Institut für Genetik, Heinrich Heine Universität, Düsseldorf, Germany
* Author for correspondence (e-mail: muellear{at}uni-duesseldorf.de)
Accepted 28 February 2004
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SUMMARY |
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In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.
Key words: Drosophila, Cell migration, Gastrulation, FGF-receptor, RhoGEF
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Introduction |
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How are signals from FGFR activation transduced to the cytoskeletal and
adhesive systems of the cell to elicit the migratory behavior of the cell? One
of the best-characterized FGFR signaling pathways involves the Ras GTPase,
which among other targets promotes activation of MAP kinase
(Campbell et al., 1998). MAP
kinase phosphorylates a variety of protein targets including transcription
factors, other protein kinases, phospholipases or cytoskeletal proteins. For
example, MAP kinase promotes phosphorylation of myosin light chain (MLC)
kinase, a regulator of the contractile actin-myosin system
(Klemke et al., 1997
).
Although many downstream components of FGFR signaling pathways have been
identified, the targets that regulate cell migration are not well
understood.
Migratory cells form a variety of characteristic cellular protrusions, most
prominently filopodia and lamellipodia
(Lauffenburger and Horwitz,
1996). The formation of these protrusions in response to
extracellular stimuli is controlled by small GTPases of the Rho family: Rho,
Rac and CDC42 (Hall, 1998
).
Mammalian cell culture systems have provided ample evidence that Rho GTPases
represent the key molecules in transducing extracellular signals to the actin
cytoskeleton (Schmidt and Hall,
1998
). Guanine nucleotide exchange factors (GEFs) act upstream of
Rho GTPases and promote their local activation in the cell. Although FGFRs
might be required and sufficient for the formation of filopodial protrusions,
as shown for tracheal cell migration in Drosophila
(Ribeiro et al., 2002
), the
molecules that connect FGFR signaling pathways to the regulators of the
cytoskeleton during morphogenesis are yet to be defined.
Mesoderm migration in the early Drosophila embryo provides a good
model system to study FGFR-dependent cell migration
(Wilson and Leptin, 2000).
Migration of the mesoderm is an important precondition for the regional
specification of different mesodermal derivatives. The most dorsal mesodermal
fates are controlled by combinatorial action of multiple signaling pathways
and intrinsic factors. These inputs govern the formation of a subset of dorsal
mesodermal derivatives, marked by the expression of even skipped
(eve) (Carmena et al.,
1998
; Halfon et al.,
2000
; Knirr and Frasch,
2001
). In mutants in which mesoderm migration is affected, e.g. in
embryos mutant for the FGFR homolog Heartless (HTL), mesoderm cells
are unable to receive appropriate signals. As a consequence, the dorsal
eve-expressing mesoderm cells are not specified in htl
mutant embryos (Beiman et al.,
1996
; Gisselbrecht et al.,
1996
; Shishido et al.,
1997
).
Although the ligand of HTL is yet unknown, the synthesis of proteoglycans
is required for HTL activation (Lin et
al., 1999). The signal transduction pathway downstream of HTL
involves the product of the downstream of FGF (dof;
stumps FlyBase) gene
(Imam et al., 1999
;
Michelson et al., 1998a
;
Vincent et al., 1998
). Genetic
experiments suggest that DOF functions as adaptor protein and operates
downstream of the receptor and upstream of signals triggered by RAS1.
Furthermore, activation of MAP kinase was observed in a subgroup of migrating
mesoderm cells and was shown to depend on HTL and DOF function
(Gabay et al., 1997
;
Vincent et al., 1998
).
We show that HTL is necessary for protrusive activity of migrating mesoderm
cells. The early attachment of mesoderm cells to the ectoderm is affected in
htl mutants, suggesting that HTL is required for the adhesion of
mesoderm cells to the ectoderm. Our data provide evidence that HTL exerts a
permissive function in mesoderm migration. In a genetic screen, we identified
the Rho GEF Pebble (PBL) as an essential molecular component of mesoderm
migration. Like HTL, PBL is required for specific cell shape changes and
protrusive activity of the mesoderm cells during migration. The function of
PBL in cell migration is independent of the well-known function of PBL in
cytokinesis (Hime and Saint,
1992; Lehner,
1992
; Prokopenko et al.,
1999
). Overexpression of HTL or a constitutively active form of
HTL using the twist (twi) promoter, are sufficient to
trigger late cell shape changes in the mesoderm, but are unable to initiate
early cell shape changes in pbl mutants. These results indicate a
novel function of PBL in HTL triggered cell migration in the
Drosophila gastrula.
