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RESEARCH COMMUNICATION
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
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Abstract |
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 Top Abstract Results and DiscussionMaterials and methodsAcknowledgmentsReferences |
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[Keywords: zebrafish; endothelial; artery; Plcg1; Vegf]
Received January 3, 2003; revised version accepted April 14, 2003.
). However, recent work demonstrates that a number
of signaling pathways specifically induce the differentiation of arterial
endothelial cells before circulation commences (Lawson et al.
2001,
2002;
Mukouyama et al. 2002;
Visconti et al. 2002). In
particular, vascular endothelial growth factor (Vegf) is required for the
development of arteries (Mukouyama et al.
2002; Stalmans et al.
2002) and the differentiation of arterial endothelial cells
(Lawson et al. 2002;
Mukouyama et al. 2002) during
embryonic development in both zebrafish and mouse. Despite the wealth of
information concerning components of the Vegf signaling pathway in endothelial
cell lines (Zachary and Gliki
2001), little is known about the downstream factors that are
necessary in vivo during blood vessel formation and arterial development. In
this study, we have taken advantage of transgenic zebrafish with fluorescently
labeled blood vessels (Lawson and Weinstein
2002a) in conjunction with a forward genetic screen to identify a
gene within the Vegf signaling pathway that is required for arterial
development. We find that y10 mutant zebrafish embryos display
defects in the formation of arteries, but not veins, and are deficient in the
expression of artery-specific markers such as ephrin-B2a, similar to
zebrafish and mouse embryos lacking Vegf function
(Lawson et al. 2002;
Mukouyama et al. 2002;
Stalmans et al. 2002). We show
that y10 encodes the zebrafish homolog of phospholipase C
gamma-1 (plcg1) a known downstream effector of numerous receptor
tyrosine kinases (Rhee 2001).
Using plcg1y10 mutant embryos together with microinjection
of vegf mRNA, we are able to demonstrate that Plcg1 is required for
normal Vegf function. Taken together, our observations reveal a specific
requirement for Plcg1 during formation and differentiation of arteries, and
indicate that Vegf is the primary growth factor that utilizes Plcg1 signaling
during embryonic development. |
Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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). As a
screening tool, we utilized zebrafish harboring a fli1:enhanced
green fluorescent protein transgene [TG(fli1:egfp)y1;
Lawson and Weinstein 2002a] to
enable fluorescent visualization of developing blood vessels in vivo. Using
this strategy, we identified the zebrafish y10 mutant. In both
wild-type and homozygous y10 mutant embryos, overall morphology is
normal at 30 h post fertilization (hpf;
Fig. 1a,d). The somites and
notochord, both of which are required for normal development of the trunk
blood vessels (Brown et al.
2000; Parsons et al.
2002), appear normal by light microscopy (data not shown). Primary
segmental vessels, which sprout from the dorsal aorta, are formed by 30 hpf in
wild-type TG(fli1:egfp)y1 sibling embryos
(Fig. 1b). These vessels are
absent in TG(fli1:egfp)y1 embryos mutant for y10
(Fig. 1e). This phenotype is
not a general defect in sprouting blood vessel growth, or angiogenesis, as
secondary segmental vessels, which sprout from the posterior cardinal vein by
50 hpf and contribute to parachordal vessels in wild-type embryos
(Fig. 1c), are apparent in
y10 mutant embryos (Fig.
1f). The midcerebral vein, a blood vessel that forms adjacent to
the midbrain–hindbrain boundary via angiogenesis, also forms normally in
y10 mutant embryos (Fig.
1g,h). In zebrafish embryos, the lateral dorsal aortae connect the
outflow tract of the heart to the trunk circulatory network and fuse in the
anterior trunk to give rise to a single dorsal aorta
(Isogai et al. 2001). In
wild-type embryos, the lateral dorsal aortae are lumenized by 30 hpf
(Fig. 1i), whereas in
y10 mutant embryos, these blood vessels appear thin and fail to
lumenize (Fig. 1j). In
contrast, the branches of the posterior cardinal vein are normal in mutant
embryos (Fig. 1, cf. i and j).
