Generation of a reporter-null allele of Ppap2b/Lpp3
and its expression during embryogenesis
DIANA ESCALANTE-ALCALDE*,1,2, SARA L. MORALES1 and COLIN L. STEWART2,#
1Departamento de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México and
2Cancer and Developmental Biology Laboratory, NCI-Frederick, Maryland, USA
ABSTRACT Our knowledge of how bioactive lipids participate during development has been
limited principally due to the difficulties of working with lipids. The availability of some of these
lipids is regulated by the Lipid phosphate phosphatases (LPPs). The targeted inactivation of
Ppap2b, which codes for the isoenzyme Lpp3, has profound developmental defects. Lpp3 deficient
embryos die around E9.5 due to extraembryonic vascular defects, making difficult to analyze its
participation in later stages of mouse development. To gain some predictive information regard-
ing the possible participation of Lpp3 in later stages of development, we generated a Ppap2b null
reporter allele and it was used to establish its expression pattern in E8.5-13.5 embryos. We found
that Ppap2b expression during these stages was highly dynamic with significant expression in
structures where multiple inductive interactions occur such as the limb buds, mammary gland
primordia, heart cushions and valves among others. These observations suggest that Lpp3
expression may play a key role in modulating/integrating multiple signaling pathways during
KEY WORDS: Ppap2b, Lpp3, reporter allele, expression
Participation of bioactive lipid-mediated signaling in the regu-
lation of a wide variety of cellular processes has gained extensive
attention during the past two decades. However our knowledge
on the contribution of lipid-mediated signals to developmental
processes has progressed slowly. Most of the information regard-
ing bioactive lipid participation during development has arisen
from studies involving the use of inactivating mutations of their
receptors and/or their synthesizing or metabolizing enzymes
(Brindley, 2004, Mizugishi et al., 2005, Saba, 2004, Tanaka et al.,
2006, van Meeteren et al., 2006).
Lipid phosphate phosphatases (LPPs) are a group of integral
membrane enzymes that participate in intracellular lipid metabo-
lism. Moreover, they regulate the biological activity and signaling
of several bioactive phospholipids such as phosphatidic acid
(PA), diacylglycerol (DAG), lysophosphatidic acid (LPA), sphin-
gosine-1-phosphate (S1P), ceramide-1-phosphate (C1P) and
ceramide (Brindley et al., 2002, Brindley and Waggoner, 1998)
controlling the availability of their phosphorylated substrates and
Int. J. Dev. Biol. 53: 139-147 (2009)
THE INTERNATIONAL JOURNAL OF
*Address correspondence to: Diana Escalante-Alcalde. Instituto de Fisiología Celular-UNAM, Circuito Exterior s/n Ciudad Univesitaria, México D. F. 04510,
Coyoacán. Fax: +5255-5622-5607. e-mail: email@example.com web: http://www.ifc.unam.mx/faculty/descalan.html
# Present address: Institute of Medical Biology, Agency for Science, Technology and Research, Singapore
Accepted: 14 August 2008. Published online: 28 November 2008. Edited by: Edward De Robertis.
ISSN: Online 1696-3547, Print 0214-6282
© 2008 UBC Press
Printed in Spain
Abbreviations used in this paper: LPP, lipid phosphate phosphatase; PPAP,
phosphatidic acid phosphatase.
dephosphorylated products. Subcellular localization of LPPs de-
pends on the cell type and on physiological conditions (Jia et al.,
2003, Kai et al., 2006, Sciorra and Morris, 1999, Sigal et al., 2005,
Sun et al., 2004). Intracellular LPPs control the balance between
PA and DAG therefore alter cell signaling mediated by these lipids
and the synthesis of choline- and inositol- containing phospholip-
ids. They play a fundamental role in the hydrolysis of phospholi-
pase D (PLD)-derived PA and, in consequence, modulate PLD-
mediated signaling. Some of the targets of PLD-derived PA are
PI4PK, Sos, Raf and mTOR while RasGRP1 is regulated by LPP-
derived DAG (McDermott et al., 2004, Mor et al., 2007, Zhao et al.,
2007). A role in the regulation of the intracellular pools of other
lipids has been demonstrated for S1P (Long et al., 2005) although
involvement in the conversion of C1P to ceramide is also possible.
LPPs localized in the plasma membrane serve to attenuate the
receptor-mediated signaling of extracellular LPA and S1P, which
140 D. Escalante-Alcalde et al.
in turn activate a wide variety of signaling pathways such as PLC/
Ca++, Ras/ERK/MAPK, Rac, Rho and PI3K (Brindley, 2004,
Moolenaar et al., 2004, Pyne et al., 2004). Furthermore, biological
actions independent of their catalytic activity have been proposed
for some LPPs (Escalante-Alcalde et al., 2003, Humtsoe et al.,
2003, Zhao et al., 2005).
