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First published online 21 January 2009
doi: 10.1242/dev.027904
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California Institute of Technology, Division of Biology MC114-96, 1200 East California Boulevard, Pasadena, CA 91125, USA.
* Author for correspondence (e-mail: angelike{at}caltech.edu)
Accepted 30 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cell migration and differentiation, Drosophila, FGF signaling, Ligand-receptor interactions
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Rottinger et al., 2008;
Thisse and Thisse, 2005). In
embryos, these functions include mesoderm induction and patterning, cell
growth, migration and differentiation. Later functions include organ formation
and maintenance, neuronal differentiation and survival, wound healing and
malignant transformation (Chen and Deng,
2005; Coumoul and Deng,
2003; Eswarakumar et al.,
2005). FGFs often signal directionally across
epithelial-mesenchymal boundaries and exhibit diverse and dynamic patterns of
expression that are both spatially and temporally restricted. Tightly
regulated expression is one mechanism that helps to ensure the specificity of
FGF signaling through FGFRs. An additional level of specificity is most likely
imparted by their divergent amino acid sequences, resulting in differential
binding affinity for the FGFR, heparan sulfate proteoglycan (HSPG)-interaction
specificity (a co-factor involved in FGF-FGFR interaction), and/or FGF ligand
range of action (e.g. diffusibility).
FGF and FGFR genes have been identified in organisms ranging from the
nematode and fly to mouse and human. There are two FGFs and one FGFR in
Caenorhabditis elegans, and three FGFs and two FGFRs in
Drosophila melanogaster, as compared with 24 FGFs and four FGFRs
(three of which exhibit alternative splicing) in humans and mice
(Birnbaum et al., 2005;
Huang and Stern, 2005). In
vertebrates, over 100 potential FGF-FGFR complexes are predicted
(Zhang et al., 2006). Thus,
the expectation is that with so many combinations possible, tight regulation
of FGF activity and receptor specificity must exist to regulate signaling.
Many FGF-FGFR interactions have been studied in the vertebrate system, but the
genetic redundancy can make dissection of the functional contribution of
particular FGF-FGFR interactions challenging (e.g.
Mariani et al., 2008). Often,
more than four ligands interact with a particular FGFR isoform at any one
time.
Drosophila is an excellent model system for studying FGF
signaling, especially now that it appears that the full repertoire of FGF
ligands and receptors has been identified (reviewed by
Huang and Stern, 2005).
Relatively few FGF-FGFR interactions are possible, with only three FGF ligands
[Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] and two FGF receptors
[Heartless (Htl) and Breathless (Btl)] (reviewed by
Ornitz and Itoh, 2001;
Szebenyi and Fallon, 1999).
The FGFR Btl, and its FGF ligand Bnl, control tracheal branching in the
embryo, mesoderm migration over male genital discs, and air sac formation in
the larva (Ahmad and Baker,
2002; Sato and Kornberg,
2002; Sutherland et al.,
1996). The preliminary function of the Htl FGFR is to control
mesoderm migration during gastrulation
(Beiman et al., 1996;
Gisselbrecht et al., 1996;
Shishido et al., 1997). Later
in development, among other functions, Htl is also required for the
differentiation of cells that form the heart and hindgut musculature
(Michelson et al., 1998;
San Martin and Bate, 2001).
The ligands for the Htl FGFR in Drosophila had remained elusive,
until pyr and ths were identified by genomic screens
(Stathopoulos et al., 2004;
Gryzik and Muller, 2004). Pyr
and Ths share homology with the FGF8 family of vertebrate FGFs. Genetic
evidence suggests that this pair of invertebrate FGFs functions through a
single FGFR, Htl, to control cell migration and differentiation, as a
deficiency mutant that removes both ligands phenocopies the htl
mutant (Stathopoulos et al.,
2004; Gryzik and Muller,
2004). Thus, Drosophila melanogaster provides a unique
opportunity within an invertebrate model system, amenable to genetic
approaches, to gain insights into why multiple FGF ligands are utilized to
activate the same receptor isoform, as is typically the case in
vertebrates.
