1 Department of Developmental Biology, Wenner-Gren Institute, Stockholm
University, S-10691 Stockholm, Sweden
2 Department of Medical Biochemistry, University of Gothenburg, S-405 30
Gothenburg, Sweden
3 Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden
4 Department of Anatomy, University of Cambridge, Cambridge, CB2 3DY, UK
Author for correspondence (e-mail:
christos{at}devbio.su.se)
Accepted 22 October 2002
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SUMMARY |
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Key words: Epithelia, Morphogenesis, Drosophila melanogaster, Tubulogenesis, Trachea
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INTRODUCTION |
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The development of the Drosophila trachea, a complex network of
epithelial airways that supplies oxygen to the entire animal, provides a
well-defined system for the analysis of regulatory mechanisms that control
cell migration and branching (Manning and
Krasnow, 1993; Samakovlis et
al., 1996a
). The tracheal system arises from 20 independent sacks
of approximately 80 cells that undergo a distinct sequential programme of
branch sprouting, directed branch outgrowth and branch fusion. Initially, the
actions of at least three independent signals, TGFß-like
(Decapentaplegic; Dpp), Wingless (Wg) and EGF, subdivide the cells in each
tracheal placode into branch-specific groups
(Affolter and Shilo, 2000
).
Subsequent branch sprouting and outgrowth occurs without cell division as
cells migrate towards localised sources of Branchless (Bnl), an attractant
signal of the FGF family (Sutherland et
al., 1996
). Primary branch growth entails the initial extension of
cytoplasmic processes towards the Bnl source, followed by movement of the cell
body and a concomitant increase in apical cell surface to promote lumenal
extension. The characteristic lengths and diameters of the newly formed
branches of the larval trachea are stereotyped and become specified during
distinct developmental intervals (Beitel
and Krasnow, 2000
).
Bnl is the key morphogen co-ordinating branching that acts via the receptor
tyrosine kinase Breathless (Btl) (Klambt
et al., 1992) and the adaptor protein Dof/Stumps
(Vincent et al., 1998
;
Imam et al., 1999
). This
pathway leads to phosphorylation and activation of MAPK
(Gabay et al., 1997
), which in
turn may alter the activity of regulatory proteins to control cell behaviour.
During primary branching, actin-rich basal extensions are sent by the tracheal
cells towards the sources of Bnl, a process that is likely to involve
cytoskeletal modulation by the Rho family GTPases
(Ribeiro et al., 2002
;
Wolf et al., 2002
). Bnl
signalling is also required for the expression of cell-fate determining genes
in specific subsets of tracheal cells in each primary branch. Analysis of
these genes has identified key components of the patterning and guidance of
the unicellular secondary and terminal branches
(Metzger and Krasnow, 1999
).
However, the role of Bnl in the movement of the cell bodies and the growth of
the branch lumen remains unknown.
We have investigated mechanisms that control the elongation of tracheal
tubes. We have characterised mutations in three genes that affect branch
growth, resulting in abnormally long tubes. Mutations in fasII and
Atp alter cell adhesion and the basolateral cell domains,
causing aberrations in cell shapes, excessive tubular elongation and sporadic
lumenal dilations and breaks. In contrast, the transcription factor Grainy
head (Grh) is required to specifically control tube elongation. Both loss of
function and overexpression of grh indicate that it is required to
limit lumenal growth and control tubular length. Grh selectively affects the
growth of the apical cell membrane, arguing that different genetic programmes
regulate distinct sub-cellular domains during branching morphogenesis. Grh is
uniformly expressed in the trachea, but its activity is modulated by Bnl/Btl
signalling and Grh counteracts the activity of Bnl induced branch growth.
Thus, through its regulation of Grh, Bnl regulates epithelial apical membrane
growth to accommodate its role in branching morphogenesis.
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MATERIALS AND METHODS |
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Genetic interactions were assessed by examining the tracheal phenotypes of the following mutant combinations: grhs2140/shgIG29, grhs2140shgIG29/grhs2140, grhs2140shgIG29/shgIG29.
grhs2140/grhs2140; Atp/TM3Z,
grhs2140/CyOZ; Atp
/Atp
,
grhs2140/grhs2140; Atp
/Atp
.
fasIIEB112/Y; grhs2140/+, fasIIEB112/+; grhs2140/grhs2140, fasIIEB112/+; grh s2140/+, fasIIEB112/Y; grhs2140/grhs2140.
grhs2140/grhs2140; bnlp1/+, grhs2140/grhs2140; bnl44026/+
esg-lacZ (Fusion-1), and the trh-lacZ markers
have been described previously (Samakovlis
et al., 1996a). GBE-lacZ was described by Uv et al.
(Uv et al., 1997
), two
independent transgenic strains where analysed and they were both responsive to
ectopic expression of Bnl.
The UAS-grh transgenic fly strains were generated by inserting the
grh cDNA (N'-form) (Uv et
al., 1997) into pUAST (EcoRI and NotI), and
injected into yw embryos to establish six independent lines. The
additional UAS strains used were: UAS-dpp, UAS-tkvQ253D
(Nellen et al., 1996
),
UAS-bnl (Sutherland et al.,
1996
) and UAS-btl::tor4021 (a chimeric construct
consisting of the extracellular domain of the constitutively active
tor13D mutant fused to the intracellular domain of btl
(Vincent et al., 1998
),
UAS-Act-GFP (Verkhusha et al.,
1999
) and UAS-EGFPF
(Finley et al., 1998
). The
Btl-Gal4 and the SRF-Gal4 where described previously
(Shiga et al., 1996
;
Jarecki et al., 1999
).