Most of the information regarding the participation of LPPs in
specific cellular processes has stemmed from in vitro experi-
ments. However, research performed during the last ten years
has demonstrated their important contribution to development. In
Drosophila, wunen and wunen2 are required for proper germ cell
migration and survival (Hanyu-Nakamura et al., 2004, Renault et
al., 2004, Starz-Gaiano et al., 2001, Zhang et al., 1997). Further-
more, wunen also participates in proper neuron synaptogenesis
and in the establishment of intestinal left-right laterality (Kraut et
al., 2001, Ligoxygakis et al., 2001). Additionally lazaro is involved
in the turnover of phosphatidylinositol during the process of
phototransduction (Garcia-Murillas et al., 2006). In the mouse,
out of the three Lpps identified, Ppap2c/Lpp2 null-gene inactiva-
tion and a gene trap of Ppap2a/Lpp1 are viable and fertile without
any apparent phenotype (Zhang et al., 2000, Lynch, pers comm.).
In contrast, Ppap2b/Lpp3 targeted inactivation results in profound
developmental defects indicating the essential participation of
Lpp3 in mouse embryo development. Lpp3 deficient embryos die
around E9.5 due to extraembryonic vascular defects. With lower
penetrance, earlier lethality is due to anterior-posterior patterning
alterations related to the antagonistic effect of Lpp3 on the Wnt/
β-catenin signaling pathway by a still poorly understood mecha-
nism (Escalante-Alcalde et al., 2003).
The early embryonic lethality of Lpp3 deficiency makes difficult
the analysis of its participation in later stages of mouse develop-
ment. One way to gain some predictive information regarding the
possible participation of Lpp3 in later stages of development is
through the detailed analysis of its expression pattern. In this
study we describe the generation of a Ppap2b null reporter allele,
which was used to establish its expression pattern in E8.5-13.5
embryos. Remarkably, Ppap2b expression during these stages
was highly dynamic with significant expression in structures
where multiple inductive interactions occur such as the limb buds,
mammary gland primordia, heart cushions and valves among
Despite the wide range of available information in databases
regarding mRNA expression using microarray analysis, little is
known about the tissue-specific expression pattern of Lpp3 during
development. Here we describe the generation of a reporter null
allele of Ppap2b by inserting the bacterial β-galactosidase gene
(lacZ) in exon 3 (Fig. 1) by homologous recombination in embry-
onic stem (ES) cells, which was designated Ppap2btm2Stw. For
convenience, we will refer to this allele as Ppap2blacZ. Heterozy-
gous Ppap2blacZ mice were viable and fertile. Mating of heterozy-
gous Ppap2blacZ on either 129/SvJ x C57BL/6J or a pure 129Sv/
J background yielded animals only of wild type and heterozygous
genotype indicating that this allele is embryonic lethal (57% +/-;
42% +/+; n= 218). The phenotype of homozygous Ppap2blacZ
embryos from heterozygous intercrosses, was essentially identi-
cal to our formerly described null alleles Ppap2btm1Stw and
Ppap2btm3.1Stw (Escalante-Alcalde et al., 2003, Escalante-Alcalde
et al., 2007). This was characterized by a delay in development
when compared to wild type or heterozygous littermates (12 to 24
hours), absence of chorioallantoic fusion at the 6 somite stage,
allantois compaction (Fig. 1E-F), impaired remodeling of the
primary capillary plexus of the yolk sac and gastrulation defects
with low penetrance (not shown). Furthermore, in the best-
developed mutant embryos, persistence of open neural tube was
frequent (Fig. 1G). The use of an IRES sequence in the targeting
design allowed us to detect the endogenous transcriptional activ-
ity of Ppap2b. Intensity of β-galactosidase staining recorded
before embryo death, correlated with the genotype being more
intense in homozygous than in heterozygous embryos. The
expression of Ppap2b during early mouse development (E6.5-
E8.5) using this allele has been shown previously (Escalante-
Alcalde et al., 2003). In this work we described, in detail, the
expression pattern of Ppap2b in E8.5-E13.5 embryos to gain
some predictive information regarding the participation of Lpp3
during these developmental stages. We used heterozygous em-
bryos to reveal Ppap2b expression as reported by the activity of
β-galactosidase (β-gal) and compare these patterns with those
obtained by whole mount in situ hybridizations and in some
stages by whole mount immunohistochemistry to test the fidelity
Fig. 1. Generation of the Ppap2blacZ allele. (A) Genomic structure of the
11 kb BamHI fragment containing exons 3 and 4 of Ppap2b (gray boxes).