These observations indicate that y10 mutant embryos display a
specific defect in the formation of arteries, whereas the development of veins
appears unaffected. Consistent with these defects in blood vessel morphology,
y10 mutant embryos fail to exhibit head or trunk circulation and have
severe peri-cardial edema by 2 d postfertilization (dpf; data not shown).
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In addition to defects in artery formation, y10 mutant embryos
exhibit a loss of artery-specific gene expression in the dorsal aorta. By 24
hpf, wild-type embryos express ephrin-B2a in the dorsal aorta
(Fig. 2a), whereas in
approximately half of y10 mutant embryos, ephrin-B2a
expression is strongly reduced or absent (Figs.
2b,
4g, below). Dorsal
aorta-specific expression of notch5
(Fig. 2c), an important
regulator of arterial differentiation downstream of Vegf (Lawson et al.
2001,
2002), is also reduced or
absent at a frequency similar to ephrin-B2a
(Fig. 2d; data not shown). We
find that general endothelial cell markers, such as flk1 and
tie1 are expressed in the major trunk blood vessels, and their
expression appears to be mildly reduced in y10 mutant embryos
(Fig. 2e–h). Expression
level of fli1 transcript
(Thompson et al. 1998) in the
dorsal aorta and posterior cardinal vein does not appear to be affected in
y10 mutant embryos (Fig.
2i,j), consistent with the equivalent level of fli1:egfp
transgene expression in wild-type and mutant embryos
(Fig. 1). The semipenetrant
nature of the loss in artery marker gene expression is consistent with our
previous observations that numerous pathways likely contribute to the
differentiation of arterial endothelial cells in the zebrafish dorsal aorta
during embryogenesis (Lawson et al.
2002). In addition, the appearance of endothelial cells in the
position of the dorsal aorta indicates a failure of these cells to undergo
arterial differentiation rather than their inability to migrate to the correct
location.
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The phenotype of y10 mutant embryos is similar to zebrafish or
mouse embryos lacking Vegf function
(Lawson et al. 2002;
Mukouyama et al. 2002;
Stalmans et al. 2002).
However, y10 does not map to vegf or its receptor,
flk1 (data not shown). To identify the gene responsible for the
y10 mutant phenotype, we performed bulk segregant analysis on
wild-type and mutant embryos and mapped y10 to within 0.9 cM of Z6376
on linkage group 23 (Fig. 3a).
By comparing the available zebrafish meiotic, radiation hybrid, and heat-shock
maps
(http://zfin.org/cgi-bin/webdriver?MIval=aa-mapperselect.apg),
we found that several expressed sequence tags (EST) with homology to rat
plcg1 were located in this region (data not shown). Because Plcg1
activity can be induced through the Vegf receptor
(Takahashi et al. 2001) and
the artery defect in y10 mutant embryos is reminiscent of loss of
Vegf phenotypes in zebrafish (Lawson et
al. 2002) and mouse (Mukouyama
et al. 2002; Stalmans et al.
2002) embryos, plcg1 represented a viable candidate gene
for y10. Mapping of polymorphic markers in the plcg1 gene
indicated that it is linked tightly to y10
(Fig. 3a). In addition,
plcg1 mRNA is expressed in the vasculature of zebrafish embryos at 24
hpf (Fig. 3b,c) in both the
dorsal aorta and posterior cardinal vein, as well as in segmental vessel
sprouts (Fig. 3c).