A question in the FGF field is whether the specificity of receptor-ligand
interactions is accomplished through the differential expression of ligands or
through differences in the signaling properties of the ligand proteins
themselves. An impressive analysis of all vertebrate FGF-FGFR interactions was
recently completed, in which the binding specificities of ligand-receptor
interactions were examined in tissue culture
(Ornitz et al., 1996;
Zhang et al., 2006).
Specificity of receptor-ligand interactions was demonstrated in this system;
however, how this relates to in vivo processes is, for the most part,
undetermined. Only a limited number of in vivo studies have been conducted to
analyze the function of particular FGF-FGFR interactions (e.g.
Rentzsch et al., 2008;
Yang et al., 2002). In this
study, we examine the individual functions of the FGF ligands Pyr and Ths in
Drosophila in order to define whether these ligands have distinct
functions and/or function redundantly, and to gain insights into why multiple
ligands are typically involved in activating a particular FGFR coordinately.
We find that the regulated expression of ligands and their divergent protein
sequence both contribute to the specificity of FGF-FGFR interactions.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The Gal4 drivers zenVRE.Kr-Gal4 and
sim-Gal4 were obtained from M. Frasch (University of
Erlangen-Nuremberg, Germany) and Stephen Crews (University of North Carolina,
Chapel Hill, NC, USA), respectively. Two PiggyBac insertion stocks were
obtained from the Exelixis Collection Stock Center at Harvard Medical School
(Thibault et al., 2004); we
determined that P02915 is located in the first intron of pyr, and
confirmed that P02026 is located in the second intron of ths.
Df(2R)ths238 is a deletion of the ths gene that was
created in the course of this study through a genetic screen for male-specific
recombination (see Preston et al.,
1996) using P9.1.2, a viable P-element insertion located 
400
bp upstream from the ths transcription start site, as mapped by RACE
(see Fig. 2A). Line P9.1.2
resulted from mobilization of P10004, a viable insertion located 900 bp
upstream from the ths transcription start site, to remove 500 bp of
ths upstream sequence. Deletion breakpoints were confirmed using
inverse PCR, in the case that the P-element was retained [i.e.
Df(2R)BSC25 and Df(2R)ths238].
Df(2R)pyr36 is a deletion of the pyr gene that was created by mobilization of the P9.1.2 insertion and screening for excision events. The breakpoints of this deficiency were mapped by complementation using available zygotic lethal insertions (including walEY09961 and walk14026); we contend that the Df(2R)pyr36 does not extend past the walrus (wal) and ths genes. The 500 bp of ths upstream sequence that was removed in the creation of line P9.1.2 is also absent from Df(2R)pyr36. Yet, ths transcript levels and domains of expression appear similar to those of the wild type in Df(2R)pyr36/Df(2R)pyr36 mutant embryos (expression up to stage 15 was examined; see Fig. 2F and data not shown). It is formally possible that cis-regulatory sequences controlling expression at later stages were removed. However, as the phenotypes exhibited by Df(2R)pyr36 are subtle relative to Df(2R)BSC25, and as the FGF-homologous portion of Ths is located at its N-terminus, it is likely that Df(2R)pyr36 does not remove ths coding sequence.
UAS-bnl and UAS-ths have been described previously
(Stathopoulos et al., 2004;
Sutherland et al., 1996).
UAS-pyr was generated during the course of this work. No EST is
available for pyr. Therefore, the 5' and 3' ends of the
pyr gene were determined by RACE
(Stathopoulos et al., 2004).
Primers based on this sequence were used to PCR amplify the intact
pyr gene. PCR of the intact full-length gene from the RACE cDNA with
a single primer pair was not possible, probably owing to repeats within the
gene. Instead, two separate PCR reactions were performed to isolate sequence
spanning the full-length gene (one to amplify the 5' end from cDNA and
the other to obtain sequence corresponding to the last exon from genomic DNA).
The two products were ligated in a three-way ligation to reconstitute the
full-length pyr coding sequence.