SRF-Gal4 expresses Gal4 in tracheal terminal cells from
stage 14. Embryos with one copy of the GAL4 driver and the UAS constructs were
collected at room temperature for 6 hours and then aged at 29°C for 10
hours before fixation. In all experiments CyO, TM3 and FM7c balancer
strains carrying GFP or lacZ transgenes were used as
necessary, to unambiguously identify embryos with the desired genotypes.
Immunostaining and TEM
Immunostainings of embryos were performed as described previously
(Samakovlis et al., 1996b).
Embryos were fixed in 6% paraformaldehyde-saturated heptane and rinsed in
ethanol before staining with Alexa Flour-568 and -488 conjugated phalloidin
(Molecular Probes). To prevent bleaching the ProLong kit was used (Molecular
Probes). The following primary antibodies were used: tracheal lumen-specific
mouse IgM antibody mAb2A12 (1:3), mouse monoclonal anti-DSRF (1:1000), rabbit
anti-ß-gal (1:1500; Cappel), mouse monoclonal anti-Grh (1:5), rabbit
anti-Dlg (1:400) (Budnik et al.,
1996
), rat anti-DE-cad (1:100)
(Oda et al., 1994
), guinea pig
anti-Cor (1:1000), rabbit anti-Nrx IV (1:500), mouse monoclonal anti-Crb
(1:20), rabbit anti-ß-heavy-spectrin (ßH-spectrin;
1:300), mouse monoclonal anti-FasII (1:10), mouse monoclonal anti-Atp
(1:100) (Lebivitz et al., 1989), rat anti-Trh (1:500) (Wappner et al., 1997),
rabbit anti-GFP (1:500; Molecular Probes). Secondary antibodies conjugated to
Biotin, Cy2 or Cy3 (Jackson Immunochemicals) or Alexa Fluor-568 and -488
(Molecular Probes) were diluted and used as recommended. Tyramide Signal
Amplification (NEN) was used to enhance tracheal Grh and Crb signal detection.
Confocal images where obtained with a Leica SP2 confocal microscope and
processed in Adobe Photoshop. Embryo preparation and analysis by TEM was as
described previously (Englund et al.,
1999
).
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RESULTS |
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Nuclear Grh is detected in all tracheal cells, appearing first at stage 11,
just after they have invaginated from the epidermis, and persisting throughout
embryogenesis (Fig. 1L). To
investigate its function, an antibody that specifically stains the tracheal
lumen (mAb2A12) and several cell fate markers
(Samakovlis et al., 1996a)
were used to analyse the tracheal phenotype of three strong loss-of-function
grh alleles (one EMS allele, grhB37; two
P-element insertions, grhs2140 and
grh0685). None of the grh mutations affect the
patterning, outgrowth and connection of branches or the expression of terminal
cell markers (Guillemin et al.,
1996
) (DSRF; Fig.
1I) and fusion cell markers (fusion-3, not shown). It is
only when primary and secondary branching is completed (during stage 16), that
grh mutant embryos begin to display tubular irregularities. The first
signs of a defect are that the dorsal trunk (main airway) appears convoluted
and elongated compared to the wild type
(Fig. 1B,C; early stage 16).
This phenotype subsequently becomes exaggerated, and is also seen in
additional branches, including the lateral trunk, transverse connectives and
ganglionic branches (Fig.
1D-G). These convoluted branches represent an overgrowth in
tracheal tube length, as indicated by an increase of 40% in the tube length of
grh mutants (based on measurements of dorsal trunk metameres 4-6 from
8 embryos of each genotype at stage 16.4). Despite this substantial increase
in tubular lengths, the tubular continuity is not affected in grh
mutant embryos. Grh is therefore required for the restriction or maintenance
of tubular length.
|
Grh mutants show irregular apical cell shapes
The excessive branch elongation in grh mutants could be associated
with an increase in cell numbers. We therefore counted the number of cells in
hemisegments 4, 5 and 6 of the dorsal trunk (DT) in stage 16 wild-type and
grh mutant embryos. The tracheal cells normally stop dividing after
invagination and each tracheal hemisegment consists of about 80 cells, of
which approximately 20 make up the DT
(Samakovlis et al., 1996a).
Using an antibody against the Trachealess transcription factor, which is
expressed in all tracheal cells (Wilk et
al., 1996
), we found that grh mutants contain similar
number of cells as the wild type (Fig.
1J,K). Thus, Grh restricts tube length without affecting cell
division or the number of cells that become allocated to individual
branches.
Tubular growth is accompanied by an immense increase in lumenal surface,
and although it has been proposed that the expansion of apical cell surface is
an essential cellular process underlying branching morphogenesis, its
regulation is poorly understood (Beitel and
Krasnow, 2000). To investigate the cellular activities controlled
by Grh during tubular morphogenesis, we used antibodies against different
membrane-associated proteins to visualise tracheal cell shapes and to monitor
the apical basal polarity of the cells. Labelling for DE-cadherin (DE-cad), a
protein localised at the apical adherence junctions (AJs)
(Oda et al., 1994
), shows that
the apical circumference of cells in the dorsal trunk of grh mutants
is highly irregular at stage 16 (compare
Fig. 2A and D). In particular,
the cells positioned at the outer edge of each curve become excessively
elongated (Fig. 2D) compared to
the cobblestone-shaped cells that line the lumen of wild-type embryos. The
anomalies in cell shapes are first detected at stage 16, and are therefore
coincident with the abnormal tube elongation.
|
The stretched and expanded tracheal cell shapes in grh embryos do
not appear related to alterations in cell polarity. The transmembrane protein
Crumbs (Crb), which confers apical character on the plasma membrane of
epithelial cells (Wodarz et al.,
1995), is present in the same punctate staining along the lumenal
surface of the dorsal trunk in both wild-type and grh mutant embryos.