|
Sequence analysis of the plcg1 gene from wild-type and mutant
embryos revealed that a consensus splice acceptor site at the intron
1–exon 2 boundary of plcg1 is eliminated by a G-to-A transition
in mutants (Fig. 3d) and is
associated with variable size deletions in the plcg1 transcript
(Fig. 3e). The majority of
these deletions result in a frame shift into a premature stop codon and
truncate the Plcg1 protein at amino acid 73
(Fig. 3e). Less than 30% of the
transcripts lack only three nucleotides and result in loss of I72
and change of D73 to N. Both of these amino acids are identical in
rat, human, and zebrafish Plcg1 (data not shown) and are located in the
pleckstrin homology domain, which is important for growth factor-induced
membrane localization of Plcg1 (Falasca et
al. 1998). To confirm that the plcg1 exon 1–exon 2
junction was affected in plcg1y10 mutant embryos, we
designed an antisense Morpholino oligonucleotide against the exon
1–intron 1 boundary sequence to interfere with plcg1 pre-mRNA
splicing (Draper et al. 2001).
Wild-type TG(fli1:egfp)y1 embryos injected with a
scrambled control Morpholino display a fully formed primary segmental blood
vessel network by 32 hpf (Fig.
3f; 111 of 111 in 3 experiments) and normal circulation (86 of 89
in 3 experiments; data not shown). In contrast,
TG(fli1:egfp)y1 embryos injected with the plcg1
Morpholino display reduced numbers of segmental vessel sprouts
(Fig. 3g; 52 of 106 with <5
segmental vessels in 3 experiments) and loss of circulation (62 of 83 without
circulation in 3 experiments; data not shown). To confirm that loss of
plcg1 function was responsible for the vascular defects in
y10 mutant embryos, we injected mRNA encoding a myc-tagged form of
Plcg1 into embryos derived from a plcg1y10 incross. The
resulting embryos were then scored for presence or absence of segmental blood
vessels or circulation at 30 hpf and subsequently genotyped using the
2182–2185 CA marker. In uninjected embryos, all wild-type siblings
display fully formed segmental blood vessels or active circulation by 30 hpf,
whereas mutants do not (data not shown;
Fig. 3i,j). In contrast, we
find that 73% of mutant embryos that were injected with mtplcg1 mRNA
form segmental blood vessels (Fig.
3h,i) and 85% of embryos with a mutant genotype display
circulation at 30 hpf (Fig. 3j;
data not shown). Together, these results establish definitively that the gene
responsible for the y10 mutant phenotype encodes the zebrafish
homolog of Plcg1.
Biochemical studies have demonstrated that Plcg1 can be an effector of Vegf
signaling in endothelial cell lines
(Takahashi and Shibuya 1997;
Takahashi et al. 2001), and
evidence from mice lacking plcg1 indicates that it is required for
blood vessel development (Liao et al.
2002). However, there is no definitive genetic evidence to confirm
that Plcg1 functions downstream of Vegf in vivo. The phenotype of zebrafish
plcg1y10 mutant embryos and the similarity to mice
(Mukouyama et al. 2002;
Stalmans et al. 2002) or
zebrafish (Nasevicius et al.
2000; Lawson et al.
2002) lacking Vegf function, suggests that Plcg1 is an important
downstream effector of Vegf during artery development. The availability of
plcg1y10 mutant zebrafish allowed us to address whether or
not this was the case. To determine whether Plcg1 was required for Vegf
function, we injected vegf121 mRNA into embryos
derived from plcg1y10 heterozygous carriers. We then
assayed for flk1 or ephrin-B2a expression in injected
embryos, followed by PCR analysis to identify wild-type and mutant embryos. We
have found previously that exogenous Vegf induces the expression of both of
these markers, although induction of ephrin-B2a requires the Notch
signaling pathway, whereas induction of flk1 does not
(Lawson et al. 2002).
Consistent with previous observations
(Liang et al. 2001;
Lawson et al. 2002), we find
that nearly all wild-type embryos injected with
vegf121 mRNA display increased levels of
flk1 expression in their trunk blood vessels
(Fig. 4a,c). In contrast,
ectopic Vegf121 fails to induce flk1 expression in
plcg1y10 mutant embryos
(Fig. 4b,c). Similarly, nearly
all wild-type embryos injected with vegf121 mRNA
also exhibit ectopic expression of ephrin-B2a in the posterior
cardinal vein (Fig. 4f,h).
