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First published online 29 August 2007
doi: 10.1242/dev.006080
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Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Author for correspondence (e-mail:
lgvolk{at}weizmann.ac.il)
Accepted 17 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mesoderm, RNA regulation, HOW, Miple, HTL, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
which includes the Caenorhabditis elegans homolog GLD-1, and the
mammalian quaking (QKI, QK) protein. The STAR RBPs are essential for the
control of transitional differentiation states - including the transition from
mitosis to meiosis and sex-determination mediated by GLD-1 in C.
elegans (Crittenden et al.,
2002; Crittenden et al.,
2003; Hansen and Schedl,
2006), and the maturation of Schwann cells in the PNS and
oligodendrocytes in the CNS mediated by QKI in mammalian species
(Ebersole et al., 1996;
Hardy, 1998;
Larocque and Richard, 2005).
In the Drosophila embryo, HOW regulates heart-beat rate, mesoderm
invagination, muscle-dependent tendon cell differentiation and glial
maturation (Baehrecke, 1997;
Nabel-Rosen et al., 1999;
Zaffran et al., 1997). The
how gene is differentially spliced into two isoforms, HOW(L) and
HOW(S), which share the same signature of the RNA-binding domain but differ at
their C-terminal region. Although both HOW(L) and HOW(S) can bind the same
mRNA target, they act in opposing directions; binding of HOW(L) to
stripe mRNA at the 3' UTR leads to mRNA degradation, whereas
the binding of HOW(S) leads to the stabilization of stripe mRNA
(Nabel-Rosen et al., 2002). In
addition to mRNA stabilization/degradation, HOW proteins regulate the splicing
of specific targets (Edenfeld et al.,
2006; Volohonsky et al.,
2007).
Previous analysis showed that how mutant germline clone embryos
exhibit defects in mesoderm invagination during the beginning of gastrulation
(Nabel-Rosen et al., 2005).
This defect stems from extra mesodermal cell divisions, due to the elevation
of string (also known as cdc25) mRNA. Despite the defects in
mesoderm invagination in how mutant germline clone embryos,
gastrulation is only delayed and, eventually, all mesoderm cells invaginate
beneath the internal surface of the ventral ectoderm. However, in addition to
this phenotype, these how mutant embryos exhibit abnormalities in
mesoderm spreading (Nabel-Rosen et al.,
2005). Mesoderm spreading over the ectoderm in the
Drosophila embryo is essential for the correct specification of the
distinct subpopulations of cells of the mesoderm lineage, including somatic,
visceral and heart muscles, fat body, gonadal mesoderm, and others
(Baylies et al., 1998;
Frasch, 1999). Following their
invagination from the ectoderm, the mesodermal cells undergo an
epithelial-to-mesenchymal transition, adhere to the basal surface of the
overlying ectoderm and spread dorsally. Then, differentiation signals from the
ectoderm subdivide the mesoderm layer into distinct domains. Proper spreading
of the mesoderm depends on FGF signaling, in which the FGF receptor, Heartless
(HTL), is expressed by mesodermal cells and is activated by two FGF8-like
ligands [Thisbe (THS) and Pyramus (PYR)] produced by the ectoderm layer
(Beiman et al., 1996;
Gisselbrecht et al., 1996;
Gryzik and Muller, 2004;
Shishido et al., 1997;
Stathopoulos et al., 2004).
Despite the ubiquitous expression of HTL and its adaptor protein, Downstream
of FGF (DOF, also known as Stumps - FlyBase), in the mesoderm, MAPK activation
in the mesoderm layer is spatially restricted. Initially, it is expressed in
the cells that adhere to the ectoderm, and later it is elevated in dorsally
located cells (Gabay et al.,
1997; Wilson et al.,
2004; Wilson and Leptin,
2000). The two FGF8-like HTL ligands Thisbe and Pyramus exhibit a
dynamic expression pattern during mesoderm spreading. thisbe mRNA is
expressed throughout the neurogenic ectoderm, and pyramus mRNA is
refined into dorsal and ventral regions of the neurogenic ectoderm
(Stathopoulos et al., 2004;
Gryzik and Muller, 2004). Both
expression patterns do not correlate with the restricted MAPK activation
detected in the spreading mesoderm. The factor(s) controlling the spatial
regulation of MAPK activation in the mesoderm are yet to be elucidated. It is
assumed that additional components, possibly of the extracellular matrix
deposited between the ectoderm and the mesoderm, might control the spatial
distribution/activation of the secreted FGF ligands, enabling spatially and
temporally restricted MAPK activation of the mesodermal cells.