No differences in the level of Crb expression between wild-type and
grh mutant embryos are evident
(Fig. 2B,E), neither are there
alterations in the minus end of the microtubules [visualized by the expression
of a UAS-NodlacZ transgene (Clark
et al., 1997
) (data not shown)]. The subcellular distribution of
two lateral membrane-associated proteins, Coracle (Cor)
(Fehon et al., 1994
) and
Neurexin IV (Nrx) (Baumgartner et al.,
1996
), which are required for the formation and function of the
laterally positioned septate junctions (SJs), is also normal in grh
mutant embryos (Fig. 2C,F,I,L).
Double labelling for Nrx and DE-cad further shows that Nrx accumulates just
basal to the AJs in both wild-type and grh embryos
(Fig. 2C,F), suggesting that
the increase in apical cell circumference in the mutants is not due to
abnormal distribution of apical domain markers into the lateral and basal cell
area.
Although the modulation of the apical cytoskeleton plays a key role in
epithelial cell shape changes and morphogenesis, we have not detected defects
in the apical cytoskeletal structures of tracheal cells in grh
embryos. Neither the subcellular localization nor the intensity of filamentous
actin staining is altered (visualised either by phalloidin or the tracheal
expression of actin-GFP; Fig.
2H,K and not shown). In addition, the distribution of two apical
cytoskeletal markers Armadillo (ß-catenin, not shown)
(Peifer and Wieschaus, 1990)
and ßH-spectrin (Zarnescu
and Thomas, 1999
) (Fig.
2G,J) is similar in grh and wild-type embryos. Thus, the
abnormal tubular extension and irregular cell shapes observed in grh
mutants are not related to the organisation or maintenance of apical and basal
cell polarity and structure, nor to the collapse of the AJs and underlying
cytoskeleton.
Grh regulates the growth of apical cell membrane
To investigate further the irregular cell shapes in grh mutants we
characterized the cellular morphology of grh embryos at stage 15 and
late stage 16 by transmission electron microscopy. Cross sections of the
dorsal trunk typically reveal 2-4 cells that span the lumen circumference. The
apical membranes are seen just beneath the secreted cuticle that lines the
lumen, and apical junctions appear as electron dense structures near the
apical cell surface while septate junctions are visible as ladder-like
structures basal to the AJs (Fig.
3E). By analysing several cross sections, we find that the
morphology of SJs and the basal part of the cells appear normal in
grh mutant embryos (not shown). The apical cell domain, however,
appears strikingly overgrown and distorted. These defects are first seen in
the tracheal dorsal trunk cells of early stage 16 embryos. The apical cell
surface continues to enlarge, becoming so expanded that it folds over
neighbouring cells, resulting in several layers of cuticle deposition (stage
17; Fig. 3B,G,F). The imbalance
in the dimensions of apical membrane thus parallels the occurrence of the
convoluted branch phenotype in grh mutant embryos. An apical membrane
overgrowth is also found in the epidermal cells, and is associated with the
production of an enlarged cuticle that lines the apical cell surface (data not
shown).
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The AJs often appear abnormal in grh mutant tracheal cells. They
are frequently misplaced, lying parallel rather than perpendicular to the
lumen and occasionally appear less electron dense
(Fig. 3H). The disruptions of
AJs could be a secondary effect, resulting from the excessive apical membrane
that forces an increase in the circumference at the apical side.
Alternatively, Grh may also directly regulate genes necessary for the
maintenance or the function of AJs. Overall the TEM analysis shows that the
major defect in grh tracheal cells is a continued expansion of the
apical membrane, which results in an enlarged and anomalous lumenal surface
with cellular protrusions that contrast to the smooth lining of the wild-type
lumen. This progressive phenotype first appears at the stage when tube
extension normally ceases, suggesting that Grh activity restricts branch
elongation by limiting apical membrane growth. Given the proposed key role of
apical cell surface expansion in branch morphogenesis
(Beitel and Krasnow, 2000), the
specific effect of Grh on this compartment argues that it has a pivotal role
in controlling the process.
Basolateral proteins modulate tube shape and integrity independently
of grh
In search for genes that are functionally related to Grh, we identified and
characterised two mutations that give rise to convoluted tubes, similar to
those of grh mutants (Fig.
4A,D). One mutation inactivates the fasciclinII gene
(fasIIEB112), encoding a homophilic cell adhesion protein
(Grenningloh et al., 1991),
and the other disrupts the gene Atp
, encoding the
sodium/potassium-transporting ATPase alpha subunit (ATP
). In addition
to the extended and curvy dorsal trunk phenotype, detection of DE-cad reveals
that both fasII and Atp
mutants display an enlarged
apical circumference (Fig. 4C and
F). Unlike grh mutant embryos, however, TEM cross
sections of the dorsal trunk in Atp
mutants show that the
apical cell domain is indistinguishable from that of wild type
(Fig. 5I). In addition
fasII and Atp
mutants also display local tubular
dilations (Fig. 4A,D) and
lumenal breaks (Fig. 4B,E).
ATP
localises to the lateral cell surface of all tracheal cells
throughout development (Fig.
5B), and the Atp
mutation appears to affect the
integrity of the lateral septate junctions since the characteristic septa
between cells are sparse (not shown) and the septate junction protein Nrx
appears more diffuse in the dorsal trunk of Atp
mutants
(Fig. 5F,H). As FasII protein
is also localised to the lateral surface of all cells in the developing
trachea, (Fig. 5A), the defects
in tube and cell shapes could arise similarly through the destabilisation of
adhesion complexes between the tracheal cells. Thus, FasII and ATP
affect cell shape and tracheal tube length, as well as tubular diameter and
epithelial integrity, most likely through their action on the lateral cell
surface.