However, plcg1y10 mutant embryos injected with mRNA
encoding Vegf121 display either normal or reduced
ephrin-B2a expression (Fig.
4d,e,h) in proportions similar to uninjected mutant embryos
(Fig. 4h). These data indicate
that Plcg1 function is required for induction of both Notch-dependent and
Notch-independent signaling downstream of Vegf in vivo and is consistent with
biochemical evidence that shows Plcg1 functions proximal to the Vegf receptor,
Flk1 (Takahashi et al.
2001).
Although Plcg1 is know to function downstream of numerous receptor tyrosine
kinases (Rhee 2001;
Wilde and Watson 2001), we
find that the primary defects in zebrafish plcg1y10
mutants are restricted to the vasculature within the embryo and are remarkably
specific to a subset of blood vessels. The similarity of the
plcg1y10 mutant phenotype to zebrafish embryos lacking
Vegf (Nasevicius et al. 2000;
Lawson et al. 2002) and the
failure of plcg1y10 mutant embryos to respond to exogenous
Vegf (this study) indicate that Plcg1 function is required downstream of Vegf
to drive arterial development. This requirement for Plcg1 to mediate Vegf
signaling during vascular development appears to be conserved in vertebrates,
as mice lacking plcg1 also display severe defects in blood vessel formation
(Liao et al. 2002) during
embryogenesis, similar to those associated with loss of Vegf
(Carmeliet et al. 1996).
However, mice lacking either Plcg1 or Vegf display a more severe phenotype,
including failure to express general endothelial cell markers indicative of an
early block in the formation of endothelial cells from mesodermal precursors,
than in zebrafish embryos with comparable loss of function
(Nasevicius et al. 2000;
Lawson et al. 2002). The
reason for these differences is unknown at this time, but may reflect
additional redundancy in the Vegf-signaling pathway in zebrafish that allows
for early stages of endothelial progenitor cell development. Therefore, only
defects in arterial development, which have been observed in mice lacking
specific isoforms of Vegf (Stalmans et al.
2002), are observed in the blood vessels of
plcg1y10 mutant embryos.
Despite apparent species-specific differences in Vegf sensitivity during
embryonic development, recent evidence indicates that Vegf is an important
signal that specifically drives development of the arterial system in both
zebrafish and mice (Lawson et al.
2002; Mukouyama et al.
2002; Stalmans et al.
2002; Visconti et al.
2002) and does so through the action of the Notch signaling
pathway (Lawson et al. 2001,
2002;
Lawson and Weinstein 2002b).
However, little is known about the downstream effectors of Vegf that mediate
this effect on the developing vasculature in vivo. With the ability to perform
screens for mutant blood vessel phenotypes using
TG(fli1:egfp)y1 embryos, we are able to demonstrate that
the zebrafish is an ideal model to dissect this signaling pathway. The studies
presented here validate this forward genetic approach by describing the
identification of plcg1 as a necessary component of the arterial
differentiation pathway downstream of Vegf. It is likely that identification
and characterization of additional mutants with similar phenotypes will yield
further insight into the components of the Vegf pathway required for this
particular aspect of blood vessel formation.
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Materials and methods |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Zebrafish were maintained and bred as described elsewhere
(Westerfield 1993). The
TG(fli1:egfp)y1 transgenic line has been described
previously (Lawson and Weinstein
2002a).
Mapping
To facilitate genetic mapping, the
TG(fli1:egfp)y1;plcg1y10 founder
female was crossed to the wild-type TL line. Identified mutant carriers
derived from this map cross were incrossed at sexual maturity, and their
embryos were scored on the basis of the presence or absence of circulation at
2 dpf. Bulk segregant analaysis was performed using a 192 marker panel of
available CA markers. The list of markers in this panel is available upon
request. Genomic DNA isolation and PCR, as well as cycle sequencing of PCR
products to identify polymorphisms were performed as described elsewhere
(Roman et al. 2002).
