Download Cellular and molecular mechanisms involved in branching

J Appl Physiol 97: 2347–2353, 2004;
doi:10.1152/japplphysiol.00435.2004.
HIGHLIGHTED TOPIC
Invited Review
Lung Growth and Repair
Cellular and molecular mechanisms involved in branching morphogenesis
of the Drosophila tracheal system
Clemens Cabernard, Marc Neumann, and Markus Affolter
Abteilung Zellbiologie, Biozentrum der Universität Basel, Basel, Switzerland
tubulogenesis; cell migration; fibroblast growth factor signaling; air sacs; zona
pellucida domain-containing proteins; adherens junction remodeling
THE QUESTION OF HOW PARTICULAR ORGANS form during development
and how these organs ultimately function during adult life has
moved to center stage in developmental genetics. The conservation of genetic regulatory cascades and networks during evolution
argues that simple model organisms might contribute important
information concerning organ formation, although the architecture
of these organs is simpler and their function more limited compared with higher organisms, such as mice and humans.
The fruit fly Drosophila melanogaster is a particularly wellcharacterized model organism. Genetic analysis over the past
century have led to the characterization of approximately onefourth of the estimated 13,500 genes present in the genome (2).
All major signaling pathways involved in cell-cell interactions
during development of higher animals are conserved in the fruit
fly, and many of the organ-specific transcriptional regulators play
similar roles in the fly compared with mice or humans (37, 57).
Several organ systems have been dissected in Drosophila using
genetic and molecular approaches (1, 5, 15, 17, 21, 47). Here, we
briefly summarize the current state of knowledge concerning the
development of the respiratory organ of the fly (the trachea) and
mention parallels between tracheal development and the development of the lung in mammals.
The larval tracheal system consists of hundreds of interconnected tubes that transport oxygen and other gases throughout
the body (3, 18, 51). Tracheal branches are simple tubes
consisting of an epithelial monolayer wrapped into a tube
around a central lumen. The development of the tracheal
system is initiated in the early embryo on the determination of
10 bilaterally symmetrical clusters of ⬃20 tracheal precursor
cells; on invagination and two additional cell divisions, each of
the tracheal sacs undergoes a similar sequence of developmental events to generate one segment of the network in the
absence of further cell divisions (Fig. 1, A–H). The transformation, in a few hours time, of a simple epithelial sac into a
highly ramified, yet stereotyped, tubular network is a fascinating phenomenon and has attracted the attention of a number of
research groups sharing the aim of better understanding the
cellular and molecular principles underlying the control and
regulation of branching morphogenesis. As it turns out, three
key cellular processes are involved in branch formation: directional cell migration, cell rearrangements, and cell shape
changes. To interconnect the individual metameres, certain
tubes ultimately fuse with corresponding tubes in neighbor
segments (38, 48).
The most insightful finding with regard to the spatial control
of tracheal branch formation in the Drosophila embryo came
from the identification and characterization of two genes,
breathless/FGFR (btl/FGFR) and branchless/FGF (bnl/FGF).
The absence of either of these two gene products leads to a
complete failure of branch outgrowth despite proper determination of tracheal cells (29, 45). btl/FGFR encodes a fibroblast
growth factor (FGF) receptor expressed in tracheal cells but not
Address for reprint requests and other correspondence: M. Affolter, Abteilung Zellbiologie, Biozentrum der Universität Basel, CH-4056 Basel, Switzerland (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
CELL MIGRATION CONTROLS BRANCH FORMATION
IN THE EMBRYO
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8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society
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Cabernard, Clemens, Marc Neumann, and Markus Affolter. Cellular and
molecular mechanisms involved in branching morphogenesis of the Drosophila
tracheal system. J Appl Physiol 97: 2347–2353, 2004; doi:10.1152/japplphysiol.