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Despite the difference in cellular phenotypes of grh, fasII and
Atp mutants, the similarity of their dorsal trunk phenotypes
prompted us to investigate a functional relationship between these genes. We
first examined the expression of FasII or ATP
in grh mutants
and in embryos overexpressing Grh in the trachea (see below). Both proteins
are expressed at normal levels and are localised correctly in these mutants
(Fig. 5C,D), and not shown). In
addition, fasII grh and Atp
grh double mutants
display an additive tracheal phenotype with increasingly fragmented tubes,
suggesting an additive effect of weakened epithelial cohesion and the physical
strains exerted by excessive apical growth. These data therefore indicate that
FasII and ATP
are not functionally related to Grh. Instead, they imply
that tubular dimensions depend on the control of distinct subcellular domains;
the growth of the apical cell surface, exemplified by Grh and the integrity
and cohesion of epithelial structure mediated by the lateral membrane proteins
FasII and ATP
.
Grh overexpression prevents lumenal growth
The apical membrane overgrowth and excessive tubular elongation phenotypes
suggest a key role for grh in the regulation of branch extension. We
therefore characterised the effects of its overexpression by directing
UAS-grh expression in all tracheal cells after invagination (late
stage 11) using the Btl-Gal4 driver strain. Detection of lumenal and
cellular tracheal markers at stage 16 reveals a pattern characteristic of the
wild type at stage 13 (Fig.
6A,E,F). This indicates that the cells have migrated towards their
targets, but they have not formed full-length primary branches or a normal
lumen. The visceral and dorsal branches only form rudimentary buds and the
dorsal and lateral trunk branches in each hemisegment remain unconnected
(Fig. 6G). Although the
specialized fusion cells that normally form unicellular anastomoses and
mediate branch fusion are in close contact and express the correct
differentiation markers (esg-lacZ;
Fig. 6D) (Samakovlis et al., 1996b),
they fail to form the interconnecting sprouts. In addition, no secondary
branch lumen is formed, but the expression of the terminal cell marker DSRF is
not affected (Fig. 6B,C). Thus,
Grh appears to inhibit branch extension without changing tracheal cell
fates.
|
To assess whether the lumen and branch growth defects are due to failure in
cellular extensions towards target tissue, we analysed embryos co-expressing
UAS-grh and the membrane marker eGFP
(Finley et al., 1998) in all
tracheal cells. Grh overexpression does not affect the basolateral projections
of the tracheal cells, since these extend towards their normal orientations
(Fig. 6F). However, the cells
appear unable to develop a lumen since their apical side, which surrounds the
cavity of the presumptive dorsal trunk and the short stumps extending from it,
seems limiting (Fig.
6F,F'). Thus, ectopic Grh appears to restrict tracheal
branch extension by directly targeting cellular activities that underlie
lumenal growth.
We also assayed the effect of Grh overexpression in single terminal cells, using the Term-Gal4 driver. In wild-type embryos, the terminal cells form unicellular branches by extending long cytoplasmic processes that subsequently become penetrated by a lumen (Fig. 6B,H). Ectopic Grh does not affect the initial extension of cytoplasmic processes, revealed by a trh-lacZ marker, but prevents the formation and elongation of the intracellular lumen (Fig. 6H,I), demonstrating that Grh can also regulate the cellular processes involved in intracellular branch formation.
Control of Grh activity by Bnl/FGF signalling
The tracheal phenotypes produced by alterations in Grh levels imply that
Grh activity must be carefully controlled during branching morphogenesis to
ensure branch extension at the right stage and to the right extent.
Consequently, tracheal Grh activity is likely to be modulated during branching
morphogenesis. To assay the in vivo activity of Grh, we used strains carrying
a transgene with four high-affinity Grh response elements (GBE-lacZ)
(Uv et al., 1997).
GBE-lacZ expression is detected in all tissues where Grh is
expressed, is absent in grh mutants, and becomes activated upon
ectopic Grh expression (data not shown). It is thus representative of Grh
transcriptional activity in vivo. During tracheal development
GBE-lacZ is expressed in all tracheal cells after invagination, and
requires Grh for its expression (Fig.
1N,Q). However, GBE-lacZ expression is not uniform, it
becomes temporarily enhanced in the fusion and terminal cells during branching
(stage 14; Fig. 7A and not
shown). As Grh itself appears to be uniform in all tracheal cells
(Fig. 1L,
Fig. 7B), the enhanced
expression of GBE-lacZ indicates that the activity of Grh is
regulated post-translationally during branching.
|
One possible mechanism for regulation of Grh activity is through Bnl signalling, which is instrumental in the formation and extension of all tracheal branches. Initially, we established that apical cell surface growth is an intrinsic component of Bnl-induced tube extension, by combining alleles of grh and bnl. This revealed that a subset of the branch outgrowth defects seen in embryos that carry only one copy of the bnl gene are partially rescued by a reduction in grh function (grhs2140/grhs2140; bnlP1/+). Thus, in embryos heterozygous for bnl 40% of the ganglionic branches (n=380) fail to reach the CNS, whereas the simultaneous removal of grh restores this phenotype so that 78% (n=380) of the branches now enter the CNS. These data therefore show that Grh-mediated modulation of the apical cell surface has an active inhibitory role on Bnl-induced branch extension.