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Material and methods |
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Microscopy, antibodies and immunocytochemistry
Embryos were obtained, staged, fixed and immunolabeled as described
(Müller and Wieschaus,
1996). For f-actin staining, embryos were fixed with 37%
formaldehyde/heptane for 5 minutes followed by devitellinization using 80%
ethanol. For cross-sections, fluorescently labeled embryos were embedded in
Technovit 7100 (Hereaus, Germany) following the manufacturer's instructions.
Sections (7 µm) were obtained with a Reichert Jung Microtome and mounted on
slides. As anti-bleaching procedure, sections were incubated for 20 minutes in
50 mM DABCO in PBS and mounted in DABCO/MOWIOL. Embryos were genotyped using
balancer chromosomes carrying lacZ transgenes. Fluorescence
microscopy was performed with a Leica-TCS NT confocal microscope. For
whole-mount staining embryos were double labeled with appropriate secondary
antibodies conjugated to alkaline phosphatase or biotin. Biotinylated
secondary antibodies were detected using the ABC-kit from Vectastain (Vector,
USA). Whole-mount stained embryos were dehydrated in ethanol and acetone and
embedded in araldite. Embryos were then either mounted and oriented in
araldite on microscope slides or embedded for sectioning. Sections were cut at
5 µm and mounted in araldite. Light microscopy was performed on a Zeiss
Axiophot. Images were processed using Adobe Photoshop on an Apple
Computer.
Preparation of embryos for transmission electron microscopy was performed
as described by Müller and Wieschaus
(Müller and Wieschaus,
1996) with few modifications. For genotyping, the embryos were
prefixed at the interphase of 25% glutaraldehyde in 0.1 M phosphate buffer pH
7.4 and heptane for 25 minutes. The embryos were then hand-peeled and rinsed
in X-Gal staining buffer (0.15 M NaCl; 1 mM MgCl2; 0.01 M sodium
phosphate buffer, pH 7.2; 3.3 mM K3Fe(CN)6; 3.3 mM
K4Fe(CN)6). For X-Gal staining, embryos were incubated
overnight with 10% 5-Br-4-Cl-3-Indolyl-ß-D-galactoside in X-Gal buffer at
16°C. The embryos were then sorted and processed as described. Micrographs
were taken at a Zeiss EM109 transmission electron microscope.
The following antibodies were used: mouse anti-dpERK (Sigma); rabbit
anti-Twist (Siegfried Roth, Cologne); rabbit anti-ßGal (Cappel); mouse
anti-ßGal (Promega); mouse anti-EVE, mouse anti-Engrailed (EN), mouse
anti-Neurotactin (NRT; DSHB, Iowa); mouse anti-CD2 (Serotec, Germany),
Phalloidin-Alexa568 (Molecular Probes, USA), rat anti-DE-Cadherin and rat
anti-D-Catenin (Hiroki Oda, Japan).
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Results |
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To follow cell shape changes of mesoderm cells, we used a transgene driving
expression of the transmembrane protein CD2 from rat under the control of the
twist (twi) promotor (twi::CD2)
(Dunin-Borkowski and Brown,
1995). twi::CD2 is already expressed during invagination
and represents a cell-surface marker specific for the mesoderm. Mesoderm
migration can be divided into three phases with characteristic cell shape
changes (Fig. 1). After
invagination, the mesoderm initially forms an epithelial tube
(Fig. 1A,D). At phase 1 of
migration, the surface of the mesoderm cells appears relatively smooth
(Fig. 1D). After disassembly of
the epithelial tube and mitosis, phase 2 begins, in which the mesodermal
aggregate migrates out in dorsolateral direction
(Fig. 1B,E). Cells at the
leading edge of the aggregate are stretched along the dorsoventral axis and
extend multiple cellular protrusions. The longest cellular protrusions often
measure half to two-thirds the size of a cell diameter (to a length of 10-15
µm in fixed samples). Cross-sections revealed that not only the leading
edge cells exhibit this polarized morphology, but that the cells immediately
following the leading edge cells frequently also extend in dorsolateral
direction (Fig. 1M). In this
work, we will be using the term `protrusive activity' to describe the
formation and/or the dynamics of the filoform and lamelliform protrusions that
we observed in our fixed preparations. The protrusive activity is specific for
the migratory phase, because when the cells have reached their final positions
(phase 3) and form a coherent monolayer, large extensions are absent and only
few filoform protrusions were observed
(Fig. 1C,F).