00435.2004.—Recent comparative studies have shown that, in many instances, the
genetic network underlying the development of distinct organ systems is similar in
invertebrate and vertebrate organisms. Genetically well-characterized, simple invertebrate model systems, such as Caenorhabditis elegans and Drosophila melanogaster, can thus provide useful insight for understanding more complex organ
systems in vertebrates. Here, we summarize recent progress in the genetic analysis
of tracheal development in Drosophila and compare the results to studies aimed at
a better understanding of lung development in mouse and man. Clearly, both
striking similarities and important differences are apparent, but it might still be too
early to conclude whether the former or the latter prevail.
Invited Review
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DROSOPHILA TRACHEAL SYSTEM MORPHOGENESIS
in surrounding epithelial cells (29), whereas bnl/FGF encodes
the FGF ligand of Btl/FGFR and is expressed in individual
cells or cell clusters surrounding the invaginating tracheal
placode (50). Gain-of-function and loss-of-function studies
(50) combined with live imaging (46) revealed that the Bnl/
FGF ligand induces a migratory behavior by promoting cytoskeletal dynamics in a few cells at the tip of the tracheal
branches (see Fig.1, K–N). The stalk cells appear to follow
these tip cells passively; during branch formation, all tracheal
cells maintain their epithelial character and remain attached to
each other via their subapical adherens junctions (AJ).
It is not clear yet whether Bnl/FGF acts as a true chemoattractant or as a motogen. A function as a chemoattractant is
supported by the finding that bnl/FGF expression in ectopic
positions induces branch outgrowth toward such sites (49, 50).
However, it was shown that the direction of migration is
influenced by other signaling systems, such as Dpp/BMP (46,
52). In the absence of Dpp/BMP signaling, prospective dorsal
branch cells start to migrate dorsally but ultimately reintegrate
into the dorsal trunk and thus never form a definitive dorsal
branch (for nomenclature, see Fig. 1I) (46). Conversely, ectopic Dpp/BMP signaling is able to direct cells from migration
along the anterior-posterior axis toward migration along the
dorsal-ventral axis (52). How Dpp/BMP influences the direction of migration is not known and remains to be investigated.
What are the molecular consequences of FGF signaling in
tracheal cells, or, in other words, how are cells instructed to
become motile and follow directions under the control of
Bnl/FGF? Genetic studies have led to the identification of a
mutation in a gene called downstream-of-FGFR (dof), which
displays a tracheal phenotype identical to bnl/FGF and btl/
FGFR (25, 40, 53). Dof turns out to be a cytoplasmic signaling
J Appl Physiol • VOL
adaptor protein, present in only those cells of the embryo that
express FGF receptors. Dof interacts constitutively with the
Drosophila FGF receptors and becomes phosphorylated by the
latter on activation, leading to the recruitment of the Corkscrew
phosphatase (44, 59). Corkscrew recruitment represents an
essential step in Bnl/FGF-induced cell migration and in the
activation of the Ras/MAPK pathway. However, it appears that
Ras activation is not sufficient to activate the migration machinery in tracheal cells (44). Additional proteins binding
either to the FGFRs, to Dof, or to Corkscrew appear to be
crucial for a migratory response, and it will be important to
identify these missing links. Somewhat surprisingly, tracheal
cells also migrate properly when the spatial information of
Bnl/FGF ligand expression is transmitted to the cell interior by
the kinase domain of a different receptor tyrosine kinase; this
was shown via the expression of chimeric receptor molecules
carrying the extracellular domain of Btl/FGFR and the intracellular kinase domain of unrelated receptor tyrosine kinases,
such as epidermal growth factor receptor or Torso (16). These
results demonstrate that tracheal cells respond with directed
migration to receptor tyrosine kinase signaling (whereas other
cells in the developing embryos respond differently). Therefore, it will be important to decipher the regulatory program
that leads to the determination of epithelial cells toward the
tracheal fate; identification and characterization of genes that
are specifically activated or repressed in tracheal cells might
shed light on why and how tracheal cells respond to FGF
signaling with migration.
