Supplementary Figure Legends - Word file

Supplementary Figure S1. Targeted disruption of LPA3. a. Genomic map of the wild-type
(WT) LPA3 locus, the targeting construct (in which a 1286 bp fragment was replaced with ~1600
bp neomycin resistance cassette), the LPA3 locus after homologous recombination, and locations
of probes for Southern blot (501O/N and 501T/5KO1). b. Southern blot analysis of DraI/EcoRIdigested genomic tail DNA (10 g/lane) from WT (+/+), Het (+/-), and LPA3-deficient (-/-) mice.
The blot was initially hybridized with probe 501T/5KO1, then stripped and hybridized with
probe 501O/501N (located within the deleted region). The 501T/5KO1 probe detected a 1.3 kb
(EcoRI/DraI) and a ~3.4 kb (DraI/DraI) bands from the mutated allele and a ~3.1 kb
(DraI/DraI) band from the WT allele. c. PCR assay with three primers (A3e1b, A3e1c, and
Neo1b) detecting the inheritance of WT LPA3 allele (216 bp) and mutant LPA3 allele (170 bp).
Supplementary Figure S2. Verification of loss of LPA3 function upon targeted deletion of
LPA3. a. Two types of truncated LPA3 transcripts detected in the putative LPA3-deficient mice
by RT-PCR using RNA derived from testis (first two lanes for each primer pair) or kidney (last
two lanes for each primer pair). Primers were annealed to exons I, II (undeleted region) and III
as indicated. Red arrow, type 1 transcript; green arrow, type 2 transcript. b. Schematic view of
the two truncated LPA3 transcripts. Type 1 truncated transcript retained the undeleted region of
exon II and full-length exon III. Type 2 truncated transcript had the entire exons I and III but no
exon II. Three in frame ATGs, including the authentic start codon, are indicated in the LPA3
transcripts. c. Schematic view of the WT and the predicated truncated forms of LPA3. If
properly translated from the non-authentic ATGs, type 1 truncated protein would lack
transmembrane domains (TMD) 1 and 2 (30.5 kD) and type 2 truncated protein would have only
the carboxyl-terminus of LPA3 plus 4 amino acids of TMD7 (7.5 kD). d. Western blot detection
of LPA3 in the testis of WT and the putative LPA3-deficient mice. An LPA3 monoclonal
antibody to the carboxyl-terminus of LPA3 detected LPA3 in WT testis (~40 kD, red arrowhead).
However, no corresponding bands around 30 kD or 7.5 kD were detected in the LPA3-deficient
testis, indicating that type 1 and type 2 truncated transcripts could not be properly translated. A
band of ~70 kD (black arrowhead) was also detected in WT testis suggesting a possible dimer or
glycosylated species. e. Calcium response of HeLa cells expressing full-length, the two
truncated forms of LPA3, and empty vector, respectively. HeLa cells were transfected with
cDNAs encoding full-length LPA3, putative truncated LPA3 variants, or empty vector and tested
for an LPA-induced calcium response. Data demonstrated that only the full-length LPA3 was
functional. Both Western (d) and LPA-induced calcium response (e) confirmed that LPA3 was
functionally deleted in the putative LPA3-deficient mice.
Supplementary Figure S3. Expression of LPA3 mRNA in WT uterus. a. During postnatal
development using real time RT-PCR. b. At different stages of the estrous cycle using real time
RT-PCR. c. In E3.5 uterus using in situ hybridization with an LPA3 sense probe. Pro:
proestrus; Est: estrous; Met: metestrus (diestrus I); Die: diestrus; Epi: luminal endometrial
epithelium; Str: stroma; Myo: myometrium. Scale bar: 100 m. -actin was used to normalize
LPA3 mRNA expression levels. The error bars indicate standard deviations.
Supplementary Figure S4. Identification/characterization of defects in LPA3-deficient female
reproduction. a. Number of superovulated eggs per oviduct. b. Cleavage rate. c. Representative
blastocysts flushed from WT/Het and LPA3-deficient uteri at E3.5 and E4.5. There was no
difference in morphology of blastocysts between those recovered from WT/Het uteri and those
from LPA3-deficient uteri at E3.5. The blastocyst recovered from E4.5 LPA3-deficient uteri was
already hatched. d. Decidualization. There was no difference between Het and LPA3-deficient
mice in decidualization expressed as fold increases in uterine weight. The error bars indicate
standard deviations.
Supplementary Figure S5. Comparison of placentas from E18.5 control (WT and Het) and
LPA3-deficient females. Placentas from control females (left panel) were uniform in size and
shape, while those from LPA3-deficient females (right panel) had variable sizes and shapes.
Arrows indicate placentas connected but separable, or shared by three or two embryos, or being
absorbed. Scale bar: 8 mm.
Supplementary Figure S6. Confirmation of maternal defects for implantation phenotypes by
transferring WT blastocysts to WT or LPA3-deficient pseudopregnant females, and LPA3deficient blastocysts to WT pseudopregnant females. a. Implantation in E4.5 and E5.5 uteri
transferred with WT blastocysts. Delayed implantation window, aberrant spacing, and reduced
implantation sites were shown in LPA3-deficient but not WT uteri. b. Embryos from E10.5 uteri
transferred with WT blastocysts. Retarded postimplantational development and embryonic death
(red arrows) were shown in WT embryos recovered from LPA3-deficient uteri. c. Weight of
embryos from E18.5 uteri transferred with WT blastocysts. Delayed development of WT
embryos was shown in LPA3-deficient uteri. * P<0.002. d. Implantation in E4.5 WT uteri
transferred with LPA3-deficient blastocysts. On-time implantation and even embryo spacing
were observed.
Supplementary Figure S7. Expression of LIF, Hoxa-10, and cPLA2 in E3.5 uteri. a. No
changes of mRNA levels of LIF, Hoxa10, and cPLA2 in E3.5 LPA3-deficient uteri. b. No
change of cPLA2 protein level in E3.5 LPA3-deficient uteri. Five samples each from WT and
LPA3-deficient uteri are shown. The anti-cPLA2 monoclonal antibody (mouse IgG) was
obtained from Santa Cruz Biotechnology, Inc. c. No change of cPLA2 activity in E3.5 LPA3deficient uteri. Three samples each from WT and LPA3-deficient uteri were examined. The
error bars indicate standard deviations.
Supplementary Figure S8. Additional information about potential dominant negative effect
and genetic background. a. No difference in the numbers of implantation sites at E4.5 and E5.5
between WT and LPA3 heterozygote females. b. No difference in embryo spacing at E4.5
between WT and LPA3 heterozygote females. c. LPA-induced Ca2+ response in cells cotransfected with full-length LPA3 and type 1 or type 2 LPA3 truncated forms, or vector
(supplementary Fig. S2). No effect of the truncated forms of LPA3 was observed. Fig. S8a, S8b
and S8c indicate that dominant negative effect was not present upon targeted deletion of LPA3 in
mice. d. LPA3-deficient females with mixed background (C57BL/6 and129SvJ) or C57BL/6
background (N=7-8, 99.2-99.6% pure) showing similar phenotypes, delayed implantation and
reduced implantation sites.