|
Our observations suggest that HTL is required for the early interaction of the mesoderm with the ectoderm. By analyzing sections, we observed that wild-type cells at the base of the mesodermal tube are attached to the basal surfaces of the ectoderm (Fig. 2A). By contrast, htl mutant mesoderm cells at the respective stage and position failed to establish contact to the ectoderm (Fig. 2C). This phenotype correlates well with a misalignment of the mesodermal tube in htl mutants (Fig. 2B,D; Fig. 1J). We conclude that HTL is required for the effective attachment of mesoderm cells to the ectoderm, which might promote the protrusive activity of mesoderm cells during migration.
|
Three loci mapped to the third chromosome and were characterized using chromosomal deletions and available point mutations. Two loci corresponded to the genes htl and dof, respectively. Embryos lacking the chromosomal interval 61A to 68 (based on breakpoints of the chromosomal translocation T(2;3)C309) displayed defects in mesoderm migration (Fig. 3A-C). Genetic mapping revealed that small overlapping chromosomal deletions, which exhibited the phenotype, all removed the gene pbl (data not shown). Analysis of a strong loss-of-function point mutation in pbl, pbl3, indicated that pbl is required for mesoderm morphogenesis. Embryos homo- or hemizygously mutant for pbl3 show a dramatic reduction in the number of EVE-positive mesoderm cells at the extended germband stage (Fig. 3D,E). These results demonstrate a thus far unrecognized function for pbl in mesoderm differentiation.
|
It remained possible that the defects in pbl mutants in cell
migration might be a secondary consequence of a failure in the
epithelial/mesenchymal transition of the mesoderm after invagination. During
epithelial/mesenchymal transition cells loose their epithelial polarity and
gain mesenchymal character. We compared the presence of adherens junctions in
the mesoderm cells in wild type, htl and pbl mutant embryos
by electron microscopy. Apical adherens junctions, a hallmark of apicobasal
polarity in epithelial cells, are lost from the mesoderm cells during phase 1
in the wild type as well as in embryos homozygous for htl or
pbl (see Fig. S1 at
http://dev.biologists.org/supplemental).
Similarly, immunolabeling with antibodies against the ectodermal epithelial
marker DE-cadherin or the adherens junction marker D-Catenin
showed that downregulation of epithelial characteristics occurs normally in
pbl and htl mutants (see Fig. S2 at
http://dev.biologists.org/supplemental).
In summary these results indicate that PBL is not required for the loss of
epithelial character of mesoderm cells, but that PBL is essential for the gain
of mesenchymal characteristics of mesoderm cells after invagination.
The requirement of PBL for cell migration is independent of its function in cytokinesis
pbl encodes a RHO1-GEF most similar to the vertebrate
ect2 proto-oncogene (Miki et al.,
1993; Prokopenko et al.,
1999
; Tatsumoto et al.,
1999
). Both pbl and ect2 are required for the
assembly of the contractile actin ring during cytokinesis. Interfering with
the function of PBL or ECT2 results in a failure of cytokinesis and the
generation of multinucleate cells (Fig.
3G,H). Because mutations in pbl affect cell shape changes
before mitoses in the mesoderm occur, we suspected that the requirement of
pbl for mesoderm migration might be independent from its cytokinesis
function. To determine, whether the defects in mesoderm migration in
pbl mutants are direct rather than a secondary consequence of the
failure in cytokinesis, we analyzed the pbl phenotype in
division-defective embryos.
Postblastoderm mitotic divisions are controlled by zygotic expression of
the cell cycle regulator String (STG)
(Edgar and O'Farrell, 1990). As
mesoderm migration and specification of EVE-positive mesoderm cells occur
normally in stg mutant embryos, this mutation provides a genetic
condition to assay cytokinesis-independent functions of pbl
(Fig. 4B,E)
(Carmena et al., 1998
;
Leptin and Grunewald, 1990
).