UAS-pyr and UAS-ths fly stocks were constructed using
standard methods (Spralding and Rubin,
1982). For the genetic rescue experiments, virgins of the
following genetic backgrounds (#1) Df(2R)BSC25 sim-Gal4/CyO
ftz-lacZ, (#2) Df(2R)BSC25/CyO wg-lacZ; zenVRE.Kr-Gal4
and (#3) Df(2R)BSC25/CyO wg-lacZ; 69B-Gal4 were crossed with
males from (#4) Df(2R)BSC25/CyO wg-lacZ; UAS-pyr,
(#5) Df(2R)BSC25/CyO wg-lacZ; UAS-ths, (#6)
Df(2R)BSC25/CyO wg-lacZ; UAS-bnl, (#7)
Df(2R)BSC25/CyO ftz-lacZ; UAS-pyr, (#8) Df(2R)BSC25/CyO
ftz-lacZ; UAS-ths or (#9) Df(2R)BSC25/CyO
ftz-lacZ; UAS-bnl.
Quantitative PCR
Embryos were manually separated and flash frozen using liquid nitrogen.
Three biological replicates of each sample (i.e. 20 dechorionated embryos of
stage 9/10) were obtained. Total RNA isolation was carried out using the
RNeasy Micro Kit (Qiagen) and quantified using a ND-1000 spectrophotometer
(NanoDrop Technologies). One microgram of total RNA was used for reverse
transcription to synthesize cDNA using the Gene Racer Superscript III RT
Module Kit (Invitrogen). The quantitative PCR (qPCR) reaction was performed in
96-well format using LightCycler SYBR Green 480 (Roche). The qPCR was set up
as follows: 95°C for 5 minutes; 35 cycles of 95°C for 20 seconds,
55°C for 20 seconds, and 72°C for 30 seconds; and a final 72°C for
10 minutes. For quantifying the transcript levels in ectopically expressing
pyr and ths lines, the data were normalized to the
transcript level of a housekeeping gene, Elongation factor 1
(Ishimoto et al., 2005). In
order to quantify the levels of `ectopic' pyr and ths
expression supported by the Gal4 driver and transgenes containing UAS, we used
twist-Gal4 and UASlacZ as a control and subtracted the endogenous
pyr and ths transcript levels; the domain of ectopic
expression supported by twist-Gal4 is significant, as compared with
expression supported by zenVRE.Kr-Gal4 and sim-Gal4, and
thereby allowed for better resolution of the levels of ectopic expression.
The lines chosen [pyrline1: UAS-pyr:AMS330(III) and thsline1: UAS-ths:AMS289.22(III)] exhibit comparable levels of ectopic expression as assayed by qPCR (see Fig. S4 in the supplementary material) and support equivalent Eve+ expression when driven by 69B-Gal4 (see Fig. S3D,E in the supplementary material). In another set of insertions, approximately twice as much ectopic ths was supported, relative to the levels of pyr (see Fig. S4 in the supplementary material, thsline2 and pyrline2); nevertheless, the same results were obtained with this other set of lines in the zenVRE.Kr-Gal4-driven rescue experiments (data not shown). Therefore, we contend that the ability of pyr alone to support the migration of mesoderm cells at a distance is not due to differences in expression levels resulting from positional effects on transgene expression.
Immunohistochemistry, in situ hybridization and sectioning
The following antibodies were used: guinea pig anti-Twist (1:200; from M.
Levine, UC Berkeley, CA, USA), mouse anti-dpERK (1:200; Sigma), rabbit
anti-β-galactosidase (1:200; Molecular Probes), rabbit anti-Eve (1:400;
from M. Frasch) and monoclonal 2A12 (1:3; Developmental Studies Hybridoma
Bank). In situ hybridization (Kosman et
al., 2004; Lehmann and Tautz,
1994), double antibody plus in situ hybridization detection of
protein and transcript levels (Frasch,
1995), and staining of tracheal branches using 2A12 antibody
(Sutherland et al., 1996) were
conducted as previously described.
For sectioning, embryos were sorted for appropriate stage and genotype
(i.e. using balancer chromosomes containing reporters), and embedded in
acetone-Araldite (Electron Microscopy Sciences) as described
(Leptin and Grunewald, 1990).