To elucidate the basis for the abnormal mesoderm spreading in how mutant embryos, we performed a microarray screen for putative HOW target genes. We expected that the mRNA levels of these genes would be elevated in the mesoderm of the mutant embryos, because only the repressor, HOW(L), is normally expressed at this stage. Four out of 32 potential targets identified in this screen were further analyzed and shown to be specifically elevated in the mesoderm in how germline clone embryos, and to bind HOW via their 3' UTRs. One of these genes, the midkine and pleiotrophin heparin-binding growth factor miple, exhibited abnormal mesoderm spreading following its overexpression in the mesoderm, which was correlated with scattered MAPK activation in mesodermal cells. This might explain the aberrant mesoderm spreading observed in how mutant embryos.
We therefore suggest that the HOW-dependent negative regulation of mRNA levels of several targets during gastrulation is essential for proper mesoderm spreading.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The GFP-miple 3' UTR was constructed by insertion of
miple 3' UTR into pUAST vector containing EGFP. The
two point mutations in the HOW response element ACUAA were inserted by
standard mutagenesis, creating the sequence ACGGA.
For the production of germline clones
(Nabel-Rosen et al., 2005),
males carrying FRT82B,OvoD/TM3Sb (Bloomington Stock Center) were
crossed to females carrying hs-flp;Dr/TM3,Sb (Umea Stock Center), and
the progeny males carrying hs-flp;FRT82B,OvoD/TM3,Sb were
crossed to females carrying FRT82B,howe44/TM3,Sb (produced
in our laboratory by recombination). Larvae of this cross were heat-shocked
daily over 3 days for 50 minutes at 37°C, and adult females carrying the
OvoD construct that had wing blisters were crossed to males carrying
howstru/TM3,2Xtwistgal4,UAS-GFP
(Prout et al., 1997).
Gal4 lines: Mef2-gal4 (Bloomington Stock Center) and twist-gal4 (A. Muller, University of Dundee, UK).
For the rescue experiments, flies carrying htl/TM6;UAS-miple/Cyo were crossed to flies carrying htl/TM6;mef2-gal4/Cyo. Both balancers were marked, so the htl mutants were identified by negative ß-gal expression.
Antibodies used include rat anti-HOW
(Nabel-Rosen et al.,1999),
anti-Myosin heavy chain (-MHC) (P. Fisher, Stony Brook, NY), mouse anti-dpERK
(Sigma), rabbit anti-Twist (obtained from S. Roth, Cologne, Germany) mouse
anti-HA (Roche), rabbit anti-Even skipped (-EVE) and rabbit anti-Tinman (M.
Frasch, Mount Sini, NY). For embryos double stained with anti-dpERK and
anti-Twist, fixation was carried out in 8% formaldehyde/PBS and 50 mM EGTA for
25 minutes, anti-dpERK primary antibody incubated for 2 hours at room
temperature (RT) in 0.1% Tween/PBS, and secondary antibody (goat anti-mouse
biotin; Chemicon) incubated for 60 minutes at RT. It was amplified by
streptavidin-HRP for 30 minutes at RT, followed by incubation with tyramide
biotin for 20 minutes (both from Perkin Elmer TSA biotin system). Finally,
embryos were incubated with streptavidin-Cy3 for 30 minutes at RT (Jackson
ImmunoResearch) (Melen et al.,
2005). Following this staining, embryos were then stained with
anti-Twist antibodies. Secondary antibodies included Cy3, Fluoresceine, or
HRP-conjugated anti-rabbit or anti-rat, or-anti mouse (Jackson). For embryos
double-labeled with MEF2 and EVE, the embryos were fixed and stained with
anti-EVE, and secondary Cy2-conjugated anti-rabbit antibody. Then, the embryos
were labeled with anti-MEF2 followed by labeling with Cy3-conjugated
anti-rabbit antibody, which also recognized the EVE antibody. This resulted
with yellow labeling of the EVE cells and red labeling of the MEF2 cells.