The cytokinesis defect of pbl was completely blocked by stg
(Fig. 4F). In pbl stg
double mutant embryos, migration of the mesoderm and specification of dorsal
mesodermal derivatives was impaired similar to pbl single mutants
(Fig. 4C,F). Moreover, cell
shape changes in phase 2 occur normally in stg mutant embryos, but
protrusive activity of the mesoderm cells was blocked in pbl stg
double mutants (Fig. 4E,F).
These results indicate that the activity of pbl is required for
mesoderm migration even in the absence of mitosis and thus in the absence of
cytokinesis defects. We therefore conclude that PBL has independent functions
in cytokinesis and cell migration, respectively.
|
Genetic data indicate different requirements of the PBL protein for its two
functions. We detected a significant difference in the strength of the
migration phenotype of two distinct mutant alleles of pbl. Embryos
homozygous for the strong loss of function allele pbl3
exhibit on average only one hemisegment that contains EVE-positive mesoderm
cells, compared with 22 EVE-positive hemisegments in the wild type
(Fig. 5A,B;
Table 1). By contrast, embryos
mutant for pbl11D exhibit an average of 12 hemisegments
that contain EVE-positive cells and few pbl11D homozygous
embryos (2/45) even contained the wild-type number of EVE-positive cells
(Fig. 5C; Table 1). Importantly,
pbl3 and pbl11D homozygotes exhibit
equally penetrant defects in cytokinesis
(Fig. 5D,E) (see also
Cui and Doe, 1995;
Weigmann and Lehner, 1995
).
These allele-specific differences demonstrate distinct requirements of the PBL
protein in cell migration and cytokinesis, and suggest that the migratory
function of PBL might be acting through a mechanism distinct from the
cytokinesis function.
|
|
These data support our conclusion that the defects in cell migration in pbl mutants are not a secondary consequence of a failure in cytokinesis. The finding that cell migration is not affected in Rho1N19-expressing cells suggests that RHO1 function is dispensable for mesoderm migration. In summary, we propose that the two functions of PBL are independent and might involve distinct molecular mechanisms in cytokinesis and cell migration, respectively.
Requirement of PBL for cell migration is specific for mesoderm cells
Mesoderm migration depends on the mesoderm-specific expression and activity
of both the HTL receptor and its putative cytoplasmic adaptor DOF
(Beiman et al., 1996;
Gisselbrecht et al., 1996
;
Michelson et al., 1998a
;
Shishido et al., 1993
;
Vincent et al., 1998
). By
contrast, pbl is expressed in all cells of the embryo and is required
in all cells for cytokinesis in postblastoderm embryos
(Hime and Saint, 1992
;
Lehner, 1992
;
Prokopenko et al., 2000a
).
We sought to assess the cell-type specific requirements of PBL during mesoderm migration by rescuing the pbl mutant phenotype in a tissue-specific manner. A UAS::pbl transgene containing the full-length pbl cDNA was expressed in the mesoderm cells in a pbl mutant background using twi::Gal4. The cytokinesis defect and the mesoderm migration defect of pbl mutants was completely rescued by mesoderm-specific expression of pbl (Fig. 6A-C). Moreover, expression of EVE in dorsal mesoderm cells was restored (Fig. 6D,E). The cells in the ectoderm of such embryos still exhibited cytokinesis defects, indicating that the activity of the transgene was specific for mesoderm cells and that the function of PBL in the ectoderm is not essential for mesoderm migration (Fig. 6B). We therefore conclude that pbl acts in a mesoderm autonomous fashion to allow proper migration of the cells.
|
|
To test whether pbl is required for HTL triggered cell shape
changes in mesoderm migration, we overexpressed wild-type or constitutively
active forms of HTL in a pbl mutant background. Both, expression of
HTL or HTL, were unable to trigger early cell shape changes in
pbl homozygous embryos (Fig.
7E and data not shown). Although HTL or
HTL expression
failed to rescue phase 1, some mesoderm spreading was observed in phase 3 and
later (Fig. 7G). Because the
rescue in phase 3 was rather moderate and variable, we examined mesoderm
differentiation by analyzing the number of EVE-positive hemisegments. Mesoderm
differentiation defects in pbl homozygotes are rescued significantly
by expression of
HTL but not by expression of HTL
(Fig. 7F;
Table 1). This result suggests
that mesoderm migration might be delayed in these embryos, in a way that might
render signaling events leading to EVE expression ineffective. In addition,
the partial rescue of pbl mutants by
HTL might reflect a
second function of the receptor in differentiation of dorsal mesodermal
derivatives (Michelson et al.,
1998b
). These results are consistent with a role of PBL for early
cell shape changes triggered by HTL. The fact that
HTL rescues some
late defects in pbl mutants suggests the presence of additional
mechanisms required for mesoderm migration, which might be independent of
pbl.