Sections (10 µm) were cut using a Historange 2218 microtome.
Because variability is present in the Df(2R)BSC25 mutant background, a system of quantitation of mutant phenotype was devised. We scored mesoderm spreading phenotypes in the deficiency and the ectopically expressing lines (see Figs S5 and S6 in the supplementary material). Clumping was used as a measure of the ability of ligands to attract cells to the ventral midline; the multilayer phenotype was scored by assessing the ability of ligands to attract cells to the dorsal-most ectoderm.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
During gastrulation, pyr and ths are expressed in distinct
domains within the ectoderm at the surface of the embryo (see
Fig. 1A-C). Early in
development, ths is expressed in ventral regions
(Fig. 1F,G) and pyr is
expressed in dorsal regions (Fig.
1D,E). Once invagination is complete and all mesoderm cells have
entered the interior of the embryo, these cells proceed to migrate along the
inner ectodermal surface, forming a monolayer. Activation of the FGFR Htl is
thought to trigger the spreading of the mesoderm across the underlying
ectoderm (Fig. 1A). The dynamic
expression domains of the ligands suggested a possible mechanism by which
directional information could be imparted to the mesoderm cells moving
beneath, causing these cells to move in the dorsal direction.
Ligand choice and expression domain are important for FGF signaling
To determine whether the distinct domains of ligand expression are
important for proper mesoderm migration, we altered their expression domains
and assayed effects on mesoderm spreading. Using the 69B-Gal4 driver,
the bnl, pyr and ths FGF-encoding genes were ectopically
expressed throughout the entire ectoderm
(Fig. 1H-M). For pyr
and ths, several transgenic lines were first compared with the
control for positional effects that might influence the levels of gene
expression, and a set of lines was chosen that exhibited comparable levels of
ectopic expression by Gal4 as assayed by qPCR (see Fig. S4 in the
supplementary material). Ectopic expression of Pyr throughout the ectoderm
resulted in mesoderm migration defects
(Fig. 1J,K; n=9). The
pyr ectopic expression defect was specific; mesoderm spreading
occurred, but a cell monolayer was not formed
(Fig. 1K). By contrast, ectopic
expression of Bnl or Ths in the same manner had little to no effect on
mesoderm monolayer formation (Fig.
1G,I and Fig. 1L,M;
n=6 and n=10, respectively).
The MAPK pathway is activated in migrating mesodermal cells as a result of
FGFR receptor tyrosine kinase activation, and leads to dual phosphorylation of
MAPK [also known as ERK (Gabay et al.,
1997) and Rolled - FlyBase]. A monoclonal antibody that recognizes
this activated form of MAPK (dpERK) can be used to monitor activation of this
pathway within cells. dpERK staining is observed within gastrulating embryos
at the leading edge of the migrating mesoderm cells (see Fig. S1A in the
supplementary material), but is lost in the absence of Htl activation
(Michelson et al., 1998;
Stathopoulos et al., 2004). In
embryos ectopically expressing pyr or ths via
twist-Gal4, which supports their expression throughout the mesoderm,
dpERK is ectopically expressed throughout all mesoderm cells (see Fig. S1C,E
in the supplementary material)
(Stathopoulos et al., 2004).
However, when the 69B-Gal4 driver was used to ectopically express
pyr or ths, only the ectopic expression of pyr
supported ectopic activation of ERK throughout the entire mesoderm (compare
Fig. S1D with Fig. S1F in the supplementary material). Ectopic expression of
bnl had no effect: dpERK remained localized at the leading edge of
the migrating mesoderm [compare Fig. S1B with Fig. S1A (see arrowheads) in the
supplementary material]. Depending on where the ligands were expressed,
differences in the capacity of Pyr versus Ths to activate signaling were
observed, whereas Bnl had no effect. Furthermore, the ability of these ligands
to support ectopic dpERK expression was found to correlate with the inhibition
of mesoderm migration (see Fig.
1; data not shown).
We then assayed the ability of Pyr and Ths to influence trachea formation,
a function that is supported by Bnl-Btl. Ectopic expression of bnl
via 69B-Gal4 leads to a substantial increase in tracheal branching
(compare Fig. S2B with Fig. S2A in the supplementary material)
(Sutherland et al., 1996).