In order to analyse whether tracheal Grh activity could be targeted by
Bnl/Btl signal transduction, we analysed GBE-lacZ expression in
embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically
expressed in all tracheal cells, GBE-lacZ expression becomes
significantly upregulated (compare Fig. 7A
and C), although the levels of Grh protein are not altered
(Fig. 7B,D). This suggests that
Bnl controls Grh activity post-translationally, and surprisingly, upregulates
the expression of this artificial Grh target. Nevertheless, the effects of Btl
appear specific since with more limited Bnl expression using the
Term-Gal4 driver, GBE-lacZ expression becomes enhanced
specifically in the cells that respond to Bnl by ectopically expressing the
terminal marker DSRF (compare Fig. 7F and
H). Similar enhancement of GBE-lacZ expression is evident
upon tracheal expression of an activated form of the Btl receptor itself
(UASBtl-Tor) (Vincent et al.,
1998) (data not shown). In all instances the augmented
GBE-lacZ expression is dependent on Grh, as embryos that express
ectopic Bnl or the activated form of Btl, but lack Grh activity, do not
express GBE-lacZ (not shown). Furthermore, ectopic activation of Dpp,
another signalling pathway that promotes the growth of dorsal and ganglionic
branches during tracheal development
(Ribeiro et al., 2002
), has no
effect on GBE-lacZ (data not shown), indicating that the effects on
GBE-lacZ are specific for Bnl/Btl.
We next tested whether Bnl signalling is a prerequisite for the
transcriptional activity of Grh, by analysing the levels of GBE-lacZ
expression in mutants for bnl, btl or pointed (pnt)
(Klambt, 1993). Tracheal
GBE-lacZ expression is both reduced and uniform in bnl and
btl mutant embryos (Fig.
7K,L and not shown), but is unchanged in pnt embryos
(Fig. 7I, J) that lack the
activity of a downstream transcriptional effector of the ETS family
(Samakovlis et al., 1996a
).
Since Grh is a substrate for activated MAPK (ERK2) in vitro
(Liaw et al., 1995
), its
activity could be modulated directly during branching by Bnl-induced
phosphorylation. This would account for the fact that GBE-lacZ
expression is affected by mutations in bnl and btl, but not
by mutations in the nuclear effector pnt.
The apparent upregulation of Grh activity by Bnl signalling and the fact that Grh and Bnl exert opposing effects on branch extension suggests that there are two possible models of Grh activity. The first assumes a two-step process, where upregulation of Grh activity represents a second function of Bnl to prevent excessive tube extension. Alternatively, the Bnl signalling augments some aspects of Grh function (e.g. activation of GBE-lacZ) but inhibits others (e.g. the restriction of apical membrane growth) allowing for branch extension. These two models are discussed below.
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DISCUSSION |
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During tracheal development, Grh is required to restrict apical membrane growth, thereby preventing excess elongation of tracheal tubes. Loss of zygotic Grh protein produces branches that are too long, whereas ectopic Grh has the converse effect. Tracheal branching is initiated in cells receiving the FGF signal, which respond by extending basal projections towards the FGF source, and subsequently move the cell body. Concurrently, a lumen is generated in the extending branch, facilitated by the enlargement of the apical cell membranes to accommodate the necessary expansion in lumenal surface. In embryos that lack grh, branch extension is initiated and proceeds as in wild-type embryos. However, when individual tubes have reached their approximate length and their extension is supposed to halt, the apical membrane growth continues in grh mutants embryos resulting in an enormous apical cell surface that folds over neighbouring cells and forms tortuous tubes. Overexpression of Grh, in contrast, does not affect the basal cytoplasmic extensions towards the Bnl source or cell motility, but specifically prevents lumen extension. Thus, Grh activity is necessary and sufficient to terminate apical membrane growth and tubular extension. Grh may exert its function as an activator of genes that promote homeostasis of apical cell membrane, or a repressor of genes that enhance apical membrane growth.
Several lines of evidence argue that Grh has a specific and restricted
function in apical membrane growth control. Firstly, epithelial polarity is
not altered in grh mutants. TEM analysis of grh mutants and
embryos overexpressing Grh showed that cuticular and lumenal components are
made and secreted and the expression and subcelluar localisation of Crb,
DE-cad, Nrx, Cor and Disc large (not shown) is normal in grh mutant
trachea. In addition, no genetic interaction was detected in embryos carrying
different combinations of grh and shg (encoding DE-cad)
mutant alleles (data not shown). Secondly, no cytoskeletal defects were
detected in grh mutant embryos. The filamentous apical actin
cytoskeleton, and the expression of Cadherin, Armadillo and
ßH-spectrin are unchanged by overexpression or inactivation of
grh. Likewise, no anomalies were evident in the minus-end
microtubules visualised by the apical distribution of Nod-lacZ in the
trachea of grh and wild-type embryos. Finally, changes in the
basolateral cell domain influence tracheal tube size and integrity
independently of grh. FasII and ATP localize to the lateral
cell membrane, and disruption of either gene function causes distinct
irregularities in tubular diameter, length and continuity. No regulatory or
functional relationship between Grh and these two lateral proteins were
detected in spite the fact that all three proteins influence tubular
length.
There are specific differences between the tracheal phenotypes caused by
the expansion of apical membrane, seen in grh, and the ones caused by
disruption of lateral domain functions, seen in fasII and
Atp mutants. The increase in apical cell surface in
grh embryos has no apparent effect on the diameter of the tracheal
tubes, whereas mutations in fasII and Atp
result in a
longer lumen with local dilations. The additional tubular breaks observed in
the latter suggest that they function in the lateral cell compartment to
maintain epithelial cohesion and structure. The programmed changes in tube
diameter that take place in the different tracheal branches during development
occur by expansion of the inner lumenal diameter, whereas the outer diameter
remains the same (Beitel and Krasnow,
2000
), requiring a decrease in the distance from the apical to
basal surface and major remodelling of the lateral cell compartment.