Regions used as probes are indicated with black bars (5’, 3’). Arrows with
lower case letters show the position of oligonucleotides used for PCR
genotyping. (B) Structure of the targeting construct. An IRES-lacZ-loxP-
PGKneo-loxP cassette was introduced into the unique MunI site. (C)
Structure of the targeted allele. (D) Southern blot of ES cells genomic DNA
digested with BamHI and hybridized with the 5’ and 3’ probes. (E) E9.5
Ppap2blacZ heterozygous and homozygous embryos. (F) Higher magnifica-
tion of the mutant embryo in panel (E) showing allantois compaction and
incomplete turning. (G) Dorsal view of the mutant embryo in panel (E)
showing open neural tube at the midbrain and hindbrain (arrow). B, BamHI;
H, HindIII; K, KpnI; M, MunI; S, SalI; X, XmaI; Xb, XbaI; a, allantois; nf, neural
folds. Scale bar, 200 µm in (E,F); 100 µm in (G).
5’ probe 3’ probe
Ppap2b embryonic expression 141
of the patterns obtained. As shown in Figures 2 and 4, mRNA,
protein and β-gal expression patterns were essentially identical
with few differences in β-gal intensity (likely due to its stability) and
to the amount of Lpp3 protein in some neural structures (see
below). As discussed in the following section, due to the great
sensitivity of this reporter, we detected structures showing even
low levels of Ppap2b expression. Cells expressing low-expres-
sion levels showed one (or a few) cytoplasmic granule while cells
expressing higher levels of the enzyme showed a more homoge-
neous cytoplasmic staining.
Expression of Ppap2b in E 8.5 mouse embryos
In embryos Theiler stages (TS) 12-13, Lpp3 expression was
abundant in the allantois and paraxial mesoderm but absent or
weak in the forebrain and posterior embryonic domain respec-
tively (Fig. 2A-B). Cells expressing β-gal were more abundant in
the head and cervical mesoderm. The pericadial-peritoneal me-
sothelial cells and the somites showed conspicuous staining (Fig.
2C-E). β-gal activity was detected in cells along the gut endoderm
being more intense at the level of the foregut diverticulum en-
trance (Fig. 2C-D). Expression was detectable in the neuroepithe-
lium such as in the prospective midbrain, hindbrain and the ventral
portion of the future spinal cord (Fig. 2 B-E). Furthermore, β-gal
was expressed in the notochord (Fig. 2E) and in the region where
it is organized at the level of the primitive streak. Reporter activity
was found in the amnion and at lower activity in the visceral
endoderm and vascular mesodermal cells of the yolk sac (not
Expression of Ppap2b in E9.5 mouse embryos
In TS 14, regions exhibiting the strongest expression levels
were the endoderm and ectoderm epithelia of the future tympanic
membrane region followed by the allantois, somites, paraxial
tissue at the cervical region and the developing intraembryonic
coelomic cavity area (Fig. 2F-G). Sections through the embryos
showed expression in the cephalic and cervical mesenchymal
cells (Fig. 2H) with the exception of the mesenchyme in the
telencephalic area. The otic vesicle epithelium and migratory
neural crest tissue showed detectable expression (not shown). In
the heart, cells in the wall of the outflow tract (Fig. 2I), the posterior
wall of the common atrial chamber and few cells of the endocardial
lining in the bulbo-ventricular canal showed reporter activity.
Along the gut, expression was variable, with higher levels in the
thyroid primordium, the ventral aspect of the foregut endoderm
and lower levels present in the hepatic diverticulum, at the
foregut-midgut junction and hindgut diverticulum (Fig. 2I-J). The
strongest intraembryonic expression was in the mesothelial cells
forming the pericardial-peritoneal coelomic cavity at the level of
the developing forelimb (Fig. 2J). Expression in the notochord and
developing nervous system followed the same expression pattern
Fig. 2. Ppap2b expression in E8.5-
10.5 embryos. Whole mount in situ
hybridization (mRNA) or β-galactosi-
dase staining (β-gal) of Ppap2blacZ het-
erozygous mouse embryos at E8.5 (A-
E), E9.5 (F-J) and E10.5 (K-T). (C,H)
Transverse sections at equivalent lev-
els shown in (B,G) respectively (dashed
lines) and photographed under
Nomarski optics, β-gal staining appears
in blue. (D-E, I-J, M-T) Transverse sec-
tions photographed under dark field
illumination, β-gal staining appears pink
or reddish. In some cases, strong β-gal
staining is clearly observed in blue or
purple color (N-P). a, allantois; ba, 1st
branchial arch; cm, coelomic mesothe-
lium; cr, cervical region mesoderm; da,
dorsal aorta; dm, dorsal mesentery;
dmy, dermomyotome; drg, dorsal root
ganglion; f, forebrain; fd. foregut diver-
ticulum; fg, foregut epithelium; fl, fore-
limb; fp, floor plate; GnV, trigeminal
ganglion; hc, heart cushions; hd, he-
patic diverticulum; hm, head mesen-
chyme; me, mesoderm; m/h, midbrain/
hindbrain region; mn, motor neurons;
n, notochord; nt, neural tube; otr, out-
flow tract; ov, otic vesicle; ppc, pericar-
dial-peritoneal canal; psm, presomitic
mesoderm; s, somite; t, thyroid pri-
mordium; tm, future tympanic mem-
brane; ugc, urogenital crest; Viii, acous-
tic neural crest cells. Asterisk and arrowhead in (K,L) show expression in the maxillary component of the first branchial arch and in the second branchial
arch respectively. Scale bars, 100 µm in (A,B,F,G); 200 µm in (K,L); 20 µm in (C-E, N,R) and 40 µm in (H-J, M,O,P,Q,S,T).