However, no such effect on the trachea was observed upon ectopic expression of
pyr or ths (see Fig. S2C,D in the supplementary material),
suggesting that neither Pyr nor Ths can activate the Btl FGFR.
Isolation and initial characterization of pyr and ths single mutants
Mesoderm spreading defects are observed in htl mutants and in a
deficiency mutant, Df(2R)BSC25, that removes both pyr and
ths (Fig. 2A)
(Beiman et al., 1996;
Gisselbrecht et al., 1996;
Stathopoulos et al., 2004). We
sought to test further the requirement for Pyr or Ths in activation of the Htl
FGFR by analysis of single-mutant phenotypes. First, we identified two
PiggyBac insertions located in proximity to pyr and ths (see
Fig. 2A). The line containing
the pyr insertion, pyr02915, is zygotically
lethal, whereas the line containing the ths insertion,
ths02026, is only semi-lethal [i.e. homozygous flies are
obtained at very low frequencies (0.5%) and are short-lived]. Each
insertion disrupts expression of the respective gene, and we detect no effect
on expression of the other ligand (see Fig.
2E,G, compare with Fig.
2A-C). As phenotypes associated with insertions can vary, we chose
to identify true null alleles.
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Complementation tests were conducted to determine genetic interactions
between our allelic series of pyr and ths mutants. We
believe that the pyr02915 allele represents a pyr
null, as no expression of pyr was detected at any time-point
examined. It is unclear whether the ths02026 allele
represents a ths null, as expression could be detected in the
visceral mesoderm at later stages (data not shown). Df(2R)pyr36 and
Df(2R)ths238 represent null alleles of pyr and ths,
respectively, because the entire coding region of each gene is absent;
however, of note is the fact that other genes are also removed by these
100 kb deletions (see Materials and methods for details). This allelic
series (i.e. PiggyBac insertions: weak alleles; Df(2R)pyr36 and
Df(2R)ths238 deficiencies: strong alleles; Df(2R)BSC25
deficiency: double mutant; see Fig.
2A) provided the first opportunity to analyze transheterozygous
combinations in order to gain insights into pyr and ths
single-mutant phenotypes. In both pyr and ths single
mutants, the mesoderm spreading was aberrant
(Fig. 3G-J, compare with
Fig. 3A,B). The mesoderm was
multilayered and a monolayer rarely formed. However, neither mutant exhibited
a phenotype as severe as that of the Df(2R)BSC25 or htl
mutants (Fig. 3C-F), suggesting
a role for both Pyr and Ths in guiding mesoderm spreading.
Importance of positional information in the function of ligands during mesoderm spreading
To determine whether the localized expression of each ligand is important
for its function, we took an ectopic expression approach using Gal4 to promote
expression of a given ligand in the pyr and ths
double-mutant background. Df(2R)BSC25 removes both pyr and
ths, and 16 other genes, none of which has known embryonic phenotypes
(Fig. 2A)
(Stathopoulos et al., 2004).
The sim-Gal4 driver (Xiao et al.,
1996) delivers ligands at the ventral midline where
single-minded (sim) is normally expressed (see
Fig. 4F). Alternatively, the
zenVRE.Kr-Gal4 driver can be used to deliver ligands to the dorsal
domain of the ectoderm within the lower half of gastrulating embryos (see
Fig. 4K)
(Frasch, 1995).
When the sim-Gal4 driver was used to express either pyr or ths in the Df(2R)BSC25 mutant background (Fig. 4F), no rescue of mesoderm spreading was observed (Fig. 4G,H). In fact, clumps of mesoderm cells were observed overlying the site of Pyr ectopic expression, suggesting that this ligand had attracted mesoderm cells to this position (Fig. 4G). This phenotype appeared to be more severe than that of Df(2R)BSC25 mutants (Fig. 4G,H, compare with Fig. 4B,C; see Fig. S5 in the supplementary material; P<0.01). Ectopic expression of ths in this manner also had a weak effect, as cells tended to pool at the ventral midline (Fig. 4H), but this effect was not supported by statistical significance as the Df(2R)BSC25 mutants themselves exhibit variability (Fig. 4B,C and see Fig. S5 in the supplementary material).