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Discussion |
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Although the ligand of the HTL receptor is unknown, it seems unlikely that
signals mediated by HTL provide the only directional cues for mesoderm
migration. Localization of activated forms of MAP kinase in the leading edge
cells during mesoderm migration suggests that local activation of the receptor
might be important for proper migration
(Gabay et al., 1997). In
addition, activation of HTL in a ligand-independent fashion throughout the
mesoderm is unable to completely rescue the htl mutant phenotype
(Michelson et al., 1998b
)
(this paper). Thus local activation of the receptor occurs in vivo and is
essential for proper mesoderm migration. We show that HTL is required for
protrusive activity only during phase 1 and 2 of mesoderm migration. However,
HTL activity is not essential for the protrusive activity of the cells per se,
because cells do extend dorsolaterally during phase 3 in htl mutant
embryos. These data demonstrate that HTL activation is unlikely to provide the
only directional cue in mesoderm migration. The results presented in this
paper suggest that HTL signaling provides temporal information for protrusion
formation during phases 1 and 2, and might be therefore acting as a permissive
factor during mesoderm migration.
PBL is required for protrusive activity of mesoderm cells also in phase 3 and later. It is therefore possible that PBL function might be required in a more general way for the cell to extend protrusions. The specificity of PBL for protrusive activity is also supported by the fact that loss of epithelial characteristics is unaffected in pbl mutant embryos. Although the specific mechanism of PBL function in cell migration is currently unknown, it is important to note that not all morphogenetic movements are compromised in pbl mutants. For example, cephalic furrow formation, invagination of the ventral furrow and germband extension movements, which all depend on a functional cytoskeleton are normal in pbl mutant embryos (data not shown). We therefore propose that PBL might constitute an important component for cytoskeletal changes, which are triggered by FGFR signaling events.
The relation of PBL function to HTL signal transduction
Of the multiple responses generated downstream of FGFR activation, only
little is known of the molecular pathways by which FGFRs trigger cell shape
changes in vivo. The Rho GEF PBL represents a good candidate for mediating
cell shape changes triggered by HTL signaling. Importantly, the early
phenotypes of htl and pbl mutants are almost identical,
indicating that both gene products are required in a narrow time window for
early cell shape changes after invagination of the mesoderm. Furthermore, in
both mutants this phenotype is completely penetrant, indicating that the gene
products do not act in a redundant fashion.
The function of PBL for mesoderm migration is specific for mesoderm cells. Because htl is expressed only in the mesoderm at this stage of development, PBL might be involved in the presentation of the receptor or its unknown ligand and thus acting upstream, or PBL might be involved in downstream events triggered by the HTL signaling cascade. If PBL was acting upstream of HTL, signaling events downstream of HTL should be blocked in such mutants. By contrast, here we show that PBL is dispensable for activation of MAP kinase in the early mesoderm cells. These results suggest that PBL does not act upstream of HTL and favor a model in which PBL acts downstream of the HTL signaling cascade.
The present results render it unlikely that PBL is directly involved in a
signaling pathway downstream of HTL FGFR. In contrast to htl mutants,
no cell shape changes and no protrusive activity was observed in pbl
mutant mesoderm cells in phase 3. In addition, the pbl null mutant
phenotype still allows a few cells to undergo eve expression,
probably owing to the larger cells and abnormal cytoarchitecture in the
division defective embryos. This is in contrast to htl loss of
function mutants where EVE-positive mesoderm cells are never observed. If
pbl was essential for signaling downstream of the HTL receptor, the
phenotype of htl and pbl mutants should be more similar with
respect to mesoderm differentiation; for example, the phenotypes of
htl and dof mutant embryos are identical
(Vincent et al., 1998;
Michelson et al., 1998a
). We
therefore propose that PBL might represent a regulator of the cytoskeleton or
adhesive mechanisms of the cell, which provide targets of the HTL signaling
cascade to trigger cell shape changes.