Therefore, the size and shape of epithelial tubular structures appears to
depend on the modulation of distinct subcellular domains during morphogenesis.
In addition to the regulation of apical membrane growth by grh, there
may be separate regulatory programmes modulating the dynamics of the lateral
cell surface. Together these two aspects of regulation co-ordinately determine
tubular dimensions.
Most of the well-studied examples, in which morphogenesis involves changes
in the apical membrane of polarised epithelia, are mediated through the
function of the apical membrane determinant Crb, an EGF-repeat-containing
transmembrane protein. In the Drosophila embryo overexpression of the
Crb intracellular domain causes cytoskeletal re-organisation and apical
membrane expansion. Crb also functions during photoreceptor morphogenesis,
where its intracellular domain is required to maintain the integrity of zonula
adherens during rhabdomere elongation
(Izaddoost et al., 2002), and
its extracellular domain has an additional and distinct function in the
extension of the stalk, by stabilizing the membrane-associated
ßH-spectrin cytoskeleton to facilitate apical membrane growth
(Pellikka et al., 2002
). As
neither the expression nor the localisation of Crb and
ßH-spectrin is detectably affected in the tracheal cells where
the apical membrane is altered by loss of or overexpression of grh, an
alternative mechanism for apical membrane growth must be involved in mediating
the grh function on branching morphogenesis. One possibility is via
an effect on membrane trafficking, which is highly regulated in polarised
epithelial cells (Mostov et al.,
2000
; Lipschutz and Mostov,
2002
). Apical membrane growth during branching may be achieved by
directly modulating the relative rates of exocytosis or endocytosis and
membrane metabolism.
Regulation of Grh activity by Bnl
Grh levels appear uniform in all tracheal cells throughout development.
However, Grh functions in the regulation of branch extension and apical
membrane growth, allowing branch extension during a certain time frame and to
different extents in the various branches. Grh activity must therefore be
modulated post-translationally as branch growth proceeds. Such a regulation of
Grh activity could be exerted through extracellular signals or by
branch-specific co-factors, modulating its ability to regulate gene expression
in a branch and time specific manner.
Using a lacZ reporter gene for Grh activity that reflects the in
vivo ability of Grh to activate transcription (GBE-lacZ), we find
that Grh activity is controlled by FGF signalling during tracheal development.
Ectopic expression of Bnl or the activated form of its receptor Btl
up-regulates the expression of GBE-lacZ, whereas GBE-lacZ
expression is reduced in mutants for bnl or btl. Thus, Bnl
signalling converts Grh to a more potent activator of its GBE-lacZ
target. Since Grh becomes phosphorylated by MAPK in vitro
(Liaw et al., 1995), and MAPK
is a downstream effector of Btl signal transduction, the alteration in Grh
activity may be brought about by MAPK-mediated phosphorylation of the Grh
protein.
Currently, we see two ways of explaining the biological consequence of the regulation of Grh. In the first model, the regulation of Grh by Bnl increases its activity, and thereby delimits lumen growth. This invokes a hierarchical two step function for Bnl in which it first promotes branching and tube elongation and it then activates Grh to halt excess apical surface growth and establish a functional lumen. In this model active restriction of morphogenetic processes is required to achieve stereotyped tube dimensions and is an intrinsic part of the program that induces branching morphogenesis. In the second model, regulation by Bnl has differential consequences on Grh, activating some functions (like the one necessary for GBE-lacZ expression) and inactivating others, necessary for inhibiting apical membrane growth. In this model, high levels of Btl signalling would temporarily inactivate Grh, in order to allow for apical membrane expansion during the process of branch extension. Both models are consistent with the genetic interactions, which indicate an antagonistic relationship between grh and bnl, and add the control of apical membrane growth to the repertoire of cellular activities regulated by FGF signalling during morphogenesis.
Of the two models we currently favour the former, where Btl coordinates
branching through a sequence of activities, since this model is consistent
with the activation of the GBE-lacZ reporter. It can also be well
integrated with the apical overgrowth phenotype of grh mutants, which
becomes apparent first in the branches that have reached their final length
and only after the completion of branch elongation at stage 16. If Grh were
acting to restrict membrane growth continuously, the grh mutant
phenotype would be expected to appear at earlier stages. A two step model
could also explain the inhibiting effect on tube elongation that is seen upon
expression of activated forms of Btl receptors in all tracheal cells of
wild-type embryos (Lee et al.,
1996).
As restriction of apical membrane growth depends on Grh-mediated
alterations in transcriptional activity, the induction of apical membrane
expansion upon branch elongation may also rely on changes in gene expression.
The nuclear factor Ribbon (Rib) is required for branch elongation
(Bradley and Andrew, 2001), and
may act as an activator of apical membrane growth. In rib mutants,
the extension of basal cytoplasmic processes towards the Bnl source appears
normal, but the movement of the cell body fails and the apical membrane does
not expand, causing a tracheal phenotype that is reminiscent of that seen with
ectopic Grh expression (Shim et al.,
2001
). It is thus conceivable that a balance between Rib and Grh
activity determines the extent of apical membrane growth and is coordinated by
Bnl through direct modulation of Grh, and perhaps also of the Rib protein.
Such a regulation of apical cell surface size by signals deriving from the
target tissue could coordinate branch elongation, and would provide an elegant
allometric control of organ size depending on the signal strength, size and
respiratory demand of the target tissue.