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FGF ligand specificity during mesoderm differentiation
After mesoderm cell spreading has been completed, both Pyr and Ths are
expressed in regions of the dorsal ectoderm that abut a subset of the mesoderm
cells (Fig. 2D and see Fig.
S3A,B in the supplementary material). pyr is present in more-dorsal
regions of the ectoderm, overlying sites of dorsal mesoderm specification;
limited expression of ths is also seen in this same region
(Fig. 2D). The mesoderm cells
that come into contact with the dorsal ectoderm receive an inductive signal
from Decapentaplegic (Dpp), a Drosophila TGFβ homolog (reviewed
by Frasch, 1999). Thereby,
expression of genes such as even skipped (eve) and
tinman, which are required for the differentiation of cardiac and
pericardial tissues, depend on Dpp as well as on Htl-dependent FGF
signaling.
We sought to define the role of each ligand in the specification of dorsal
mesoderm cell lineages within germ-band elongated embryos. Normally, Eve is
expressed within 12 hemisegments. In pyr single mutants, often only a
few hemisegments exhibited Eve expression
(Fig. 5D,E, compare with
Fig. 5A), whereas within
ths single-mutant embryos, only subtle defects in Eve+
cell specification were observed (Fig.
5G,H, compare with Fig.
5A). The pyr mutant phenotype was much stronger than the
phenotype exhibited by ths single mutants at all stages examined, and
even when comparisons were made between the weakest pyr allele
(pyr02915) and the strongest ths allele
[Df(2R)ths238] (Fig.
5C,F, compare with Fig.
5I). Moreover, the phenotype of Df(2R)pyr36 was
consistently stronger than that of pyr02915, suggesting
that either pyr02915 is not a null allele or that
ths gene function also supports Eve+ cell specification.
Although Pyr has the more dominant role, Ths probably also plays a role in
supporting Eve expression because the Df(2R)BSC25 mutant phenotype is
more severe than that of Df(2R)pyr36 mutants. No Eve+
cells are present within the dorsal mesoderm of Df(2R)BSC25 mutants,
a phenotype similar to that of htl mutants
(Fig. 6B)
(Stathopoulos et al.,
2004).
To investigate the roles of Pyr and Ths in controlling cell
differentiation, which might differ from their roles during cell migration, we
focused on how ectopic expression of these ligands affects specification of
Eve+ cells within the dorsal mesoderm of the embryo. We have
demonstrated previously that when Ths is ectopically expressed in the ectoderm
using the 69B-Gal4 driver, ectopic Eve+ cells result; this
is similar to the phenotype observed when constitutively activated Htl is
ectopically expressed in the same manner (see Fig. S3E in the supplementary
material) (Stathopoulos et al.,
2004). We found that ectopic expression of Pyr driven by
69B-Gal4 also increases the number of Eve+ cells (see Fig.
S3D in the supplementary material). Normally, three to four Eve+
cells are specified in 12 hemisegments (see Fig. S3C in the supplementary
material). In the presence of excess levels of Pyr or Ths, 6-15 cells
were specified and Eve expression was expanded to 14 hemisegments (compare
Fig. S3C with Fig. S3D in the supplementary material). When activated Ras is
ectopically expressed in the ectoderm, a similar increase in the number of
Eve+ cells is observed (Carmena
et al., 1998).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). However, in this study, we assayed whether the expression
supported in distinct domains would support Htl activation. By a series of
`rescue' experiments, through ectopic expression of one ligand in the
Df(2R)BSC25 mutant background, we obtained evidence that localized
expression of the ligands is important for proper mesoderm spreading. We find,
surprisingly, that the ligands exhibit differences in their functional range
of action (Fig. 4). In
addition, using this same approach, we find that either Pyr or Ths can support
Eve+ cell specification within the dorsal mesoderm, but that Bnl
cannot (Fig. 6). Collectively,
these data suggest that the Pyr and Ths FGFs function as ligands for the Htl
FGFR and that specificity of FGF-FGFR interactions exists in
Drosophila.