For in situ hybridization, a probe was prepared using T7/T3/SP6 RNA polymerase (according to the relevant ESTs) and the Roche RNA DIG labeling mix. Fixation, hybridization and detection were performed according to http://www.biology.ucsd.edu/~davek/.
Microarray experiments
Sample preparation
Embryo collections were performed every 2 hours from either y w or
from how germline clone mutant females on apple juice/yeast plates at
25°C. Plates were removed and the embryos were aged for an additional 3
hours at 25°C. Because the how mutation was established in trans
to a balancer chromosome carrying GFP,the how/GFP-balancer
collections contained a mixed population of embryos. Homozygous how
mutant embryos were separated from their siblings using a fluorescent
binocular. Carefully staged embryos that had been aged 3-5 hours in this
manner were collected and dechorionated. Total RNA was extracted from
sufficient amounts of embryos (
100 embryos) using the Macherey-Nagel
Nucleospin RNA II mini kit, following the protocol, and then kept at
-70°C. Total RNA was prepared independently five times from embryos of
each genetic background in order to better normalize the age of these embryo
populations. The RNA samples were then collected and concentrated to give 1 mg
of total RNA using the RNA cleanup RNeasy mini kit (Qiagen). The probe
preparation, cDNA synthesis, cRNA reactions and hybridization with Affymetrix
high-density oligonucleotide arrays for Drosophila melanogaster was
carried out in the Weizmann Institute microarray unit.
Normalization and statistics
More than 13,500 gene sequences predicted from the annotation of the
Drosophila genome are represented on the Drosophila
affymetrix array. Signals were pre-processed using Robust Multichip Average
(RMA) algorithms with the default parameters (i.e. RMA model-based background
adjustment, quantile normalization, median polish summation of the probe
intensities). A total of 690 probe sets on the chip were differentially
expressed with P<0.05 (t-test). Among them, 147 probe
sets exhibited a 1.5-fold or greater upregulation in how germline
clones.
All microarray data were submitted to Gene Expression Omnibus (GEO). The accession number is GSE7772.
Protein-RNA binding assay and western analysis
The protein-RNA binding assay was performed essentially as described
(Nabel-Rosen et al., 1999).
The entire miple, CG31638, falten, lap or punt cDNAs (ESTs
obtained from DGRC) were used as templates to produce biotin-labeled RNAs
(Biotin labeling mix, Roche, and T7/T3 or SP6 polymerase, Promega). The
biotin-labeled RNA was purified on a G-50 Sephadex Quick Spin Column (Roche)
and then mixed with in vitro-translated HOW(L) or HOW(L)R185 to
C-HA-tagged proteins (TNT T7 quick coupled transcription/translation
system, Promega) and precipitated with magnetic streptavidin beads. Binding
was performed by adding approximately 1 µg of biotin-labeled RNA to 5 µl
of the translated HOW proteins. Streptavidin magnetic beads were first washed
with binding buffer, and 300 µl of the beads was added to each binding
reaction for 25 minutes at RT. The magnetic beads were then isolated, washed,
and boiled in sample buffer, and the supernatant was loaded on 10%
SDS-polyacrylamide gel and reacted with mouse anti-HA antibodies (1:2000
dilution). Blocking, hybridization and detection were performed using standard
protocols.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To further define the effects of abnormal mesoderm
spreading on later stages of mesoderm development, we analyzed the expression
pattern of various markers characteristic of the heart, pericardial cells and
somatic musculature precursors in how germline clone mutant embryos.