These studies on the formation of the tracheal tree provide
the first insights into molecular events of guided cell migration
controlled by FGF signaling. However, the developing embryo
of Drosophila is not an ideal model system to investigate these
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Fig. 1. Tracheal development in the Drosophila embryo. A–D:
development of the embryonic tracheal system. The tracheal
system arises from 10 placodes on each side of the embryo that
bud in from the epidermis. On invagination of the tracheal cells
and after 2 rounds of cell division, the individual tracheal
metameres consist of ⬃80 cells (A). From this placode, 6
branches grow out into different directions (B–D). Subsequently, some of these branches fuse to form a continuous
network that will be used for oxygen supply in the hatching
larva. E–H: schematic drawing of the development of an individual (boxed) tracheal metamere. Lateral views show the
stereotyped branching pattern of the tracheal epithelium (TR;
red) as well as the expression of the fibroblast growth factor
(FGF) ligand bnl (blue). Light blue indicates fading bnl/FGF
expression. Top views (e– h) indicate the connection to the
epidermis (EP) and the flattening of the tracheal sac on progression of development. Note that embryonic branching morphogenesis does not rely on cell division but on cell migration,
cell intercalation, and cell shape changes. I: nomenclature of the
tracheal branches. DB, dorsal branch; DTa/p, dorsal trunk
anterior/posterior; VB, visceral branch; SB, spiracular branch;
LTa/p, lateral trunk anterior/posterior. K–N: live images of a
dorsal branch expressing actin tagged with green fluorescent
protein. Branch outgrowth is driven by Bnl/FGF, which induces
filopodia in tracheal tip cells (arrows). During branch outgrowth, the cells are rearranged from a side-by-side (K) to an
end-to-end (N) arrangement, visualized here by the rearrangement of the nuclei (*).
Invited Review
DROSOPHILA TRACHEAL SYSTEM MORPHOGENESIS
CELL REARRANGEMENTS AND CELL SHAPE CHANGES
LEAD TO BRANCH ELONGATION IN THE EMBRYO
Quite obviously, the active migration of a small number of
tracheal tip cells away from the saclike structure of the invaginated tracheal placode has to be accompanied by cell rearrangements and cell shape changes to generate elongated
tracheal branches. Because tracheal cells are at all time part of
a tightly sealed epithelium, epithelial AJs must be remodeled to
a significant extent during branch formation. Indeed, extensive
AJ remodeling is required for the generation of most tracheal
branches of a late Drosophila embryo, and individual steps in
the remodeling process have been described in dorsal branch
formation (27, 48). During the early stages of branch formation, tracheal cells are initially arranged in a side-by-side-side
fashion (see Fig. 1K). Through cell intercalation (Fig. 1, L–M),
this conformation is transformed into an end-to-end arrangement (Fig. 1N). The dorsal branch (as well as most other
branches) finally consists of single cells wrapped around the
lumen and closed up by autocellular AJs (27, 48).
The molecular events underlying AJ remodeling have not
yet been elucidated, but it appears that the coordination of cell
rearrangements with cell migration requires additional developmental signaling systems. For example, the outgrowth of a
particular branch, the dorsal branch, required Dpp/BMP signaling in addition to Bnl/FGF signaling (46). The lack of
Dpp/BMP leads to the absence of dorsal branches, despite the
presence of filopodia formation and initial dorsal branch outgrowth under the control of Bnl/FGF signaling; in the absence
of Dpp/BMP signaling, cells in the initial dorsal bud reintegrate into the major tracheal branch, the dorsal trunk (46). Thus
Dpp/BMP signaling appears to be required for proper cell
rearrangements to take place, and in the absence of concomitant cell rearrangements cell migration is not sufficient to bring
about the formation of distinct tracheal branches. It also appears that the activity level of the small GTPase Rac has a
strong impact on cell rearrangements; reduced Rac activity
inhibits tracheal cell rearrangements, whereas hyperactivation
leads to the loss of tracheal cell adhesion (12). These effects
J Appl Physiol • VOL
might be due to a regulation of the levels of E-cadherin in
tracheal cells, but how exactly Rac activity levels and Ecadherin protein levels are coordinated remains to be determined.
Interestingly, a recent study has made a link between tracheal cell rearrangements and the presence of proteins secreted
apically into the luminal space (27). Piopio and Dumpy, two
proteins containing a zona pellucida domain (28), have been
shown to contribute to the apical extracellular matrix and to
play an important role during the intercalation process (see Fig.