Non-tracheal Grh expression and function of mammalian homologues
Apart from its tracheal expression, Grh is found in the embryonic epidermis
and all primary epithelial tissues. The epidermal expression of grh
is also essential as grh mutant embryos show a `blimp' phenotype,
where the embryonic cuticle stretches to a much greater extent than the
wild-type cuticle upon removal of the vitelline membrane
(Ostrowski et al., 2002). We
find that the epidermal cells in grh embryos also show an abnormal
apical membrane expansion (data not shown). This is associated with the
production of an enlarged cuticle that lines the apical cell surface. Grh may
therefore have a common biological function in the epithelial tissues where it
is expressed, being required to regulate apical cell membrane growth. Grh
protein is continuously expressed in epithelial tissues during larval life
(Uv et al., 1997
), a period of
extensive organ growth to accommodate the dramatic increase in animal size.
Thus, Grh is likely to be required not only for organogenesis, but also for
the continuous modulations in organ size and shape that occurs throughout the
animals life. The temporal and spatial control of Grh activity must however be
accomplished through distinct mechanisms in different tissues, as Bnl
signalling does not operate in the epidermis.
Grh belongs to a small family of transcription factors that is found only
in higher eukaryotes. The specific, but basic function of Grh in the
regulation of epithelial apical cell membrane growth raises intriguing
questions as to its functional conservation in higher organisms. Two mammalian
Grh homologues, MGR and BOM have been recently identified
(Wilanowski et al., 2002).
Like Grh, MGR and BOM form dimers and MGR interacts specifically with Grh DNA
binding sites in vitro. Intriguingly, these mammalian homologues display
similar expression patterns to that of Grh. During mouse development MGR is
expressed predominantly in the epidermis, and BOM is expressed in the
epidermis as well as in several internal tubular organs including the kidney
and lung. Thus the biological function of Grh may be conserved in its murine
homologues. Given the functional conservation of FGF signalling in tracheal
and lung morphogenesis, it will be of great interest to test whether the
mammalian homologues of Grh participate in the growth of the lung and to
investigate their functional relationship with FGF signalling.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Affolter, M. and Shilo, B. Z. (2000). Genetic control of branching morphogenesis during Drosophila tracheal development. Curr. Opin. Cell Biol. 12,731 -735.[CrossRef][Medline]
Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A., Harbecke, R., Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H. J. (1996). A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87,1059 -1068.[Medline]
Beitel, G. J. and Krasnow, M. A. (2000).
Genetic control of epithelial tube size in the Drosophila tracheal system.
Development 127,3271
-3282.
Bradley, P. L. and Andrew, D. J. (2001). ribbon
encodes a novel BTB/POZ protein required for directed cell migration in
Drosophila melanogaster. Development
128,3001
-3015.
Bray, S. J. and Kafatos, F. C. (1991). Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila. Genes Dev. 5,1672 -1683.[Abstract]
Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C., Woods, D. and Gorczyca, M. (1996). Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron 17,627 -640.[Medline]
Cheng, Y., Endo, K., Wu, K., Rodan, A. R., Heberlein, U. and Davis, R. L. (2001). Drosophila fasciclin II is required for the formation of odor memories and for normal sensitivity to alcohol. Cell 105,757 -768.[CrossRef][Medline]
Clark, I. E., Jan, L. Y. and Jan, Y. N. (1997).
Reciprocal localization of Nod and kinesin fusion proteins indicates
microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle.
Development 124,461
-470.
Deak, P., Omar, M. M., Saunders, R. D., Pal, M., Komonyi, O.,
Szidonya, J., Maroy, P., Zhang, Y., Ashburner, M., Benos, P. et al.
(1997). P-element insertion alleles of essential genes on the
third chromosome of Drosophila melanogaster: correlation of physical and
cytogenetic maps in chromosomal region 86E-87F.
Genetics 147,1697
-1722.
Englund, C., Uv, A. E., Cantera, R., Mathies, L. D., Krasnow, M.
A. and Samakovlis, C. (1999). adrift, a novel bnl-induced
Drosophila gene, required for tracheal pathfinding into the CNS.
Development 126,1505
-1514.
Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S.
(1994). A Drosophila homologue of membrane-skeleton protein 4.1
is associated with septate junctions and is encoded by the coracle
gene. Development 120,545
-557.
Finley, K. D., Edeen, P. T., Foss, M., Gross, E., Ghbeish, N., Palmer, R. H., Taylor, B. J. and McKeown, M. (1998). Dissatisfaction encodes a tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Neuron 21,1363 -1374.[Medline]
Gabay, L., Seger, R. and Shilo, B. Z. (1997).
In situ activation pattern of Drosophila EGF receptor pathway during
development. Science
277,1103
-1106.
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell 67,45 -57.[Medline]
Guillemin, K., Groppe, J., Ducker, K., Treisman, R., Hafen, E.,
Affolter, M. and Krasnow, M. A. (1996). The pruned
gene encodes the Drosophila serum response factor and regulates
cytoplasmic outgrowth during terminal branching of the tracheal system.
Development 122,1353
-1362.
Hogan, B. L. and Kolodziej, P. A. (2002). Organogenesis: molecular mechanisms of tubulogenesis. Nat. Rev. Genet. 3,513 -523.[CrossRef][Medline]
Imam, F., Sutherland, D., Huang, W. and Krasnow, M. A.
(1999). stumps, a Drosophila gene required for fibroblast growth
factor (FGF)-directed migrations of tracheal and mesodermal cells.
Genetics 152,307
-318.