We first analyzed mesoderm spreading in how mutant embryos at stages
7-9, which we labeled with anti-Twist (anti-TWI) antibodies. In contrast to a
homogenous distribution of mesodermal cells in wild-type embryos, the
distribution of the mesoderm Twist-expressing cells in how mutant
embryos was uneven. Although in some regions the mesodermal cells migrated
dorsally, in other regions, the cells were retarded and did not reach the
dorsal ectoderm domain (Fig.
1F). Consistent with abnormal mesoderm spreading, staining for the
dorsally located heart cells by anti-Tinman antibodies at later stages (e.g.
stages 13-14) revealed 1-2 segments that lacked cardiac and pericardial cells
in how germline clone mutant embryos
(Fig. 1G). In addition, such
embryos exhibited 1-2 segments that lacked EVE-positive cells in the dorsal
mesoderm (Fig. 1H,I'),
and staining for Myosin heavy chain (MHC) revealed 1-2 segments in which some
of the more dorsal muscles were missing
(Fig. 1J). Analysis of embryos
labeled simultaneously for both EVE and MEF2 revealed that, in regions in
which cardioblasts (the most dorsal MEF-2-positive cells) were missing, the
distribution of EVE-positive pericardial cells was also deranged, but the
pattern of more-ventral somatic muscles was normal, supporting a defect in
dorsal spreading. However, cells that did migrate dorsally expressed EVE,
indicating that the cells were capable of responding to patterning signals
(Fig. 1I,I'). The double
labeling for EVE and MEF2 was performed sequentially (see Materials and
methods for details), because both antibodies were raised in rabbits.
Additionally, the cardioblasts of the how germline clone embryos were
often not ordered as a single line of cells and the EVE-positive cells were
not spaced-out evenly, as in wild-type embryos
(Fig. 1I, arrow). These
phenotypes might stem from later requirements for HOW in this tissue. Taken together, these results suggest that, in embryos lacking HOW, the mesoderm does not spread correctly and evenly over the ectoderm following gastrulation, resulting in embryos in which 1-2 segments lack dorsal mesoderm structures.
The repressor isoform HOW(L) is expressed during mesoderm spreading
To analyze which of the HOW isoforms is responsible for the phenotype of
mesoderm spreading, we stained wild-type embryos at different developmental
stages with anti-HOW antibodies that recognize both HOW isoforms, and detected
positive staining in the mesoderm that persisted during mesoderm spreading
(Fig. 2). Western analysis of
3-5-hour-old embryos showed that the larger isoform, HOW(L), was predominantly
expressed during the time period of mesoderm spreading (stages 7-9, which
occur during the time period of 3-5 hours), and this was confirmed by mRNA in
situ hybridization with each of the how splice variants
(Fig. 2D-F). This suggests that
the mesoderm phenotype described above resulted from lack of the HOW(L)
isoform, which was previously demonstrated to repress levels of various mRNA
species.
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;
Nabel-Rosen et al., 2002).
Therefore, the mRNA levels of direct HOW targets are expected to be increased
in the how germline clone mutant embryos. To identify mRNA targets of
HOW, we used cDNA microarray hybridization to compare the abundance of mRNA
species extracted from 3-5-hour-old how germline clone embryos with
that of stage-matched wild-type embryos. We used a GFP-marked balancer to
identify homozygous (non-fluorescent) how mutant embryos (which also
lacked maternal HOW). The RNA was extracted from five independent embryo
collections, which were sorted separately each time. This was done to reduce
fluctuations in RNA levels due to slight changes in the age of the collected
embryos. Statistical analysis of the results obtained in the microarray
experiment identified 145 mRNAs that showed increased expression equal to or
above 1.5-fold. To further select for putative direct HOW target mRNAs, we
screened for the presence of a HOW binding sequence at the 3' UTR of
each of the 145 genes (HOW response element, HRE=ACUAA)
(Israeli et al., 2007). This
analysis reduced the number of putative targets to 49 mRNAs. Further sorting
was performed based on the conservation of the HRE sites in Drosophila
pseudoobscura. A database of the 3' UTRs present in D.