1, K–N) and autocellular AJ formation in tracheal development
(27). In the absence of these two proteins, all fine tracheal
branches form cysts instead of tubes, leading to a disruption of
the tracheal network. Although the precise role of the two
proteins remains to be firmly established, this study demonstrates for the first time an important requirement for luminal
matrix proteins in tubulogenesis.
DEVELOPMENTAL CONTROL OF TUBE SIZE, TUBE
DIAMETER, AND LIQUID CLEARANCE
The size and shape of the branches making up a tubular
organ are fundamental parameters in the control of transport
demands. Like in other tubular organs, tracheal tubes increase
in size and diameter as the animal grows during development.
At the end of larval life, tracheal tubes have expanded up to 40
times their initial size. This increase in tube size does not
depend on cell division or on the number of cells in the
branches but is controlled mostly at the apical surface of
tracheal cells; although there is a dramatic increase in the inner
tube diameter, little change is observed in the outer tube
diameter (7).
Tube size is controlled genetically and is not modified under
high- or low-oxygen conditions. A number of mutants, in
which the length as well as the diameter of tracheal branches is
altered, have been described (7). The two Drosophila Claudin
family members Megatrachea (6, 7) and Sinuous (60) as well
as the cell surface protein Lachesin (36) are components of
septate junctions, and mutations in these genes show overgrown tubes. Other components of septate junctions, such as
Coracle and Neurexin, also show similar phenotypes in the
tracheal system when absent (6, 43). However, whether the
developmental control of tube shape, e.g., the increase in tube
diameter in larval stages, is exerted via these genes remains to
be investigated.
Apart from septate junction components, the control of the
growth of the apical membrane seems to be equally important
for maintaining a balanced tube size and length. The transcription factor Grainy head (Grh) limits apical membrane growth;
in grh mutants, several branches are convoluted, and grh
tracheal cells show an expansion of the apical membrane in late
embryonic stages, resulting in an enlarged and anomalous
luminal surface (22). Neither Grh target genes nor other effector proteins involved in the control of the apical surface of
tracheal cells have been identified so far.
Liquid clearance of the tracheal system provides a developmentally controlled physiological aspect of this tubular organ.
At the end of embryogenesis, 2 h before larvae emerge, the
liquid in the tracheal system is cleared and replaced by air (38).
Clearance occurs by active epithelial absorption, and epithelial
Na⫹ channels appear to be involved in this process. Several
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molecular events at a much more detailed level using a genetic
approach. This is due to the fact that many proteins essential
for either cell signaling (i.e., Ras) and/or cytoskeletal rearrangements (i.e., Rac) are maternally provided (large amounts
of RNA and/or protein products of numerous genes are transferred from the polyploid nurse cells of the mother to the
oocyte and are sufficient for the early development of the
embryo) and thus often fail to be detected in standard genetic
screens for zygotic effects. In addition, the lack of proteins
involved in important, basic cellular events might lead to
dramatic defects in embryonic stages before tracheal branching, making analysis of their contribution to branching morphogenesis more difficult. However, and as we will outline
below, FGF signaling via Bnl/FGF and Btl/FGFR is again used
during larval and pupal stages to induce branching morphogenesis in a growing tracheal epithelium, the developing air sac
(49). This system allows the identification of genes involved in
branching morphogenesis in a genetically more amenable context, and major progress toward a better molecular understanding of FGF signaling in branching morphogenesis is expected
to be obtained by studying this system.
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Invited Review
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DROSOPHILA TRACHEAL SYSTEM MORPHOGENESIS
Degenerin/epithelial Na⫹ channel family members are expressed quite specifically in the tracheal system. Inhibitor as
well as RNAi studies targeting the products of two of these
genes (PPK4, PPK11) inhibited liquid clearance (34), suggesting that the regulated activity of these channels contributes to
the proper differentiation of tracheal cells.