Specificity of FGFR activation: only three FGF-FGFR combinations function in Drosophila
Our results demonstrate that both Pyr and Ths FGF ligands can activate the
Htl FGFR, whereas only the Bnl FGF ligand can activate the Btl FGFR
(Fig. 6 and see Fig. S2 in the
supplementary material). Specificity of FGFR activation was observed:
pyr or ths, but not bnl, expression is able to
activate Htl to affect expression of Eve, and bnl, but neither
pyr nor ths, is able to support tracheal specification. No
evidence was obtained that other cross-interactions occur (i.e. Pyr-Btl,
Ths-Btl or Bnl-Htl), which demonstrates that Gal4-mediated ectopic expression
does not simply `swamp the system'. This experimental approach also `levels
the playing field', as expression of each ligand is driven at the same time
and place and presumably at similar levels. We conclude that only three
FGF-FGFR combinations function in Drosophila (i.e. Pyr-Htl, Ths-Htl
and Bnl-Btl), which supports the idea that FGFRs exhibit ligand-binding
preferences. Previous studies have investigated FGF signaling specificity by
analyzing the ability of other receptor tyrosine kinases to support cell
migration or by activating particular intracellular signaling pathways to
examine which are required to effect FGFR-dependent cell migration versus cell
differentiation (Dossenbach et al.,
2001; Wilson et al.,
2005). In this work, we analyzed the specificity of FGF
ligand-receptor interactions and how they contribute to particular
developmental processes.
Different FGF ligand activities regulate particular developmental processes
When ligand expression is supported by twist-Gal4, Htl FGFRs
presumably become saturated because dpERK is ectopically activated in all
cells and spreading is negatively affected (see Fig. S1 in the supplementary
material). One explanation for why this might affect mesoderm cell spreading
is that these FGF-saturated mesoderm cells may no longer be competent to
respond to endogenous ligands that provide directional cues. Recently, we have
shown that movement of the mesoderm cells during gastrulation is in fact
directional (McMahon et al.,
2008). Pyr and Ths ligands are differentially expressed during
gastrulation and this might provide the necessary positional information
required to direct migration of the mesoderm (see
Fig. 7A).
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) of single-mutant phenotypes will provide insights. With regard to the FGF-dependent cell differentiation, our `rescue' experiments suggest that ectopic expression of either Pyr or Ths is sufficient to support Eve+ cell specification (Fig. 6D,E). The reason why loss of ths has less of an effect on Eve+ cell specification is most likely because pyr is prominently expressed in the vicinity of the future Eve+ cells; normally, Pyr supports this function, but Ths can support this activity if presented at sufficient levels within the correct domain. Furthermore, we propose that FGF signaling might not play an instructive role in supporting eve expression (Fig. 7B). Other signaling pathways already provide positional information required for the specification of Eve+ cells; FGF signaling pathway activation might simply serve a permissive role, and in this context either ligand would suffice.
Conclusion: implications for vertebrate biology?
We have used Drosophila to study FGF signaling and determine why
multiple ligands are utilized to activate the same receptor. It will be
informative to obtain additional insights into how these ligands differ in
their activities. The expression domains of the ligands do confer information
that is important for controlling developmental processes, but their
individual protein sequences also impart differential functionality. For
instance, the ligands might exhibit different affinities for specific HSPGs
that influence their range of diffusion, or the proteins themselves may have
different stabilities. Future experiments will also define the FGF ligand
preferences that exist to support FGF signaling at later stages of
development.
At least 15 human genetic diseases result from mutations within FGFR genes
and each disease is caused by a different mutation shown to affect receptor
activation (Chen and Deng,
2005). Several mutations in FGFR lead to an expansion of FGF
ligand-binding preference (Ornitz,
2005); however, it is still not clear why different mutations
yield different syndromes. Continuing this work in order to understand how
different FGF ligands activate the same receptor to effect different outcomes
is an important goal, as this may provide insights into why different
mutations in the same FGFR lead to various dysplasias and diseases
(Wilkie, 2005).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/739/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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