Although the activation of MAP kinase in the mesoderm depends on HTL, it is
not known whether this is a direct response or whether it is indirect, i.e.
MAP kinase may not be directly activated by HTL itself, but through
interactions of the mesoderm with the ectoderm. In this case, activation of
MAP kinase would be a response rather than a cause of the cell shape changes.
The phenotype of pbl mutants, however, argues against the latter
possibility, because it shows that in the absence of cell shape changes, MAP
kinase can still be activated. This result also suggests that activation of
MAP kinase alone cannot account for the cell shape changes that occur. This
idea is supported by the fact that activated forms of RAS1 are unable to
completely rescue the defects in mesoderm migration of htl or
dof mutant embryos, including the defects in cell shape changes in
phase 1 and 2 (data not shown) (Vincent et
al., 1998; Michelson et al.,
1998a
; Michelson et al.,
1998b
; Iman et al., 1999). These results suggest the presence of a
signaling pathway acting in parallel to the Ras/Raf MAP kinase pathway to be
involved in mesoderm migration.
A novel function for PBL in cell migration
The pbl gene was originally identified and characterized as an
essential factor for cytokinesis (Hime and
Saint, 1992; Lehner,
1992
; Prokopenko et al.,
1999
). We describe a function of PBL in interphase cells that can
be genetically separated from its requirement for cytokinesis.
Two lines of evidence indicate that the function of PBL in cell migration is mediated through a pathway different from the cytokinesis pathway. First, expression of a dominant-negative form of RHO1, which blocks cytokinesis in the mesoderm has no effect on mesoderm migration, cell shape changes associated with it or expression of differentiation markers specific for dorsal mesoderm derivatives. Second, a mutation in pbl, pbl11D exhibits significantly weaker defects in mesoderm differentiation compared to the strong loss of function mutation pbl3. These allele-specific differences indicate distinct requirements of the PBL protein for its two functions, because both alleles exhibit identical cytokinesis defects and only differ in mesoderm differentiation defects significantly. We therefore propose that the function of PBL for cell migration might not involve RHO1 and might therefore be using another mechanism.
How does PBL act in cell migration and which GTPase represents its
substrate? Both PBL and its mammalian orthologs belong to the Dbl family of
Rho-GEFs, which promote activation of Rho GTPases through a conserved
Dbl-homology (DH) domain (Prokopenko et
al., 2000b). The DH domain is required for both functions of PBL,
because a missense mutation in pbl, called pbl5,
in which an amino acid exchange renders the DH domain inactive, exhibits
equally strong defects in cell migration and cytokinesis (S.S. and H.A.J.M.,
unpublished) (M. Smallhorne, M. Murray and R. Saint, personal communication).
Data from yeast two-hybrid assays, as well as genetic interactions indicate
that PBL binds to and interacts with RHO1
(Prokopenko et al., 1999
).
During cytokinesis, PBL is proposed to locally activate RHO1, which then
interacts with its effector Diaphanous, a Drosophila homologue of the
Formin family of actin regulators (O'Keefe
et al., 2001
; Prokopenko et
al., 2000b
; Somers and Saint,
2003
). Although PBL appears to interact with RHO1, but not with
CDC42 or RAC1, mammalian homologs of PBL promote GTP/GDP exchange of the
GTPases RHO1, RAC1 and CDC42 (Tatsumoto et
al., 1999
). Because these discrepancies might reflect differences
in the sensitivity of the assays applied, it remains to be determined which
substrate PBL uses for its function in cell migration.
Although we have detected a role of PBL in FGFR triggered cell migration,
it is currently unclear how general the requirement of PBL is for the
protrusive activity of migrating cells. Interestingly, mutations in
pbl have been discovered in a screen for genes required for the
development of the peripheral nervous system
(Salzberg et al., 1994). These
mutants affected the correct migration of the axons in the PNS without obvious
defects in cytokinesis. It will therefore be interesting to assess the
function of PBL in a variety of migrating cells to further characterize its
potential role as a mediator of cell shape changes triggered by extracellular
signals.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
In this paper, we state that the ligand of HTL FGFR is unknown. While this
paper was in press, two papers were published describing the identification of
two novel genes encoding FGF-like growth factors in Drosophila,
consistent with being ligands of HTL
(Stathopoulos et al., 2004;
Gryzik and Müller,
2004
).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beiman, M., Shilo, B. Z. and Volk, T. (1996). Heartless, a Drosophila FGF receptor homolog, is essential for cell migration and establishment of several mesodermal lineages. Genes Dev. 10,2993 -3002.[Abstract]
Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J. and Der, C. J. (1998). Increasing complexity of Ras signaling. Oncogene 17,1395 -1413.[CrossRef][Medline]
Carmena, A., Gisselbrecht, S., Harrison, J., Jimenez, F. and
Michelson, A. M. (1998). Combinatorial signaling codes
for the progressive determination of cell fates in the Drosophila
embryonic mesoderm. Genes Dev.