PHYSIOLOGICAL ASPECTS OF TRACHEAL
DEVELOPMENT AND FUNCTION
COORDINATION BETWEEN CELL MIGRATION AND CELL
DIVISION DURING AIR SAC FORMATION IN THE LARVA
A striking difference between the development of branched
organs in higher vertebrates and the formation of the tracheal
system in the Drosophila embryo lies in the fact that cells do
not divide during the branching process in the embryonic
tracheal system of the fly; the increase in the three-dimensional
complexity of the network is due to cell migration, cell rearrangements, and cell shape changes. During larval development, however, the tracheal system is remodelled tremendously to satisfy the needs in the adult fly, and the formation of
new structures includes, and presumably relies on, controlled
cell proliferation (38).
Recently, the formation of the thoracic air sacs of the adult
fly has been investigated (49). The thoracic air sac is an
offshoot of an existing tracheal branch, the transverse connective, which attaches to the wing imaginal disc (see Fig. 2). In
the early-third instar larva, some 50 h before pupation, a few
tracheal cells of the transverse connective branch start to
proliferate and migrate out in a stereotypical manner. Both
btl/FGFR and dof are expressed in the tracheal cells of this
growing air sac, suggesting that the FGF signaling system,
Fig. 2. Air sac development in the third instar larva of Drosophila. A: cross section through a wing imaginal disc of a
Drosophila third instar larva. The transverse connective branch
(TC) attaches to the columnar epithelial (CE) of the wing
imaginal disc. A group of CE cells, lying underneath the
outgrowing air sac, secrete FGF/Bnl (depicted in blue). PM,
peripodial membrane. B: schematic drawings of three stages of
air sac development corresponding to early, mid, and late third
instar larval stages. The tracheal cells of the TC as well as the
air sac tracheoblast cells express the Bnl/FGF receptor Btl/
FGFR (red), whereas cells from CE express Bnl/FGF (blue).
Air sac tracheoblast cells follow the Bnl/FGF-secreting CE cells
via cell migration, and the air sac increases in size through cell
proliferation. C: projection of a wing imaginal disc from a late
third instar larva. D: close up of the boxed areas in B. A
membrane-targeted GFP protein outlines all wing imaginal disc
cells, whereas the tracheal cells express Moesin tagged with real
fluorescent protein, under the control of the trachea-specific
enhancer of btl/FGFR.
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Quite in contrast to the stereotyped, developmentally controlled organization of the major branches of the larval tracheal
system, the organization and ramification of the so-called
terminal branches are under physiological control. Terminal
branch formation is regulated by the gene DSRF/blistered,
which encodes the Drosophila homolog of serum response
factor (4, 20, 42). DSRF/blistered is specifically expressed in
terminal tracheal cells and required for the formation of tens to
hundreds of long cytoplasmic processes by each of these cells
during the larval stages. These cytoplasmic processes form an
intracellular lumen, which connects up to the lumen of the
main tracheal network. Larvae grown under nonphysiological,
low-oxygen conditions show a dramatic increase in terminal
branching; conversely, larvae grown under high-oxygen tension show the opposite phenotype, having relatively few terminal branches (26).
A link between terminal branching and oxygen conditions
has been established through the analysis of bnl/FGF, which is
expressed broadly in the larva and in all tissues that become
heavily tracheated. Strikingly, bnl/FGF transcription is enhanced in many cells under low hypoxic conditions. 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. This implies that, in
Drosophila, Bnl/FGF is either a or possibly the key mediator of
the hypoxic response (26).
These experiments suggested that an oxygen-sensing pathway exists in Drosophila. Indeed, it has been shown more
recently that the hypoxia-inducing factor-␣, encoded by the
gene similar (sima), as well as the hypoxia-inducing factor-␤
subunit encoded by tango, are required for the hypoxic response. Sima and tango code for basic helix-loop-helix PAS
transcription factors that form heterodimers. It was also proposed that Sima protein stability is controlled by oxygen
through the Prolyl 4-hydroxylase homolog encoded by
CG1114; a mutant in which CG1114 protein expression is
reduced shows an upregulation of Sima under normal physiological oxygen conditions (31). Thus fine terminal branches are
formed from terminal tracheal cells as a result of oxygen needs
in target tissues, but how the outgrowth of terminal branches is
controlled and coordinated by FGF signaling remains to be
established.