Oligonucleotide sequences for Z130, Z6376, and Z4003 are available at
http://zebrafish.mgh.harvard.edu/mapping/ssr_map_index.html.
Oligonucleotide primers for PCR to identify additional polymorphisms were
designed on the basis of available zebrafish genomic sequence obtained through
Blastn searches of trace data from the Sanger Institute
(http://trace.ensembl.org/perl/ssahaview?server=danio_rerio)
using zebrafish plcg1 coding sequence. The oligonucleotide sequences
are as follows: 2182, 5'-GTTATGGC TAAATTGAGACTCA-3'; 2185,
5'-CTTACACTCGATGCATCTGC-3'; 2115,
5'-GATGACAATGTAGACATGCAAT-3'; and 2116, 5'-GTCT
GCTCGGGTGGACTTTAA-3'. The sequence of the plcg1 intron
1–exon 1 junction was obtained using the Universal Genomewalker kit
according to manufacturer's instructions (BD Biosciences Clontech) with
wild-type and mutant genomic DNA as starting material and the following
oligonucleotides: 5'-TAGAGGATGACGAAGCAGTGGGCCTGGT-3' and
5'-GGCCGGGTCGTGACGTCCTCCACATAT-3' for PCR. Resultant PCR fragments
were sequenced directly. Polymorphism analyses and sequence comparisons were
performed using SeqMan alignment software (DNASTAR, Inc.).
Injections
Morpholinos (Gene Tools) and mRNA were injected into embryos as described
elsewhere (Lawson et al.
2002). Sequence for the plcg1 Morpholino is
5'-ATTAGCATAGGGAACTTACTTTCG-3'. Genomic DNA from injected embryos
was isolated as described elsewhere (Roman
et al. 2002) and genotyped using oligonucleotide primers 2182 and
2185.
Imaging
Imaging of blood vessels in TG(fli1:egfp)y1 embryos was
performed using a multiphoton laser or confocal laser microscope as described
previously (Lawson and Weinstein
2002a). Transmitted light images were obtained with a Leica MZ12
or Zeiss Axiophot2 microscope equipped with a Pro-gRes mF digital camera
(Jenoptik).
Cloning
Sequences encompassing the plcg1 3' UTR and start codon were obtained using available zebrafish EST (GenBank accession nos. AW510269 [GenBank] , BI979386 [GenBank] , AW281801 [GenBank] , BM778133 [GenBank] , AW279908 [GenBank] , AI385081 [GenBank] , and AI397215 [GenBank] ) and genomic trace sequence. The plcg1 start codon was obtained through a tBlastn search of zebrafish genomic traces using the rat Plcg1 amino acid sequence. The full-length coding sequence of zebrafish plcg1 was amplified by PCR using cDNA from wild-type embryos using the following oligonucleotides: 5'-TTACTAGTGAACAAACAGGGGAAATG-GCT-3' and 5'-TTTCTAGACTGCTCGGTTTACGCTCGGTTAT-3' and TOPO-cloned into pCR2.1 (Invitrogen) to give pCRplcg1CDS. Additional plcg1 cDNA sequence was obtained or confirmed by cycle sequencing of reverse transcriptase PCR (RT–PCR) products using the Big-Dye sequencing kit and an ABI 310 capillary sequencer according to manufacturer's instructions (Applied Biosystems). A fragment containing the full-length plcg1 coding sequence was digested from pCRplcg1CDS and cloned into pCS2+ to give pCSplcg1CDS. Subsequently, a 6x myc epitope tag was cloned in frame of the 5' end of plcg1 coding sequence in pCSplcg1 CDS to give pCSMTplcg1. pCSMTplcg1 was digested with NotI and used as template for mRNA synthesis using the mMessage mMachine kit according to manufacturer's protocols (Ambion).