Izaddoost, S., Nam, S. C., Bhat, M. A., Bellen, H. J. and Choi, K. W. (2002). Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416,178 -183.[CrossRef][Medline]
Jarecki, J., Johnson, E. and Krasnow, M. A. (1999). Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF. Cell 99,211 -220.[Medline]
Klambt, C. (1993). The Drosophila gene
pointed encodes two ETS-like proteins which are involved in the
development of the midline glial cells. Development
117,163
-176.
Klambt, C., Glazer, L. and Shilo, B. Z. (1992). breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and specific midline glial cells. Genes Dev. 6,1668 -1678.[Abstract]
Lebovitz, R. M., Takeyasu, K. and Fambrough, D. M. (1989). Molecular characterization and expression of the (Na++K+)-ATPase alpha-subunit in Drosophila melanogaster. EMBO J. 8,193 -202.[Abstract]
Lee, T., Hacohen, N., Krasnow, M. and Montell, D. J. (1996). Regulated Breathless receptor tyrosine kinase activity required to pattern cell migration and branching in the Drosophila tracheal system. Genes Dev. 10,2912 -2921.[Abstract]
Liaw, G. J., Rudolph, K. M., Huang, J. D., Dubnicoff, T., Courey, A. J. and Lengyel, J. A. (1995). The torso response element binds GAGA and NTF-1/Elf-1, and regulates tailless by relief of repression. Genes Dev. 9,3163 -3176.[Abstract]
Lipschutz, J. H. and Mostov, K. E. (2002). Exocytosis: the many masters of the exocyst. Curr. Biol. 12,R212 -214.[CrossRef][Medline]
Manning, G. and Krasnow, M. A. (1993). Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster (ed. A. Martinez-Arias and M. Bate), pp. 609-686. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Metzger, R. J. and Krasnow, M. A. (1999).
Genetic control of branching morphogenesis. Science
284,1635
-1639.
Mostov, K. E., Verges, M. and Altschuler, Y. (2000). Membrane traffic in polarized epithelial cells. Curr. Opin. Cell Biol. 12,483 -490.[CrossRef][Medline]
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[Medline]
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994). A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165,716 -726.[CrossRef][Medline]
Ostrowski, S., Dierick, H. A. and Bejsovec, A.
(2002). Genetic control of cuticle formation during embryonic
development of Drosophila melanogaster. Genetics
161,171
-182.
Peifer, M. and Wieschaus, E. (1990). The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell 63,1167 -1176.[Medline]
Pellikka, M., Tanentzapf, G., Pinto, M., Smith, C., McGlade, C. J., Ready, D. F. and Tepass, U. (2002). Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416,143 -149.[CrossRef][Medline]
Ribeiro, C., Ebner, A. and Affolter, M. (2002). In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis. Dev. Cell 2, 677-683.[Medline]
Samakovlis, C., Hacohen, N., Manning, G., Sutherland, D. C.,
Guillemin, K. and Krasnow, M. A. (1996a). Development of the
Drosophila tracheal system occurs by a series of morphologically distinct but
genetically coupled branching events. Development
122,1395
-1407.
Samakovlis, C., Manning, G., Steneberg, P., Hacohen, N.,
Cantera, R. and Krasnow, M. A. (1996b). Genetic control of
epithelial tube fusion during Drosophila tracheal development.
Development 122,3531
-3536.
Scholz, H., Deatrick, J., Klaes, A. and Klambt, C.
(1993). Genetic dissection of pointed, a Drosophila gene encoding
two ETS-related proteins. Genetics
135,455
-468.
Shiga, Y., Tanaka-Matakutsu, M. A. and Hayashi, S. (1996). A nuclear GFP/b-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Differ. 38,99 -106.
Shim, K., Blake, K. J., Jack, J. and Krasnow, M. A.
(2001). The Drosophila ribbon gene encodes a nuclear BTB domain
protein that promotes epithelial migration and morphogenesis.
Development 128,4923
-4933.
Sutherland, D., Samakovlis, C. and Krasnow, M. A. (1996). branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87,1091 -2101.[Medline]
Tepass, U., Gruszynski-DeFeo, E., Haag, T. A., Omatyar, L., Torok, T. and Hartenstein, V. (1996). shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia. Genes Dev. 10,672 -685.[Abstract]
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35,747 -784.[CrossRef][Medline]
Uv, A. E., Harrison, E. J. and Bray, S. J. (1997). Tissue-specific splicing and functions of the Drosophila transcription factor Grainyhead. Mol. Cell Biol. 17,6727 -6735.[Abstract]
Verkhusha, V. V., Tsukita, S. and Oda, H. (1999). Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein. FEBS Lett. 445,395 -401.[CrossRef][Medline]
Vincent, S., Wilson, R., Coelho, C., Affolter, M. and Leptin, M. (1998). The Drosophila protein Dof is specifically required for FGF signaling. Mol. Cell 2, 515-525.[Medline]
Wilanowski, T., Tuckfield, A., Cerruti, L., O'Connell, S., Saint, R., Parekh, V., Tao, J., Cunningham, J. and Jane, S. (2002). A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead. Mech. Dev. 114,37 .[CrossRef][Medline]
Wilk, R., Weizman, I. and Shilo, B. Z. (1996). trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila. Genes Dev. 10, 93-102.[Abstract]
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82,67 -76.[Medline]
Wolf, C., Gerlach, N. and Schuh, R. (2002).
Drosophila tracheal system formation involves FGF-dependent cell extensions
contacting bridge-cells. EMBO Rep.
3, 563-568.
Zarnescu, D. C. and Thomas, G. H. (1999).
Apical spectrin is essential for epithelial morphogenesis but not apicobasal
polarity in Drosophila. J. Cell Biol.
146,1075
-1086.