First published online August 18, 2003
doi: 10.1242/10.1242/dev.00635
The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis and axis patterning
Diana Escalante-Alcalde1,*,
Lidia Hernandez1,
Hervé Le Stunff3,
Ryu Maeda2,
Hyun-Shik Lee2,
Jr-Gang-Cheng1,
Vicki A. Sciorra4,
Ira Daar2,
Sarah Spiegel3,
Andrew J. Morris4 and
Colin L. Stewart1,
1 Cancer and Developmental Biology Laboratory, Division of Basic Science,
National Cancer Institute, Frederick, MD 21702, USA
2 Regulation of Cell Growth Laboratory, Division of Basic Science, National
Cancer Institute, Frederick, MD 21702, USA
3 Department of Biochemistry and Molecular Biophysics, Medical College of
Virginia Campus, Virginia Commonwealth University, Richmond, VA
23298,USA
4 Department of Cell and Developmental Biology, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7090, USA
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Fig. 1. Embryonic expression pattern of LPP genes. LPP1 in situ
hybridization in an E9.5 embryo (left sense, right antisense) with a uniform
ubiquitous expression (purple color). (B) LPP2 in situ hybridization
in an E9.5 embryo (left sense, right antisense). LPP2 is weakly but
still widely expressed (purple color). (C-K) LPP3 expression in
LPP3-IRESlacZ embryos as revealed by ß-galactosidase
staining (blue). (C,D) E6.5 embryos showing expression in the extra-embryonic
ectoderm (Ex). (E,F) In E7.5 embryos LPP3 is expressed in the
anterior (a) domain of the embryo and extra-embryonic membranes. (E) lateral
and (F) frontal views. ve, visceral endoderm; ch, chorion. (G,H) E8.0 embryo
showing expression of LPP3 in (G) the chorion and anterior domain of
the embryo and (H) around (arrows) the node (n) and in the tip of the
allantois (all). (I) E8.5 embryo showing strong LPP3 expression in
the allantois (all) and paraxial mesoderm. (J) E9.5 embryo showing expression
in the somites (s), developing gut (*) and the chorio-allantoic
placenta (p). (K) E10.5 embryo showing LPP3 expression in the limb
with strongest expression in the apical ectodermal ridge (AER) and continued
expression in the umbilical cord (u).
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Fig. 2. Inactivation of LPP3 by homologous recombination in ES cells. (A)
Gene targeting strategy. The exon containing the 3rd outer loop,
containing part of the catalytic domain (box), was deleted (top). The
structure of the wild-type allele (middle) and targeted allele (bottom) after
homologous recombination are shown. The fragments used for confirming 5'
and 3' recombination as well as the location of the primers used for PCR
genotyping (arrows) are indicated. (B) LPP3 genotyping by Southern
blot. BamHI and BglI digested DNAs were tested with 5'
and 3' probes respectively. (C) Genotype of embryos by PCR. DNA samples
of yolk sacs were used for amplification of mutant and wild-type allele
products using the set of 3 primers indicated in A. Wild-type product = 302
bp; mutant product = 500 bp. (D) Northern blot of embryoid body (EBs) total
RNA shows the presence of a smaller transcript (-177 bp) in homozygous mutant
cells resulting from the deletion of the exon. (E) Western blot of primary
cultured cells revealed a reduction in the levels of LPP3 in heterozygous
cells (compare to wild-type) and the absence of any intact protein in
homozygous mutant cells. The 36 kDa upper band corresponds to the glycosylated
form of the enzyme. (F) PKC phosphorylation in heterozygous and homozygous
mutant EBs cultured for 12 days. A phospho (pan)-PKC antibody was used to
indirectly measure PKC activation. A reduction of around 50% phospho-pan PKC
was observed in the homozygous compared with the heterozygous tissues. Actin
was used as a control for amount of protein loaded.
