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Molecular Endocrinology, doi:10.1210/me.2004-0133
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Molecular Endocrinology 18 (9): 2241-2254
Copyright © 2004 by The Endocrine Society

Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor 4 Null Mice Exhibit Intrauterine Growth Retardation Associated with Embryonic and Perinatal Lethality

Sabine Mazerbourg, Donna M. Bouley, Satoko Sudo, Cynthia A. Klein, Jian V. Zhang, Kazuhiro Kawamura, Lisa V. Goodrich, Helen Rayburn, Marc Tessier-Lavigne and Aaron J. W. Hsueh

Division of Reproductive Biology, Department of Obstetrics and Gynecology (S.M., S.S., C.A.K., J.V.Z, K.K., A.J.W.H.) and Departments of Comparative Medicine (D.M.B.), and Biology (L.V.G, H.R., M.T.-L.), Stanford University, Stanford, California 94305

Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven-transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4–6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene after the insertion of a ß-galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistochemical studies in wild-type animals. Analysis of the viability of 250 newborn animals suggested a skewed inheritance pattern, indicating that only 40% of the expected LGR4 null mice were born. For the LGR4 null mice viable at birth, most of them died within 2 d. Furthermore, the LGR4 null mice showed intrauterine growth retardation as reflected by a 14% decrease in body weight at birth, together with 30% and 40% decreases in kidney and liver weights, respectively. The present findings demonstrate the widespread expression of LGR4, and an essential role of LGR4 for embryonic growth, as well as kidney and liver development. The observed pre- and postnatal lethality of LGR4 null mice illustrates the importance of the LGR4 signaling system for the survival and growth of animals during the perinatal stage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE LEUCINE-RICH REPEAT (LRR)-CONTAINING, G protein-coupled receptors (LGRs) designated as LGR4 through LGR8 are structurally similar to receptors for gonadotropins and TSH (1, 2, 3). These receptors are characterized by a large N-terminal extracellular domain-containing LRRs and a seven-transmembrane region. Phylogenetic analysis showed that there are three LGR subfamilies: the classic glycoprotein hormone receptors, the second subgroup of LGR4, LGR5, and LGR6; and a third subgroup represented by LGR7 and LGR8 recently found to be the receptors for relaxin family ligands (4, 5, 6). These three subgroups of LGRs have an ancient evolutionary origin and existed before the divergence of vertebrates and invertebrates (1, 2, 6). LGR4, also known as GPR48, is a large protein consisting of 18 extracellular LRRs together with a seven-transmembrane region (1, 7). It shares only around 20% sequence identity with the glycoprotein hormone receptors but exhibited around 45% and 35% homology with LGR5 and LGR6, respectively (1, 7). Unlike LGRs in the other two subfamilies, the ligands and functions for the LGR4/5/6 subfamily are unclear. In an attempt to elucidate the physiological roles of this subgroup of orphan LGRs, we performed gene deletion experiments of the prototypic LGR4 gene using a mouse gene deletion model.

Generation of transgenic mice using the secretory-trap approach is ideal for large-scale functional analysis of secreted and membrane-spanning proteins (8, 9). When the trap vector integrates within the intron of a target gene, a ß-galactosidase fusion protein, including the N terminus of the target gene driven by its endogenous promoter, is produced. These insertions also effectively mutate the trapped gene to create null alleles. Here, we characterized the phenotypes of LGR4 null mice generated by this secretory trap approach to investigate the physiological roles of this receptor. Taking advantage of the expression of the fusion reporter gene controlled by the endogenous LGR4 promoter, we also performed detailed analysis of the tissue expression pattern of LGR4. The LGR4 gene showed a wide tissue distribution, and its disruption was associated with intrauterine growth retardation (IUGR) and perinatal lethality.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Disruption of the LGR4 Gene in Transgenic Mice
The LGR4 gene was mapped to mouse chromosome 2 (10). The mouse genomic sequence of the LGR4 coding region starts at nucleotide 9579071 and ends at nucleotide 9673612 of the mouse chromosomal contig NT_039209 (GenBank accession number). The predicted full-length cDNA sequence of mouse LGR4 (GenBank accession no. XM_355385) is consistent with the deduced sequence after comparison between the LGR4 partial cDNA (GenBank accession no. AK044357) and the genomic contig (Fig. 1AGoGo, upper panel). The deduced amino acid sequences of the mouse LGR4 protein show 98% and 95% similarity with the rat (GenBank accession no. NP_775450) and human (GenBank accession no. NP_060960) LGR4 protein sequences, respectively (Fig. 1AGoGo, lower panel). The mouse LGR4 gene contains 18 exons. Exon 1 encodes the signal peptide (SP) for secretion and the N-terminal LRR domain (LRRNT), whereas exons 2–17 encode the 17 LRR domain, and exon 18 encodes the seven-transmembrane domain as well as the intracellular region (Fig. 1AGoGo).