142 D. Escalante-Alcalde et al.
as in the previous stage (Fig. 2H). In TS 15 embryos forelimb buds
showed a strong β-gal staining in a group of scattered ectodermal
cells in the ventral side of the developing forelimb (Fig. 3A).
Expression of Ppap2b/Lpp3 in E10.5 mouse embryos
In embryos TS 18, reporter activity was broadly found in the
mesenchyme. In the cephalic and cervical regions, expression in
the cephalic flexure mesenchyme and the one surrounding the
primary head and anterior cardinal veins and dorsal aorta was
more intense (Fig. 2K-L, N-P). Expression was also found in the
mesenchyme of the anterior-distal aspect of the maxillary compo-
nent of the first branchial arch and the second branchial arch (Fig.
2L). In the paraxial mesenchyme, expression was more abundant
in the anterior (Fig. 2S) than in the posterior part (Fig. 2T) of the
embryo correlating with de-epithelization of the somites into
sclerotome cells. Expression in the dermomyotome of epithelial-
ized somites was present in the caudal region whereas at the
cervical and thoracic levels, expression was gradually restricted
to the dorsal lip region of the dermomyotome (Fig. 2L). Expression
in the notochord was more evident at this stage (Fig. 2S).
Staining in the heart area corresponded to the most dorsal
aspect of the heart and the body wall overlaying the pericardial
cavity. Detectable expression was found in the endocardial cush-
ion tissue associated to the atrioventricular canal and in cells of
the dorsal truncus arteriosus (Fig. 2Q). At this stage, expression
in blood vessel endothelial cells was evident. The dorsal meso-
cardium as well as the mesenchyme surrounding the bronchus
epithelium and oesophageal region of the foregut showed moder-
ate β-gal activity (not shown). In the urogenital area, a bilateral
stripe of mesothelial cells contiguous to the dorsal mesentery and
the mesenchyme adjacent to the dorsal aorta showed reporter
activity (Fig. 2T).
Expression was also detected in some components of the
central and peripheral nervous systems at this stage. Forebrain
neuroepithelium was devoid of significant reporter expression.
indicated the future digital domains of the
forelimb (Fig. 3D). Expression of β-gal in
the developing mammary gland placodes
3 and 4 was evident at this stage (Fig.
3D). At the hindlimb level, the paraxial
mesenchyme ventral to the inter dorsal
root ganglia space expressed abundant
reporter activity. Expression in the uro-
genital sinus area and umbilical hernia
was stronger than in previous stages.
Dermal expression in the cervical area
became evident at this developmental
Expression of Ppap2b in E12.5 mouse
In this stage, expression in the dermis
had extended and the five pairs of mam-
mary gland placodes showed abundant
Lpp3 expression. The head mesenchyme
showed abundant expression being more
evident around the primary head vein and
the cephalic ganglia. Ppap2b/ Lpp3 ex-
pression was detected in the most ante-
Fig. 3. Ppap2b expression during limb development. β-galactosidase staining of limbs of E9.5 (A),
E10.5 (B,C), E11.5 (D), E12.5 (E) and E13.5 (F,G). Ppap2blacZ heterozygous embryos. (B,C) Dorsal and
lateral views of a forelimb respectively. (G) Longitudinal section of a forelimb at E13.5; arrow shows
expression in the central domain of the digit. (H) Higher magnification of the boxed area in (G) showing
expression in the mammary gland epithelium. AER, apical ectodermal ridge; D, dorsal view of the
limb; fl, forelimb; hl, hindlimb; mg, mammary gland; mv, mesenchyme surrounding the marginal vein;
pc, perichondrium; pz, progress zone; se, surface ectoderm; t, tendon; V, ventral view of the limb.
Scale bars, 100 µm in (A,C); 200 µm in (B, D-G) and 40 µm in (H).
Detectable expression in this and other regions of the neural tube
correlated with invading blood vessels from the perineural vascu-
lar plexus. At the midbrain, hindbrain and cervical regions, cells
expressing β-gal were found in the basal and floor plates (Fig. 2M-
P) while a group of presumptive motor neurons showed reporter
activity at the thoracic and lumbar areas (Fig. 2R). In the eye,
detectable expression was found in the central domain of the
outer layer of optic cup and lens pit (not shown). Ppap2b was also
expressed in neural crest derivatives. In the trigeminal crest
tissue, facio-acoustic (VII-VIII) neural crest complex and other
developing cervical ganglia, expression was evident in certain
groups of cells (Fig. 2O-P). Stronger expression levels were
observed in the interacting region between the otic vesicle epithe-
lia and the neural crest cells of the acoustic preganglion (Fig. 2P).