Pseudoobscura, and their alignments with the 3' UTRs of the
relevant genes in Drosophila melanogaster, was obtained from A. Stark
(Stark et al., 2005). Selected
genes contained HREs in a conserved sequence along the 3' UTR of D.
melanogaster and D. pseudoobscura or exhibited the HRE at
another location in the 3' UTR. This reduced the number of potential HOW
targets to 32 (see Table 1). We
chose to focus on five genes out of these 32; the sequences of these five
genes indicated possible roles in the process of mesoderm spreading:
miple, falten, CG31638, lap and punt. miple codes for a
secreted heparin binding domain protein (see below); falten encodes a
protein with potential GTPase activity; CG31638 exhibits partial homology to
Myosin (according to protein Blast); LAP (like AP-180) contains an ENTH domain
that binds phosphatidylinositol phosphates (PtdIns) to modulate Clathrin
adaptors in endocytosis; and punt codes for a type II TGFß
receptor. In situ hybridization with probes representing these five genes
revealed that the levels of the first four mRNAs were significantly elevated
in the how germline clone mutant embryos, consistent with the
microarray results (Fig. 3),
whereas punt mRNA did not show a significant elevation in these
mutants (data not shown). Each of these five candidates was also tested for
direct binding to HOW via their 3' UTR using a protein-RNA binding assay
(Nabel-Rosen et al., 1999).
The 3' UTR of each gene was isolated, transcribed, labeled with biotin,
purified and further tested for its ability to co-precipitate with in
vitro-translated HOW(L). The mRNAs of the four genes that were upregulated in
the mutant how embryos in the mesoderm - miple, falten,
CG31638 and LAP - exhibited specific binding to HOW via their
3' UTRs, but did not bind a point-mutated HOW (HOWR185 to C),
which molecularly mimics the severe howe44 allele, which
is incapable of RNA binding (Fig.
3I). The punt 3' UTR did not show binding to HOW,
nor did its mRNA elevate in the how mutant embryos, thus it does not
represent a direct target for HOW activity.
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If HOW repression of these genes is essential for mesoderm spreading, we would expect the aberrant mesoderm spreading phenotype detected in how germline clone mutant embryos to be phenocopied by overexpression of these genes in the mesoderm. To address this possibility, we produced transgenic flies carrying miple, falten and CG31638 under the control of gal4-binding UAS sites. Driving the expression of these proteins (without their 3' UTRs) with the twist-gal4 driver led to various degrees of mesoderm spreading defects (Fig. 4A and data not shown). We decided to further focus on the phenotype obtained by overexpressing miple, because it exhibited the most prominent and consistent mesoderm spreading phenotype (Fig. 4A, compare to wild-type Twist expression in Fig. 1A).
Consistent with a defect in the even spreading of the mesoderm, we also detected loss of pericardial and cardiac cells in 1-2 segments in the embryos overexpressing miple at later developmental stages (Fig. 4B,C). The somatic musculature also showed lack of muscles in 1-2 segments, especially at the dorsal aspects of the embryo (Fig. 4D). Double-labeling with both EVE and MEF2 antibodies (as described above) showed that, in regions of aberrant distribution of EVE-positive cells, we often detected abnormal arrangement of MEF2-positive cells (Fig. 4E,F), supporting a defect in dorsal spreading. However, cells that did migrate dorsally expressed EVE, indicating that the cells were capable of responding to patterning signals. We believe that these phenotypes originated during the early stages of mesoderm spreading when the twist-gal4 driver is expressed at high levels. However, we cannot exclude the possibility that residual expression of Miple by twist-gal4 is maintained at later stages. Although the miple construct was tagged with hemagglutinin (HA), we could not detect the overexpressed Miple-HA by staining with anti-HA antibody. To verify that UAS-miple-HA is indeed expressed following the expression of the twist-gal4 driver, we performed western analysis with embryos overexpressing HA-tagged Miple, using anti-HA antibodies. Only extracts originating from embryos carrying both UAS-miple-HA and twist-gal4 reacted with the anti-HA antibodies (Fig. 4G). We also tested the activity of the putative heparin-binding domain in Miple by incubating the embryo extract with heparin beads, followed by elution with high salt buffer. Western analysis showed that Miple is indeed a heparin-binding protein, because it binds and can be eluted from a heparin column (Fig. 4G).