12,3910
-3922.
Costa, M., Sweeton, D. and Wieschaus, E. (1993). Gastrulation in Drosophila: Cellular Mechanisms of Morphogenetic Movements. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor Laboratory Press.
Cui, X. and Doe, C. Q. (1995). The role of the
cell cycle and cytokinesis in regulating neuroblast sublineage gene expression
in the Drosophila CNS. Development
121,3233
-3243.
Dunin-Borkowski, O. M. and Brown, N. H. (1995). Mammalian CD2 is an effective heterologous marker of the cell surface in Drosophila. Dev. Biol. 168,689 -693.[CrossRef][Medline]
Edgar, B. A. and O'Farrell, P. H. (1990). The three postblastoderm cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell 62,469 -480.[Medline]
Forbes, A. and Lehmann, R. (1999). Cell migration in Drosophila. Curr. Opin. Genet. Dev. 9,473 -478.[CrossRef][Medline]
Gabay, L., Seger, R. and Shilo, B. Z. (1997).
MAP kinase in situ activation atlas during Drosophila embryogenesis.
Development 124,3535
-3541.
Gisselbrecht, S., Skeath, J. B., Doe, C. Q. and Michelson, A. M. (1996). heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo. Genes Dev. 10,3003 -3017.[Abstract]
Gryzik, T. and Müller, H. A. (2004). FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula. Curr. Biol. 14,659 -667.[CrossRef][Medline]
Halfon, M. S., Carmena, A., Gisselbrecht, S., Sackerson, C. M., Jimenez, F., Baylies, M. K. and Michelson, A. M. (2000). Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103,63 -74.[Medline]
Hall, A. (1998). Rho GTPases and the actin
cytoskeleton. Science
279,509
-514.
Hime, G. and Saint, R. (1992). Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114,165 -171.[Abstract]
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.
Klemke, R. L., Cai, S., Giannini, A. L., Gallagher, P. J., de
Lanerolle, P. and Cheresh, D. A. (1997). Regulation of cell
motility by mitogen-activated protein kinase. J. Cell
Biol. 137,481
-492.
Knirr, S. and Frasch, M. (2001). Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors. Dev. Biol. 238,13 -26.[CrossRef][Medline]
Lauffenburger, D. A. and Horwitz, A. F. (1996). Cell migration: a physically integrated molecular process. Cell 84,359 -369.[Medline]
Lehner, C. F. (1992). The pebble gene
is required for cytokinesis in Drosophila. J. Cell
Sci. 103,1021
-1030.
Leptin, M. and Grunewald, B. (1990). Cell shape changes during gastrulation in Drosophila. Development 110, 73-84.[Abstract]
Lin, X., Buff, E. M., Perrimon, N. and Michelson, A. M.
(1999). Heparan sulfate proteoglycans are essential for FGF
receptor signaling during Drosophila embryonic development.
Development 126,3715
-3723.
Michelson, A. M., Gisselbrecht, S., Buff, E. and Skeath, J.
B. (1998a). Heartbroken is a specific downstream mediator of
FGF receptor signalling in Drosophila. Development
125,4379
-4389.
Michelson, A. M., Gisselbrecht, S., Zhou, Y., Baek, K. H. and Buff, E. M. (1998b). Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm. Dev. Genet. 22,212 -229.[CrossRef][Medline]
Miki, T., Smith, C. L., Long, J. E., Eva, A. and Fleming, T. P. (1993). Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature 362,462 -465.[CrossRef][Medline]
Montell, D. J. (1999). Developmental regulation of cell migration. Insight from a genetic approach in Drosophila.Cell Biochem. Biophys. 31,219 -229.[CrossRef][Medline]
Müller, H. A. and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134,149 -163.[Abstract]
Müller, H. A., Samanta, R. and Wieschaus, E.