The dorsal root ganglia showed a low but still detectable activity
of β-gal. Unexpectedly, in contrast with the level of β-gal activity
found in neural derivatives, Lpp3 immunohistochemistry showed
a very strong staining in projecting axons of cranial ganglia and
motor neurons. Furthermore, a weak expression in the ependyma
of the spinal cord was evidenced (Fig. 4H).
In the limbs, the apical ectodermal ridge (AER) is the structure
with strongest expression of Ppap2b at this stage. In the forelimbs
moderate expression was found in the progress zone (PZ) region
and the proximal dorsomedial mesenchyme, though expression
in the PZ area was more restricted to the dorsal side (Figs. 3B-C,
Expression of Ppap2b/Lpp3 in E11.5 mouse embryos
Ppap2b expression pattern was essentially the same as that
observed in E10.5. However, modification in the expression
pattern of some structures was observed. Expression in the AER
of limbs was preserved though weaker staining was observed in
the superficial dorsomedial mesenchyme of the zeugopod. A
region expressing strong reporter activity was present in the
dorsal-central domain of the autopod and a weak expression
Ppap2b embryonic expression 143
Fig. 4. Ppap2b-Lpp3 expression patterns in E10.5 and 12.5 embryos.
Whole mount in situ hybridization (A,D), β-gal staining (B) and whole
mount immunohistochemistry (C, E-I) in E12.5 (A-G) and E10.5 (H,I)
embryos. In E12.5 embryos, differences in mRNA (A), β-gal (B) and
protein (C) expression patterns are mainly due to the great abundance of
Lpp3 protein in growing axons. mg, mammary gland; mb, midbrain; hb,
hindbrain. (D) Higher magnification of the interlimb area of the embryo in
(A) showing Ppap2b mRNA expression in the mammary gland primordia
(numbers). (E) Interlimb area of an E12.5 embryo showing Lpp3 protein
expression in the mammary gland primordia (numbers). (F) Magnification
of the boxed area in panel (C) showing a weak staining in the body of the
trigeminal ganglion (GnV), but strong Lpp3 expression in projecting axons
(ax). (G) Transverse section at the level indicated in panel (C) (dashed line)
showing Lpp3 expression in a group of dorsal interneurons (in), the
ependymal layer (ep), a subset of motor neurons (mn), dorsal root
ganglion (drg) and projecting axon (ax). m, myotome; mg, mammary
gland. In earlier embryos (E10.5), expression of Lpp3 protein in neural
derivatives (H) such as the fasiculated axons (fax) projecting to the
cervical or forelimb areas is also more evident by immunohistochemistry.
Insert: transverse section at the level indicated by the white dashed line.
In contrast, expression in non-neural tissue is essentially identical to the
expression pattern obtained using the reporter allele. For instance, in the
limb buds (I), staining was found in the apical ectodermal ridge (AER) and
progress zone mesenchyme (pzm) of the forelimb (fl). hl, hindlimb.
rior myotomes at the inter limb area and more restricted to their
dorsal lip in the rest of the embryo (Fig. A-B and D-E). Expression
was found in the tail somites but absent in its distal extremity. The
liver and heart did not show significant Ppap2b expression except
for the atrioventricular endocardial cushions (not shown). In the
genitourinary system, expression was found in the mesonephric
ducts epithelium and urethral plate epithelium in addition to the
deep mesenchyme of the genital tuberculum (not shown).
In the CNS, expression in the central domain of the optic cup
was preserved however reporter activity in the optic stalk and
developing hyaloid plexus was apparent at this stage (not shown).
Expression domains in the ventral midbrain, hindbrain, spinal
cord and peripheral structures were essentially as in the previous
stage, but new expression domains became apparent by immuno-
staining, such as a group of spinal dorsal interneurons (Fig. 4C
and G). Weak expression levels were also detected in the in-
fundibulum neuroepithelium and the thalamus ventricular area. A
graded expression of β-gal was observed in dorsal root ganglia
being stronger in posterior than in anterior ganglia.
Expression in the AER was gradually down regulated between
E11.5 and E12.5. While expression in the AER disappeared,
expression in the mesenchyme surrounding the marginal vein
was evident but stronger in the distal region of the prospective
digits. The dorsal central domain of expression in the autopod was
wider. Expression in the dorsal muscle anlagen was evident in the
developing limbs (Fig. 3E, 4A-B).