The 3' UTR of miple contains a single site for HOW binding (at position 800 after the stop codon). This site was mutated from ACUAA into ACGGA, fused to GFP and inserted into a pUAST vector suitable for expression in SR+ cells. Similarly, a wild-type 3' UTR was fused to GFP and subcloned into the pUAST vector. SR+ cells were transfected with GFP-miple 3' UTR or with GFP-miple 3' UTR* (mutated) together with HOW(L), and the GFP levels were monitored by western analysis following 2 days of transfection. Consistently, the GFP levels were reduced when the wild-type GFP-miple 3' UTR but not mutated 3' UTR was present (a representative western blot of three experiments is shown in Fig. 4H). These results favor a direct effect of HOW on miple levels via its binding to the HRE at the 3' UTR of miple mRNA.
In summary, based on microarray data, we identified three potential targets recognized by HOW that, when overexpressed in the mesoderm without their 3' UTRs, led to mesoderm spreading defects that mimic the how mutant phenotype.
Overexpression of Miple activates the MAPK pathway in the migrating mesoderm
Mesoderm spreading in the Drosophila embryo depends on the
activity of the FGF receptor, HTL, and its two FGF8-like ligands, Thisbe and
Pyramus (Gryzik and Muller,
2004; Stathopoulos et al.,
2004). In wild-type embryos at stages 7-9, HTL-dependent
activation of MAPK, as visualized by staining with anti-dpERK antibody, is
restricted to the most dorsal mesodermal cells
(Gabay et al., 1997) (and
Fig. 5B,D). The basis for this
local MAPK activation is not clear. To understand the relationship between
Miple expression and MAPK activation, Miple was overexpressed in the mesoderm
by the twist-gal4 driver. Overexpression led to uneven dpERK staining
scattered over many mesodermal cells, independent of their position along the
dorsoventral axis (Fig. 5F-H).
This scattered activation might form the basis for the defects in mesoderm
spreading observed in the embryos overexpressing Miple. Importantly, a
scattered MAPK activation was also detected in how germline clone
embryos (Fig. 5J-L). This
effect might stem from the high expression levels of miple in these
embryos.
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) (Fig.
6A). This number reflects the sum of the activation of both the
EGFR and HTL signaling pathways. Higher activation of the pathway (e.g.
following overexpression of activated RAS, activated HTL or activated EGFR, or
in argos mutants) induces elevated numbers of EVE-expressing cells in
the dorsal mesoderm, driven by mef2-gal4
(Carmena et al., 1998).
We detected five to six EVE-positive cells in most of the embryo segments
in embryos overexpressing Miple driven by the mef-2-gal4 driver
(Fig. 6B). This elevation in
the number of the EVE-positive cells is a further indication of the ability of
Miple to activate the MAPK pathway in the dorsal mesodermal cells. To further
test the possibility that MAPK activation is mediated by the HTL receptor, we
examined the ability of Miple to induce the production of a higher number of
EVE-positive cells in htl mutant embryos. In htl mutants
there was a complete loss of the EVE-positive cell clusters, and
overexpression of Miple by the mef2-gal4 driver did not restore EVE
expression (Fig. 6C). This
result is consistent with the possibility that Miple-dependent elevation of
the dpERK, as well as the elevation in the number of EVE cells, is mediated by
the HTL receptor (Gryzik and Muller,
2004). However, we cannot exclude the possibility that the
htl mutant cells do not express EVE, due to their failure to reach
dorsal positioning.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a
process that is required for the spatial patterning of the mesoderm layer by
ectoderm-derived signals. This phenotype could not result from the earlier
effect of extra cell division during gastrulation detected in the how
germline clone embryos, because mesoderm invagination and gastrulation were
eventually completed in these embryos. Furthermore, mesoderm spreading was
unaffected in tribbles (trbl) mutant embryos, which exhibit
a similar defect of extra cell divisions during gastrulation that leads to
delayed and unsynchronized mesoderm invagination (T.V., unpublished data).