(1999). Wingless signaling in the Drosophila embryo: zygotic
requirements and the role of the frizzled genes.
Development 126,577
-586.
O'Keefe, L., Somers, W. G., Harley, A. and Saint, R. (2001). The pebble GTP exchange factor and the control of cytokinesis. Cell Struct. Funct. 26,619 -626.[CrossRef][Medline]
Oda, H., Tsukita, S. and Takeichi, M. (1998). Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation. Dev. Biol. 203,435 -450.[CrossRef][Medline]
Prokopenko, S. N., Brumby, A., O'Keefe, L., Prior, L., He, Y.,
Saint, R. and Bellen, H. J. (1999). A putative
exchange factor for Rho1 GTPase is required for initiation of cytokinesis in
Drosophila. Genes Dev.
13,2301
-2314.
Prokopenko, S. N., Saint, R. and Bellen, H. J. (2000a). Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development. Mech. Dev. 90,269 -273.[CrossRef][Medline]
Prokopenko, S. N., Saint, R. and Bellen, H. J.
(2000b). Untying the Gordian knot of cytokinesis. Role of small G
proteins and their regulators. J. Cell Biol.
148,843
-848.
Ranganayakulu, G., Elliott, D. A., Harvey, R. P. and Olson, E.
N. (1998). Divergent roles for NK-2 class homeobox genes in
cardiogenesis in flies and mice. Development
125,3037
-3048.
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]
Salzberg, A., D'Evelyn, D., Schulze, K. L., Lee, J. K., Strumpf, D., Tsai, L. and Bellen, H. J. (1994). Mutations affecting the pattern of the PNS in Drosophila reveal novel aspects of neuronal development. Neuron 13,269 -287.[Medline]
Sato, M. and Kornberg, T. B. (2002). FGF is an essential mitogen and chemoattractant for the air sacs of the Drosophila tracheal system. Dev. Cell 3, 195-207.[Medline]
Schmidt, A. and Hall, M. N. (1998). Signaling to the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 14,305 -338.[CrossRef][Medline]
Shishido, E., Higashijima, S., Emori, Y. and Saigo, K.
(1993). Two FGF-receptor homologues of Drosophila: one
is expressed in mesodermal primordium in early embryos.
Development 117,751
-761.
Shishido, E., Ono, N., Kojima, T. and Saigo, K.
(1997). Requirements of DFR1/Heartless, a mesoderm-specific
Drosophila FGF-receptor, for the formation of heart, visceral and
somatic muscles, and ensheathing of longitudinal axon tracts in CNS.
Development 124,2119
-2128.
Somers, W. G. and Saint, R. (2003). A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis. Dev. Cell 4,29 -39.[Medline]
Stathopoulos, A., Tam, B., Ronshaugen, M., Frasch, M. and
Levine, M. (2004). pyramus and thisbe: FGF genes that pattern
the mesoderm of Drosophila embryos. Genes Dev.
18,687
-699.
Strutt, D. I., Weber, U. and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signalling. Nature 387,292 -295.[CrossRef][Medline]
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 -1101.[Medline]
Szebenyi, G. and Fallon, J. F. (1999). Fibroblast growth factors as multifunctional signaling factors. Int. Rev. Cytol. 185,45 -106.[Medline]
Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I. and Miki,
T. (1999). Human ECT2 is an exchange factor for Rho GTPases,
phosphorylated in G2/M phases, and involved in cytokinesis. J. Cell
Biol. 147,921
-928.
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]
Weigmann, K. and Lehner, C. F. (1995). Cell
fate specification by even-skipped expression in the Drosophila
nervous system is coupled to cell cycle progression.
Development 121,3713
-3721.
Wilson, R. and Leptin, M. (2000). Fibroblast growth factor receptor-dependent morphogenesis of the Drosophila mesoderm. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,891 -895.[CrossRef][Medline]
Winklbauer, R. and Keller, R. E. (1996). Fibronectin, mesoderm migration, and gastrulation in Xenopus. Dev. Biol. 177,413 -426.[CrossRef][Medline]
Yang, X., Dormann, D., Munsterberg, A. E. and Weijer, C. J. (2002). Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8. Dev. Cell 3,425 -437.[Medline]
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