Invited Review
DROSOPHILA TRACHEAL SYSTEM MORPHOGENESIS
SIMILARITIES BETWEEN TRACHEAL BRANCHING IN
DROSOPHILA AND LUNG BRANCHING IN MAMMALS
Clearly, the most striking similarity between tracheal development in Drosophila and lung-branching morphogenesis in
mammals is the involvement of the FGF signaling pathway as
a key regulator of branching (3, 14, 23, 54, 55). In mice,
FGF10 is clearly associated with early branching, because in
FGF10 mutant mice branching morphogenesis is completely
absent (41). In addition, FGF10 expression in the mesenchyme
surrounding the growing lung epithelium correlates well with
local branch outgrowth (8). The precise function of FGF10 in
lung development in mice is less well understood than the role
of Bnl/FGF in tracheal branching, but organ culture experiments favor a role of FGF10 as both a chemoattractant/
motogen and a mitogen (58), similar to the role of Bnl/FGF in
J Appl Physiol • VOL
larval air sac branching in the fly (49). Studying the molecular
consequences of FGF signaling in the larval tracheal system in
Drosophila will provide further insight into the molecular
events downstream of FGF signaling during branch formation.
In addition, these studies might shed light on the dual role of
FGF in mammals, the control and coordination of cell proliferation, and cell migration.
Like in Drosophila, other signaling pathways, including the
Hh and the Dpp/BMP pathway, play important roles in lung
branching in mice. It appears that, in the mouse, these signaling
pathways act in part by limiting the extent of FGF10 signaling
in the lung epithelium (13). In Drosophila, both the Wnt (11,
35) and the Dpp/BMP pathway are essential for successful
branching in the embryo but, as outlined above, Dpp/BMP
plays a major role in controlling cell rearrangements during
branch outgrowth. To our knowledge, it is not clear whether AJ
remodeling, in particular the formation of tubes with autocellular AJs, is important for the formation of fine terminal
branches in the lung. Thus it remains to be seen to what extent
similarities between Drosophila and mice or humans exist with
regard to cell rearrangements.
Recent studies have demonstrated a requirement of luminal
zona pellucida-domain proteins during the branching process
in Drosophila (27). Zona pellucida-domain proteins are also
major components of lumen-containing epithelial organs in
vertebrates (10, 24, 30, 32, 33, 39, 56). It remains to be seen
whether they contribute to the proper formation of these organs
during development or whether they play physiological roles.
Without doubt, the number of molecules linked to the process
of branching morphogenesis of specific organs will dramatically increase in the future, and it is hoped that this will lead
not only to an increase in the molecular complexity underlying
the branching process but also to an enhancement of clarity
with regard to developmental logic.
ACKNOWLEDGMENTS
We thank all members of the Affolter laboratory for discussions.
GRANTS
The authors are supported by funds from the Swiss National Science
Foundation and the Kantons Basel-Stadt and Basel-Land.
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in late larval stages opens the door for the application of
genetic mosaic analysis, namely the generation of genetically
altered (for example mutant) cells in an otherwise heterozygous organ via mitotic recombination (9, 19, 61). Because the
migration of air sac cells occurs some 70 h after egg laying, the
maternal store of RNA and/or protein of most genes has been
used up, and the effect of the removal of these genes on
tracheal cell migration can be studied in genetic mosaic analysis in these late developmental stages. Generation of mosaic
air sacs, including small clusters of mutated cells, will allow
addressing a range of open questions, such as the role of
different molecules in cell migration and cell proliferation, in
particular with regard to components of the FGFR signaling
pathway, such as Ras, Raf, or Erk (all of which are maternally
provided). These studies will allow a better understanding of
the molecular pathways that link FGF signaling to cytoskeletal
dynamics and eventually provide a more precise molecular
view of how FGF signaling controls branching morphogenesis.
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