HOW regulates the mRNA levels of maternally contributed and zygotic genes in the mesoderm
Regulation of mesoderm-specific mRNA levels by HOW might contribute to the
spatial and temporal control of gene expression during mesoderm spreading. The
genome analysis that we have performed was designed to identify mRNAs whose
levels might be directly controlled by the repressor isoform HOW(L) in the
mesoderm. Such targets should be normally repressed to enable even spreading
of the mesoderm. Three out of the four HOW targets identified in this screen,
namely falten, CG31638 and LAP (CG2520) are contributed
maternally (in situ results, H.T.-K. and T.V., unpublished results), and
therefore HOW(L)-dependent repression in the mesoderm might be essential for
reducing their levels in this tissue to enable proper mesoderm spreading. This
scenario is supported by the defective mesoderm spreading induced by
overexpression of falten and CG31638. Miple does not appear
to be maternally contributed according to expression data [from Berkeley
Drosophila Genome Project (BDGP)] and in situ analysis. It is not
clear, however, which transcription factor is responsible for miple
induction. Because miple mRNA was detected in mesoderm derivatives at
stage 11, it might be induced by mesoderm-specific transcription factors such
as Twist, MEF2 and/or Tinman, which are expressed in the mesoderm during
spreading. In that case, to abrogate the effects of Miple, it would be
necessary to block miple expression during mesoderm spreading. Our
data suggest that this is the role of HOW(L), because in its absence,
miple mRNA is significantly elevated in the mesoderm. Thus, HOW(L) in
the mesoderm of gastrulating embryos is necessary to reduce maternal mRNA
expression and, in addition, to reduce the levels of gene products whose
expression is not compatible with early mesoderm development, but might be
required shortly after the process of mesoderm spreading has been completed.
Thus, HOW(L) is essential to enable temporal morphogenetic processes in the
mesoderm during its spreading over the ectoderm (see summary in
Fig. 7).
Possible involvement of Miple in HTL-dependent MAPK activation
Miple was further analyzed because its vertebrate homologs, midkine and
pleiotropin, are involved in cell migration and are associated with receptor
tyrosine kinase (RTK) signaling (Stoica et
al., 2002). Therefore, its downregulation by HOW(L) might
contribute to the restricted dorsal activation of the HTL-dependent signaling
during mesoderm spreading. Moreover, the putative heparin-binding motif of
Miple could affect the affinity of the HTL ligands to the HTL receptor,
thereby modulating the strength of HTL-dependent signaling.
Indeed, our findings suggest that downregulation of Miple levels in the
mesoderm is essential for correct mesoderm spreading, because Miple
overexpression led to impaired mesoderm spreading. The disordered pattern of
MAPK phosphorylation (detected by anti-dpERK antibody) observed following
Miple overexpression might be the primary cause for the mesoderm spreading
defect. In wild-type embryos, MAPK activation was detected only at the most
dorsal cells of the spreading mesodermal cells. The mechanism by which this
spatial MAPK activation is achieved is not clear. It has been suggested that
MAPK activation takes place only in cells that directly contact the ectoderm
(Wilson et al., 2005). In that
case, Miple might trigger prolonged mesoderm-ectoderm cell contacts and this
could delay mesoderm spreading. Indirect evidence, especially the observation
that overexpression of an activated form of HTL does not lead to an ectopic
dpERK signal in the entire mesoderm, led to the suggestion that a constitutive
inhibitory input of MAPK activation is present in mesoderm cells
(Wilson et al., 2005). This
inhibitory activity was suggested to be overcome only in cells that form close
contact with the ectoderm. It is unlikely that the role of Miple is to
counteract this inhibitory signal, because overexpression of Miple has an
effect not only on MAPK activation in early mesoderm spreading but also on the
late HTL-dependent signaling in the dorsal EVE-positive cells, in which this
inhibitory signal has not been implicated. We therefore favor the possibility
that Miple enhances HTL signaling, and that this enhancement is reflected by
MAPK activation in both early and later stages of mesoderm development.