Expression of Ppap2b in E13.5 mouse embryos
β-gal expression was widely distributed at this stage. However,
reporter activity in the surface ectoderm, in most of the endoderm-
derived epithelia, liver, heart muscle, condensed cartilage and the
follicles of vibrissae, was very low or undetectable (Fig. 5). In the
heart, the developing atrioventricular valves, atrium and vena
cava showed reporter activity (Fig. 5 C-F). β-gal staining varied
from strong to moderate in the perichondrium tissue and in some
regions of highly differentiated cartilage (Fig. 5C, F-G). In the
limbs, expression was found in the perichondrium of developing
skeletal elements, the most central cells of the developing digits
and the tip of the digits (Fig. 3F-G). Expression was also evident
in the dorsal and ventral muscle anlagen (not shown) and devel-
oping ventral tendons (Fig. 3F). The mammary gland epithelia
showed strong reporter activity while in the surrounding mesen-
chyme was weaker (Fig. 3G-H, 4A).
In the brain, β-gal expression was restricted to some structures
such as the developing cerebral cortex and the ventricular area of
the mesencephalon, rhombencephalon and cerebellum primor-
dium (Fig. 5C and data not shown). In the spinal cord, a stripe of
β-gal positive cells running along the central domain of the
ependymal layer was observed and a weaker staining extended
to the whole ependymal layer (Fig. 5H-I). Dorsal root ganglia
showed a low but still detectable level of reporter activity (Fig. 5C,
F and H).
The mouse Lpp3 belongs to a group of enzymes with the
potential to regulate/modulate a wide variety of signaling path-
ways. This would initially suggest a ubiquitous expression for this
enzyme. Nonetheless, our previous (Escalante-Alcalde et al.,
2003) and present data show that, Lpp3 displays a very dynamic
144 D. Escalante-Alcalde et al.
and specific expression pattern during mouse embryo develop-
Our analysis revealed that the reporter allele clearly reflected
the endogenous mRNA expression pattern however the amount of
Lpp3 protein did not correlate with the relative transcription levels
found in central and peripheral nervous structures. For instance, in
dorsal root ganglia or the motor neurons in the spinal cord, while the
β-gal activity or amount of mRNA were very low, protein levels were
much more robust. Furthermore, the protein content in developing
nerves from motor neurons, cranial and dorsal root ganglia was
particularly abundant (Fig. 4C, F-H). This observation suggests a
tissue-specific translational regulation of Lpp3 in the nervous
Interestingly Ppap2b/ Lpp3 expression is abundant in sites
where multiple inductive interactions occur such as the limb bud,
mammary gland and developing heart valves. In these structures,
the convergence of several signaling pathways takes place to give
rise to proper development and patterning. In the limb bud, FGF
signaling from the AER is fundamental for proper proximal-distal
limb patterning and development (Sun et al., 2002, Yu and Ornitz,
2008). Lpp3 is abundantly expressed in the AER between E9.5 -
E12.5, fingertips at E13.5 and in the distal mesenchyme around
E10.5. It would be interesting to analyze whether expression of
Ppap2b in the AER is a consequence of Fgf8 signaling. Further-
more, it is temping to speculate that Lpp3 could function as a
modulator of Fgf8 signaling in the AER and subjacent mesen-
chyme in view of its potential impact on Ras activation, through the
control of phosphatidic acid and DAG levels. On the other hand,
Wnt7a non-canonical signaling from the dorsal ectoderm and
Engrailed-1 activity from the ventral ectoderm are both required for
D-V limb patterning. While Wnt7a induces Lmx1b expression in the
dorsal mesenchyme, β-catenin-induced En1 expression in the
ventral ectoderm is required to restrict Lmx1b expression in the
ventral mesenchyme (Loomis et al., 1996, Loomis et al., 1998). It
is worth noting that Lpp3 is expressed in the distal-dorsal limb
mesenchyme at E10.5 partially resembling the Wnt7a-dependent
expression of Lmx1b in the limb. It will be interesting to determine
whether Lpp3 expression in dorsal domains of the limb is down-
stream of Wnt7a/Lmx1b and whether it participates in dorsal-
ventral and proximal-distal limb patterning.
Ppap2b expression is very abundant in epithelial placodes and
mammary buds at E11.5-13.5 embryos. Moreover, it appears
progressively in the surrounding mesenchyme after E13.5 while
epithelial expression gradually diminishes in E15.5-16.5 (not shown).