Whole mount in situ hybridization
Antisense mRNA probes for ephrin-B2a, notch5, and fli1
were prepared as described (Lawson et al.
2001). To derive a plcg1 riboprobe, pCRplcg1CDS was
linearized with HindIII and transcribed using T7 polymerase.
Whole-mount in situ hybridization was performed as described elsewhere
(Hauptmann and Gerster
1994).
GenBank accession numbers
Zebrafish plcg1 coding sequence, AY163168 [GenBank] ; plcg1 intron/exon containing CA repeat 2182–2185, AY163169 [GenBank] ; zebrafish plcg1 intron 1–exon 2 boundary, AY163170 [GenBank] .
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18USC section 1734 solely to indicate this fact.
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Footnotes |
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2 E-MAIL
Nathan.Lawson{at}umassmed.edu;
FAX (508) 856-5460.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1072203.
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References |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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T. Xiao, T. Roeser, W. Staub, and H. Baier A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection Development, July 1, 2005; 132(13): 2955 - 2967. [Abstract] [Full Text] [PDF]
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Y. Sakurai, K. Ohgimoto, Y. Kataoka, N. Yoshida, and M. Shibuya Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice PNAS, January 25, 2005; 102(4): 1076 - 1081. [Abstract] [Full Text] [PDF]
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M. Tsang, S. Maegawa, A. Kiang, R. Habas, E. Weinberg, and I. B. Dawid A role for MKP3 in axial patterning of the zebrafish embryo Development, June 15, 2004; 131(12): 2769 - 2779. [Abstract] [Full Text] [PDF]
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S. Isogai, N. D. Lawson, S. Torrealday, M. Horiguchi, and B. M. Weinstein Angiogenic network formation in the developing vertebrate trunk Development, November 1, 2003; 130(21): 5281 - 5290. [Abstract] [Full Text] [PDF]
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A
transition in the conserved AG splice acceptor sequence is highlighted in red.
(e) Variable size deletions found within the plcg1 coding
sequence in cDNA derived from y10 mutant embryos. Numbers on the
left indicate the frequency of particular transcripts (n =
11). Green boxes denote alternating codons of wild-type plcg1 coding
sequence. Red boxes indicate premature stop codons present in transcripts
i–iii. Light blue box in transcript iv indicates a frameshift; this
transcript encodes an additional 53 amino acids before terminating in a
premature stop codon. Black vertical line indicates position of exon
1–exon 2 splice junction. (f,g) Confocal laser microscope
images; lateral views of the trunk; anterior is to the left and dorsal is up.
Red brackets indicate lumen of the dorsal aorta. (f) Wild-type
TG(fli1:egfp)y1 embryo injected with 15 ng of a control
scrambled morpholino that displays fully formed segmental vessels (arrows) and
dorsal longitudinal anastomotic vessel (arrowhead). (g)
TG(fli1:egfp)y1 embryo injected with 15 ng of
plcg1 morpholino with no segmental vessel sprouts.
(h–j) Embryos derived from a
plcg1y10/+ incross were injected with 100 pg of
myc-epitope-tagged plcg1 (mt-plcg1) mRNA, scored for the
presence or absence of trunk circulation or segmental vessels (SeV) at
30–32 hpf, and then genotyped using oligonucleotide primers 2182 and
2185. (h) Epifluorescence micrograph of a
TG(fli1:egfp)y1 embryo mutant for
plcg1y10 with partial rescue of segmental vessel (arrow)
and dorsal logitudinal anastomotic vessel (arrowhead) formation. (i)
MTplcg1 rescues SeV formation in plcg1y10 mutant embryos.
Results represent average of two experiments. Partial SeV formation was scored
as rescue. (j) MTplcg1 rescues circulation in
plcg1y10 mutant embryos. Results represent the average
from three experiments.