The elevation of the dpERK signal detected following overexpression of Miple might be mediated by HTL activation, because no other RTK has been shown to be expressed in the mesoderm at the stage of gastrulation. Although the increased number of EVE-expressing cells detected in the dorsal mesoderm clusters following overexpression of Miple is eliminated in embryos lacking active HTL receptor, we cannot exclude the possibility that the lack of EVE-positive cells in the dorsal mesoderm might stem from the failure of the htl mutant mesoderm cells to reach the most dorsal locations.
In vertebrates, midkine and pleiotrophin have been identified by phage
display as potential high-affinity ligands for the human receptor tyrosine
kinase ALK (Stoica et al.,
2002). Although we do not exclude the possible role of Miple in
ALK-dependent signaling, ALK is not expressed in the early stages of mesoderm
spreading, and does not overlap with the EVE-expressing clusters
(Lin et al., 1999); thus, it
is unlikely to affect the increased number of EVE-expressing cells. Receptor
tyrosine phosphatase-zeta has been implicated as a putative pleiotrophin
receptor (Milev et al., 1998).
If a similar receptor exists in Drosophila, it might respond to Miple
overexpression by altering MAPK levels.
It is possible that the heparin-binding domain of Miple enhances the
activity of the HTL ligands. In vertebrates, heparin-containing proteins act
as co-ligands to FGFs by inducing their dimerization
(Ornitz, 2000;
Thisse and Thisse, 2005). We
confirmed that Miple is a heparin-binding protein, because it binds
specifically to a heparin column. The contribution of heparan sulfate
proteoglycans to proper mesoderm spreading in Drosophila had been
demonstrated by the requirement of two enzymes, Sugarless and Sulfateless, for
this process (Lin et al.,
1999). Moreover, a genetic interaction between mutations in each
of these enzymes and the two FGF receptors HTL and Breathless (BTL) was
demonstrated (Lin et al.,
1999). Overexpression of Miple during mesoderm spreading might, on
the one hand, compete with endogenous heparan sulfate proteoglycan for Thisbe
and Pyramus binding, and thus could inhibit their ability to activate the
HTL-dependent signaling. On the other hand, Miple might also activate the HTL
pathway by replacing the endogenous heparan sulfate proteoglycan that is
normally involved in activation of the FGF8-like ligands. These dual
activities might interfere with the normal dorsal-restricted MAPK activation
in the mesoderm.
In wild-type embryos, miple is downregulated by HOW(L) in the
mesoderm; however, its mRNA expression is detected at later developmental
stages, including in the ventral midline and in the brain
(Englund et al., 2006). In
midline glial cells, a second FGF receptor, Breathless, has been implicated in
the promotion of cell migration at stages 12-13 of embryonic development
(Klambt et al., 1992). At this
stage, Miple might contribute to the spatial and temporal control of
Breathless activation. Such a scenario must be tested directly in
miple mutant embryos.
Although the mesoderm spreading phenotype of how germline clone
embryos is not fully penetrant and is detected in only a few segments, the
contribution of HOW activity is crucial because of the secondary effect that
non-homogenous mesoderm spreading exhibits on the development of the heart and
dorsal somatic mesoderm. HOW(L) appears to function in the mesoderm as a
safety mechanism to prevent mis-expression of either maternally contributed
genes or genes whose early transcriptional activation in the mesoderm might
interfere with the normal development of the mesoderm. An example of similar
repressive activity of HOW(L) is its activity in the reduction of
string levels in the gastrulating embryo to prevent premature cell
division during mesoderm invagination
(Nabel-Rosen et al.,
2005).
In summary, this study reveals the crucial function of the STAR family member HOW(L) in enabling proper mesoderm development via the repression of specific mRNAs provided either maternally, or expressed prematurely in a specific tissue. HOW(L) and its vertebrate homolog, QKI5, are expressed in wide range of tissues during early developmental stages, and might function in these tissues in a similar fashion to enable proper embryonic and tissue development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: IGBMC, Department of Developmental Biology, 1, Rue Laurent
Fries, Illkirch 67404, C. U. Strasbourg, France
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