In the mouse embryo, mammary gland specification is dependent
on FGF signals from the ventral-lateral dermomyotome at the level
of the milk line and Wnt signals in the milk line ectoderm. Posterior
placode formation requires the participation of the same groups of
factors but more prominently of Wnt signals (Hens and Wysolmerski,
2005). Contradicting results regarding the participation of epithelial
or mesenchymal Wnt signals during mammary bud development
exist, using two different TOPGAL reporter transgenics. In one
study, β-gal activity was found first in ectodermal cells along the
mammary line and latter restricted to epithelial cells of the mam-
Fig. 5. Ppap2b expression in E13.5 embryos. β-galactosidase staining of
Ppap2blacZ heterozygous mouse embryos. External (A) and mid sagittal
views (B,C). (A-B, G,I) Bright field microscopy showing reporter activity in
blue. (C-F, H) Dark field microscopy showing reporter activity in pink or
reddish color. Expression in the heart is restricted to the atrium (D)
atrioventricular valves (E) and superior vena cava (F). (F,G) Expression in
cartilage condensations, lung mesenchyme and dorsal root ganglia. β-gal
staining in the neural tube at the sacral (H) and thoracic (I) levels. (I) Open-
book preparation of the neural tube. Insert: cross section of the spinal cord
showing the position of the cells forming the stripe of cells with expression
of β-gal (arrow). A, anterior; at, atrium; avv, developing atrioventricular
valve; bo, cartilage primordium of the basioccipital bone; cx, developing
cortex; d, dorsal; di, diaphragm; drg, dorsal root ganglion; h, heart;
hindbrain li, liver; lu, lung; mg, mammary gland buds; nt, neural tube; pi,
pituitary; r, rib cartilage primordium; tg, tongue; v, ventral; vc, vena cava;
ve, vertebral cartilage primordium; vs, vibrissae follicles; vz, ventricular
zone. Scale bars, 40 µm in (E),; 200 µm in (B,D, F-G).
Ppap2b embryonic expression 145
mary placodes and buds, with few mesenchymal cells expressing
the reporter by E13.5 (Chu et al., 2004). In the other, β-gal
expression in the mesenchyme surrounding the mammary pla-
codes started around E11.5 and it was not until E13.5 that it was
also found in the bud epithelia, all of these in agreement with
nuclear localization of β-catenin (Boras-Granic et al., 2006). The
latter work suggests that mesenchymal cells integrate Wnt signals
during the early stages of mammary gland development and that
epithelial Wnt expression is required later to the progress of
mammary gland development. We have previously described that
Lpp3 acts as a negative regulator of the canonical Wnt/β-catenin
signaling pathway (Escalante-Alcalde et al., 2003). In this sense,
Ppap2b expression during mammary gland development fits better
with the second model where it acts as a negative feedback loop
to attenuate canonical Wnt signaling, by restricting Wnt activity in
epithelial cells during early mammary gland development and then
to restrict Wnt activity in the surrounding mesenchyme.
Ppap2b expression during cardiac valve development was
particularly interesting. Abnormalities in heart valves and associ-
ated structures are the most common subtype of cardiovascular
malformation. Heart valves develop from cardiac cushions pro-
duced by endothelial-mesenchymal transition of the endocardial
cells with subsequent proliferation and tissue remodeling in spe-
cific regions of the heart. Cushions later protrude from the under-
lying myocardium to form the valve leaflets (Armstrong and Bischoff,
2004, Lincoln et al., 2004). A complex signaling network involving
Vegf, Nfat, Notch, Wnt/β-catenin, BMP, TGFβ, ErbB and NF1/Ras
has been implicated in the control of heart valve development
(Armstrong and Bischoff, 2004). Again, due to the potential effect
of Lpp3 in modulating several of these signaling pathways, an
essential role for it in heart valve development may be predicted.
During central and peripheral nervous system development,
Ppap2b/ Lpp3 is expressed in a very particular fashion. In the
central nervous system, Lpp3 is present predominantly in ventral
domains of the neural tube as early as E8.5. In addition it is
remarkable that Ppap2b is expressed in the notochord. Gene
expression and cell type specification in ventral domains of the
neural tube are mainly controlled by the secretion of Shh by the
notochord and floor plate (Briscoe et al., 2000). In this sense, it is
provocative to speculate that Lpp3 expression in the neural tube,
notochord and perhaps paraxial mesenchyme (see below) might
be regulated by Shh signaling. In support of this idea, an in silico
analysis (NCBI DCODE.org Comparative Genomics Develop-
ments) of conserved regulatory elements in the Ppap2b promoter
of rat and mice, revealed the presence of putative binding sites for
Gli2, Pax6, Nkx2-2, Nkx6-1 and FoxA2 transcription factors, all
participating in the ventral specification of the neural tube (Briscoe
et al., 1999, Ding et al., 1998, Ericson et al., 1997, Sander et al.,
2000, Sasaki et al., 1997). Assaying whether inhibition of Shh
signaling affects the neural expression of Lpp3 and whether the
aforementioned putative binding sites are functional will be of
significance. Furthermore, it will be interesting to analyze the
participation of Lpp3 in the specification of ventral cell types in the
neural tube. Up-regulation of Ppap2b expression by the treatment
with Shh has been recently described in mesenchymal stem cells
(Ingram et al., 2008).
As shown, Lpp3 is also abundantly expressed in projecting
axons of motor neurons, cranial and dorsal root ganglia neurons.