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Fig. 3. Phenotype of LPP3-deficient embryos. (A,B) E9.5 wild-type embryo
showing the vascularization of the yolk sac (A, arrows), and normal embryonic
development at this stage (B); the embryo has turned and the allantois has
contacted the chorion. In this individual the connection was lost because of
the removal of the extra-embryonic membranes. (C,D) E9.5 homozygous null
sibling of the embryo shown in (A,B). The extra-embryonic membranes appear
thin, pale, with an anemic appearance and no indication of large blood vessel
formation (C). The embryo was smaller and developmentally delayed. The most
evident malformation is the abnormal development of the allantois (all). (E) A
unique LPP3-/- embryo (right) recovered at E10 showing an
advanced developmental progression. Despite an almost normal appearance, the
allantois (all) of this embryo formed a very compact mass of tissue. The
differentiation of allantoic endothelial cells was demonstrated by the
presence of the endothelial marker flk-1 (brown staining). A heterozygous
sibling is shown on the left. (F) Semithin section through the allantois of an
E9.5 mutant embryo showing blood vessels formation (arrows) and
differentiation of hematopoietic cells (asterisk). Scale bars, 0.5 mm.
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Fig. 4. LPP3-/- yolk sacs show abnormal vasculogenesis. (A)
External appearance of the yolksac in normal (top) and homozygous mutant
(bottom) conceptuses recovered at E10.5. In the mutant note the complete
absence of blood vessels and a very delayed embryo with its corresponding
amnion that can be observed through the yolk sac. (B,C) Development of the
vascular plexus in an E10.5 wild-type yolk sac. Formation and ramification of
large blood vessels is evident when blood cells are detected by their
endogenous peroxidase activity (B) or by staining endothelial cells using a
flk-1 probe (C). (D) PECAM-1 detection of endothelial cells in a
wild-type mouse embryo at E8.5 showing the formation of the dorsal aortas.
(E,F) An E10.5 LPP3-/- yolk sac showing a poor development
of the vascular plexus. No large blood vessels were formed. Blood cells and
endothelial cells were detected as in B and C. (G) PECAM-1 detection of
endothelial cells in a LPP3-deficient embryo at E9.5 showing the
formation of the dorsal aortas.
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Fig. 6. LPP3 deficiency results in axis duplication. (A,B) An E9.5
LPP3-/-conceptus where the anterior part (hf) of the
embryo developed outside the yolk sac (ys) and also exhibited abnormal
vasculogenesis (A). When dissected from the extra-embryonic membranes (B), the
embryo showed abnormal development of the allantois (all). (C) Cross section
through an abnormal embryo showing deficiency in mesenchymal tissue around the
paraxial mesoderm. Two large blood vessels (bv) have developed ventral to the
somites. d, dorsal. (D) Higher magnification of C showing the neural tube (nt)
clearly duplicated in the ventral region, in which a double notochord
(*) is also evident. A third somite (s) row has formed ventrally to
both notochords. (E,F) Shh and Twist double whole-mount in
situ hybridization in LPP3-/- embryos with axis
duplication. (E) In some embryos, two short Shh-positive notochords
(n) were observed without an additional somite row forming between them. (F)
In others, a clear Twist-positive extra somite row formed between the
duplicated Shh-positive notochords. (G-H) At E8.5 (G) and E7.5 (H)
LPP3-/- embryos (right) develop outside the yolk sac
unlike the nornal littermate (left). A constriction between the embryonic and
extra-embryonic tissues (arrowhead) is present. In the null embryos the
brachyury-expressing primitive streak is shorter in the mutant embryo
that in the wild type. A, anterior; P, posterior. (G) E8.5 embryos, (H) E7.5
embryos. (I) On the left, two E7.0 embryos show normal brachyury
expression in the primitive streak. On the right, two abnormal littermates
show retarded development (equivalent to E6.5), one of which shows primitive
streak duplication (arrows). (J) Posterior view of a
LPP3-/- embryo recovered at E8.5. Brachyury
expression revealed the presence of a common primitive streak-like structure
from which two axial mesoderm-like structures have formed. (K) dkk1
expression in an E7.5 LPP3 heterozygous (left) and homozygous mutant
(right) embryos. While dkk1 expression extends from the proximal AVE
to the distal visceral endoderm in the heterozygous embryo, in the
LPP3 null embryo weak dkk1 staining is restricted to a band
of cells located in the distal visceral endoderm (arrow). An abnormal
outgrowth of tissue formed exactly below the dkk1 expression domain
of this mutant embryo (small arrow). The arrowheads indicate the junction
between the embryonic and extra-embyonic tissues. A, anterior; P, posterior.
