalizedexpression of ephrin-B2 during renal glomerulardevelopment. J. Am. Soc. Nephrol. 12, 2673–2682 (2001).

74. Childs, S., Chen, J.-N., Garrity, D. M. & Fishman, M. C.Patterning of angiogenesis in the zebrafish embryo.Development 129, 973–982 (2002).This paper describes the assembly of veins in thezebrafish embryo using live imaging, a key advance.The authors also describe a novel mutation, obd, inwhich the veins are assembled in the wrong places.The obd gene product probably regulates endothelial-cell migration non-cell-autonomously.

75. Adams, R. H. et al. The cytoplasmic domain of the ligandEphrinB2 is required for vascular morphogenesis but notcranial neural crest migration. Cell 104, 57–69 (2001).This paper shows that Ephrin B2 ligands signal cellautonomously during vascular morphogenesis, butnot during neural-crest migration.

76. Uemura, T. et al. Zygotic Drosophila E-cadherin expressionis required for processes of dynamic epithelial cellrearrangement in the Drosophila embryo. Genes Dev. 10,659–671 (1996).

77. Tanaka-Matakatsu, M., Uemura, T., Oda, H., Takeichi, M. &Hayashi, S. Cadherin-mediated cell adhesion and motility inDrosophila trachea regulated by the transcription factorEscargot. Development 122, 3697–3705 (1996).

78. Bayless, K. J., Salazar, R. & Davis, G. E. RGD-dependentvacuolation and lumen formation observed duringendothelial cell morphogenesis in three-dimensional fibrinmatrices involves the α(v)β(3) and α(5)β(1) integrins. Am. J.Pathol. 156, 1673–1683 (2000).

79. Maina, F. et al. Coupling Met to specific pathways results indistinct developmental outcomes. Mol. Cell 7, 1293–1306(2001).Analysis of the effects of different mutants in the Metcytoplasmic domain on its developmental functions.Met orchestrates several distinct responses throughthe phosphorylation of specific tyrosine residues andthe recruitment of different sets of adaptor/signallingmolecules.

80. Pollack, A. L., Runyon, R. B. & Mostov, K. E. Morphogeneticmechanisms of epithelial tubulogenesis: MDCK cell polarityis transiently rearranged without loss of cell–cell contactduring scatter factor/hepatocyte growth factor-inducedtubulogenesis. Dev. Biol. 204, 64–79 (1998).

81. O’Brien, L. E. et al. Rac1 orientates epithelial apical polaritythrough effects on basolateral laminin assembly. Nature CellBiol. 3, 831–838 (2001). The authors show that cell-surface receptorsassemble laminin, an extracellular-matrix component,to set the apical–basal polarity for cells in cysts grownin culture. Similar interactions with laminin mightdefine cell polarity in developing tubes in vivo.

82. Derman, M. P., Chen, J. Y., Spokes, K. C., Songyang, Z. & Cantley, L. G. An 11-amino acid sequence from c-metinitiates epithelial chemotaxis via phosphatidylinositol 3-kinase and phospholipase C. J. Biol. Chem. 271,4251–4255 (1996).

83. Karihaloo, A., O’Rourke, D. A., Nickel, C., Spokes, K. &Cantley, L. G. Differential MAPK pathways utilized for HGF-and EGF-dependent renal epithelial morphogenesis. J. Biol.Chem. 276, 9166–9173 (2001).

84. Li, D. & Mrsny, R. J. Oncogenic Raf-1 disrupts epithelial tightjunctions via downregulation of occludin. J. Cell Biol. 148,791–800 (2000).

85. Berrier, A. L., Mastrangelo, A. M., Downward, J., Ginsberg, M.& LaFlamme, S. E. Activated R-ras, Rac1, PI 3-kinase andPKCε can each restore cell spreading inhibited by isolatedintegrin β1 cytoplasmic domains. J. Cell Biol. 151,1549–1560 (2000).

86. Boccaccio, C. et al. Induction of epithelial tubules by growthfactor HGF depends on the STAT pathway. Nature 391,285–288 (1998).

87. Beitel, G. J. & Krasnow, M. A. Genetic control of epithelialtube size in the Drosophila tracheal system. Development127, 3271–3282 (2000).

88. LeCouter, J. et al. Identification of an angiogenic mitogenselective for endocrine gland endothelium. Nature 412,868–869 (2001).The purification and characterization of a new factorthat regulates the development of a subset ofendothelial cells. This discovery indicates theexistence of several subsets of endothelial cells thatmight respond to different, perhaps spatiallyregulated, growth factors.

89. Litingtung, Y., Lei, L., Westphal, H. & Chiang, C. Sonichedgehog is essential to foregut development. NatureGenet. 20, 58–61 (1998).

90. Lindahl, P. et al. Alveogenesis failure in PDGF-A-deficientmice is coupled to lack of distal spreading of alveolarsmooth muscle cell progenitors during lung development.Development 124, 3943–3953 (1997).

91. Cho, N. K. et al. Developmental control of blood cellmigration by the Drosophila VEGF pathway. Cell 108,865–876 (2002).Reports the cloning and expression of Drosophilagenes encoding VEGF receptor and VEGF ligands and the failure of blood-cell migration in mutants. The authors speculate about the evolutionary origin of blood vessels and the interdependence of blood cells/vessels and other tubular systems.

92. Matsumoto, K., Yoshitomi, H., Rossant, J. & Zaret, K. S.Liver organogenesis promoted by endothelial cells prior tovascular function. Science 294, 559–563 (2001).

93. Lammert, E., Cleaver, O. & Melton, D. Induction ofpancreatic differentiation by signals from blood vessels.Science 294, 564–567 (2001).

AcknowledgementsWe thank M. Fishman and S. Childs for making their manuscriptavailable before publication, D. Andrew for stimulating discussionand our colleagues M. Weaver, S. Hanks, Y. Li, R. Zent and T. Zhong for comments on the manuscript. Work in our laboratoriesis supported by the Howard Hughes Medical Institute and theNational Institutes of Health. B.L.M.H. is an Investigator of theHoward Hughes Medical Institute.

Online linksDATABASESThe following terms in this article are linked online to:FlyBase: http://flybase.bio.indiana.eduRhoA | Abdominal-B | Branchless | Breathless | crumbs | Dpp | fkh |Fog | Huckebein | raw | ribbon | Scr | Semaphorin II | Short Stop |Shotgun | Sp1 | teashirt | zipperLocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkangiopoietin-1 | Cdc42 | ephrin B2 | Fgf10 | hepatocyte growthfactor | Jam | Lef1 | Msx1 | Msx2 | neuropilin-1 | p120 | Pdgfa |PLCγ | Pthrp | Roundabout | Sonic hedgehog | Stat3 | TTF2 |VegfaWormbase: http://www.wormbase.orgexc-5 | sma-1OMIM: http://www.ncbi.nlm.nih.gov/Omimfaciogenital dysplasia | spina bifidaAccess to this interactive links box is free online.


Documents

ORGANOGENESIS: MOLECULAR MECHANISMS OF TUBULOGENESIS