Given that LPA and S1P exhibit a powerful neurite-retraction
activity (Postma et al., 1996, Tigyi et al., 1996), it is temping to
suggest that Lpp3 expression in these neuronal types might serve
to counteract the retractogenic environment caused by such lipids,
contributing to the axon outgrowth during mouse development. In
support of this idea is the inability of Lpp3 deficient MN differenti-
ated in vitro from ES cells to produce neurites (R. Sánchez and D.
E-A in preparation).
As previously described, Lpp3 is expressed during embryonic
development in several structures where complex signaling net-
works occur to ensure their proper development and patterning.
These observations suggest that Lpp3 expression may play a key
role in modulating/integrating multiple signaling pathways during
development. This idea rests on the assumption of a precise
control of its temporal and spatial expression in conjunction with an
accurate regulation of its actions as an intracellular or as a plasma
membrane-associated protein. Tissue specific inactivation of
Ppap2b will help us to address the participation of Lpp3 in the
various mentioned developmental processes. Likewise the re-
porter allele described in this paper will allow us to address some
aspects of the transcriptional regulation of Ppap2b during develop-
Materials and Methods
Generation of Ppap2tm2Stw ES cells and mice
The mouse EST AA276423 IMAGE Consortium clone ID 776179
(Lennon et al., 1996) was used to screen a 129/SvJ mouse genomic BAC
library (Research Genetics). From clone BJ4123, a 5.1 XmaI fragment,
containing exons 3 and 4, was used to produce an insertion targeting vector
with the following configuration: 3 stop codons in 3 frames were introduced
upstream of an IRES-lacZ reporter cassette. A PGKneo cassette, flanked
by loxP sites, was introduced downstream of the reporter cassette for
positive selection. The reporter-positive selection cassette was introduced
in the proper orientation into the unique MunI restriction site located in exon
3. This insertion interrupts the protein between C1 and C2 domains
essential for the catalytic activity. A TK negative selection cassette was
added downstream of the 3’ arm and then the vector linearized. W9.5 ES
cells were electroporated with the targeting construct selected in the
presence of G418 and FIAU and screened for legitimate homologous
recombination by Southern (Fig.1). The insertion of the reporter cassette
introduces two additional BamHI sites, originating bands of 5.7 or 8.3 kb
when using the 5’ and 3’ probes respectively, in BamHI digested genomic
DNA. Out of 117 isolated clones, 24% resulted in the recombined allele.
After karyotyping, one clone was selected for derivation of chimeras that
were crossed to C57BL6/J females to test germ line transmission and to
establish a mouse line designated Ppap2btm2Stw.
For PCR genotyping, a set of three oligonucleotides was designed to
distinguish between the wild type and targeted alleles. Primer a is upstream
(a. 5’ CTG TGC CAT TAG CCA GTC CTT CAC 3’) and primer b is
downstream (b. 5’ TAG TTC TGA ATG TAG CCC TCG GAG 3’) of the MunI
restriction site fragment in exon 3. In the wild type configuration, these
amplify a product of 128 bp. A primer in the floxed PGKneo cassette (c. 5’
TTC TAT CGC CTT CTT GAC GAG TTC 3’) in combination with primer b
amplifies a product of around 700 bp. Alternatively born heterozygous
individuals were genotyped using lacZ specific primers or β-galactosidase
staining of ear fragments.
β β β β β-galactosidase staining
Embryos or fragments of tissues were stained as previously described
(Hogan et al., 1994). Briefly, tissues were immersed in fixative solution,
washed 3 times for 30 min with detergent rinse and then incubated
146 D. Escalante-Alcalde et al.
overnight in staining solution. The samples were embedded in paraffin,
sectioned at 6 µm. Sections were visualized and photographed using
Nomarski optics (DIC) or dark field illumination in which β-gal staining
appeared of pink or reddish color.
Whole mount in situ hybridization
Mouse embryos were fixed and processed for in situ hybridization as
described previously (Hogan et al., 1994). Full length Lpp3 cDNA was
cloned into pBluescript(KS) and then linearized with MunI. Anti-sense and
sense probes were transcribed using T3 and T7 RNA polymerases
Whole mount immunohistochemistry
Protein detection was performed as previously described (Hogan et
al., 1994). Briefly, embryos were fixed with methanol/DMSO (4:1) and
endogenous peroxidase activity was blocked by H2O2 treatment. The
incubation with the primary antibody (anti-LPP3, 1:200 Upstate) was
carried out overnight at 4˚C. Anti-rabbit coupled to peroxidase was used
at a dilution of 1:500. After color reaction embryos were post fixed with 4%
paraformaldehyde and cleared with BABB for observation and photo-
We are grateful to T. Rosenbaum and I. Velasco for comments and
carefully reading of the manuscript. This work was partially supported by
CONACyT 39995 and PAPIIT IN215605.
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