(L) Hex and Wnt3 expression are also altered in E7.0
LPP3 null embryos. Hex-positive cells accumulate in the
distal tip of the egg cylinder and Wnt3 expression is also found in
the anterior embryonic ectoderm (arrows). In normal embryos Hex is
expressed in the AVE and Wnt3 is restricted to the posterior
embryonic ectoderm. Bars: 250 µm.
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Fig. 7. LPP3 regulates ß-catenin-mediated TCF transcriptional activity. (A)
ß-catenin transcriptional activity in wild-type and LPP3 null ES
cells, as measured by luciferase levels produced from the transfected reporter
construct TOPFlash. ß-catenin-mediated TCF activity is upregulated
approx. 10- to 15-fold in the LPP3 null ES cells. (B) Transfection of
HEK293 cells (which lack endogenous LPP3 activity) with the
NH2 truncated (stabilized) ß-catenin results in high levels of
ß-catenin-mediated transcription. These levels are attenuated by
co-transfection of increasing levels of LPP3. As a control, a
ß-catenin unresponsive construct (FOPFlash) was used in these
experiments. LPP3 activity in the transfected cells was verified by
the release of 32P from labeled LPA. (C) Increasing levels of
transfected LPP3 also inhibits endogenous ß-catenin-mediated
transcription in the HEK293 cells. (D) Phosphatase-deficient LPP3 also
inhibits ß-catenin-mediated TCF transcription. HEK 293 cells transfected
with TOPFlash reporter construct and an LPP3 expression cassette carrying the
Ser197 Thr mutation that inactivates the phosphatase site inhibited
TCF/ß-catenin transcription. (E) Western analysis of extracts from the
transfected cells in B show that higher concentrations of LPP3 decreased
phosphorylation at Ser9 in GSK-3, which correlates with GSK-3 having an
increased inhibitory effect on ß-catenin. This coincides with the levels
of ß-catenin dephosphorylated at Ser37/Thr41 (the stabilized form) being
reduced by increasing LPP3 levels.
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Fig. 8. Effect of LPP3 in Wnt target gene expression. Expression analysis
of Wnt target genes during EB differentiation. Semi-quantitative RT-PCR
analysis of markers at the indicated days in culture. The loading control was
the Hprt gene. Bmp4 did not show significant differences in
expression during EB differentiation. In contrast, while brachyury
expression (a direct transcriptional target of the Wnt signaling pathway)
decreased after 6 days in culture in heterozygous EBs, its expression was
increased and prolonged in LPP3-/- EBs.
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Fig. 9. Effect of LPP3 in Xenopus axial patterning. (A-C) Effect
of murine LPP3 mRNA injection in Xenopus embryo development.
Stage 36 larvae (A) uninjected, (B) injected dorsally or (C) ventrally with 1
ng of mLPP3 mRNA. Inserts show translated LPP3 protein. Note that
only the larvae injected dorsally had abnormal anterior development. (D-H) In
situ hybridization of un-injected and dorsally injected Xenopus
embryos with markers for anterior development (stage 22). (D,E) Xotx2
detection in uninjected (bottom) and dorsally injected (top) embryos. Some
injected embryos lacked (D) or had reduced (E) Xotx2 expression.
Reduced and fused eyes can be observed in the injected embryo (E, arrow). (F)
Xpax6 detection in uninjected (bottom) and dorsally injected (top)
embryos. Injected embryos lacked distinguishable eye staining. (G) Embryos
injected ventrally with Xwnt3a show a duplicated axis. (H)
Co-injection of mLPP3 with Xwnt3a mRNA rescues secondary
axis formation but results in a weak dorsalizalized phenotype. (I) Axis
duplication induced by ventral injection of Xwnt8. (J) Co-injection
of Xwnt8 with LPP3 mRNA inhibited axis duplication, but the
embryos still retained a weak dorsalization phenotype.
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© The Company of Biologists Ltd 2003
LPP3-/- chimeric embryo recovered at
E10.5. In the presence of wild-type extra-embryonic tissue (blue), the
allantois from this embryo, entirely derived from LPP3-/-
cells, contacted the chorion (arrow). The embryo is also developmentally
delayed. (B) Rosa26