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  		Fig. 1. LGR4 Gene Targeting and Transgene Insertion in Mice as Well as Genotyping Using Genomic DNA

A, Genomic structure and corresponding coding regions of the mouse LGR4 gene. The LGR4 gene contains 18 exons with exon 1 encoding the signal peptide for secretion (SP) and the LRRNT domain. Exons 2–17 encode the 17 LRR domain, and exon 18 encodes the seven transmembrane region plus the C-terminal tail. The lower panel shows the alignment of the deduced amino acid sequences of mouse, rat (GenBank accession no. NP_775450) and human (GenBank accession no. NP_060960) LGR4 cDNAs. The full-length cDNA of mouse LGR4 has been deduced from partial cDNA (accession no. AK044357) and genomic (NT_039209) sequences. The predicted signal peptide is boxed, the seven-transmembrane domain is overlined, and the conserved amino acids are shaded. B, Transgene insertion in LGR4 mutant mice. The secretory-trap vector (11.98 kb) includes the mouse En-2 SA sequence, the CD4 transmembrane domain (CD4TM) inserted in frame with ß-geo (a fusion gene encoding ß-galactosidase, neomycin, and phosphotransferase), followed by the IRES fused to the PLAP gene and the simian virus 40 polyadenylation signal (8 11 12 ). The secretory-trap vector was inserted at the 5'-end of intron 1 of the LGR4 gene. Due to the presence of the SA site in the 5'-end of the insertion vector, transcript splicing occurred between this acceptor site and the SD site in exon 1 of the LGR4 gene. This random insertion resulted in the loss of LGR4 expression and leads to the expression of a chimeric mRNA encoding a protein composed of the LGR4 LRRNT domain and ß-galactosidase. In addition, the PLAP protein was also expressed due to the presence of the IRES. Expression of ß-galactosidase and PLAP proteins from the trap vector was under the control of the endogenous LGR4 promoter. C, PCR amplification of a LGR4 fragment and the transgene in wild-type (+/+), heterozygous (+/–), and null (–/–) mice. Triplex PCR was performed using genomic DNA as the template together with three primers. Primers A and B allowed the amplification of a LGR4 fragment (A/B, 805 bp) in the wild-type allele whereas primers A and C amplified a transgene fragment (A/C, 650 bp) in the mutant allele. Due to the insertion of the large (11.98 kb) trap vector, primers A and B could not amplify a PCR product in the mutant allele. In heterozygous animals, two PCR products were generated.

 


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  		Fig. 1A. Continued

 
Using the secretory-trap approach, the LGR4 gene was disrupted by random insertion of a secretory-trap vector (Fig. 1BGoGo). This vector includes the mouse En-2 splice acceptor sequence (SA), a fragment of CD4 containing the transmembrane domain inserted in frame with ßgeo (the LacZ-neomycin-phosphotransferase fusion gene), together with a downstream internal ribosome entry sequence (IRES), the placental alkaline phosphatase (PLAP) gene, and the simian virus 40 polyadenylation signal (8, 11, 12). PCR analyses indicated that the insertion of the secretory-trap vector resulted in the generation of a chimeric transcript containing the first exon of LGR4 fused to the ß-geo transcript, suggesting that the splicing occurred between the splice donor (SD) site in exon 1 of the LGR4 gene and the SA site of the trap vector (Fig. 1BGoGo).

To delineate the transgene insertion site, we performed PCR by using upstream primers complementary to different regions of intron 1 and a downstream primer complementary to the 5'-end of the trap vector. We localized the insertion site for the secretory-trap vector within intron 1 of the LGR4 gene at 6324 bp upstream from the 3'-end of exon 1 (nucleotide position 9585590 of the chromosome 2 genomic contig sequence, GenBank accession no. NT_039209) (Fig. 1BGoGo). By combining three primers (primers A and B, complementary to two sequences in the 5'-region of the LGR4 intron 1 and primer C, complementary to the 5'-end of the trap vector), we demonstrated the amplification by PCR of an expected fusion gene product (A/C; Fig. 1CGoGo, lower panel) and the wild-type product (A/B; Fig. 1CGoGo, lower panel) in the heterozygous mice. Only the fusion gene product A/C is present in the homozygous mice. The chimeric transcript was predicted to encode a truncated form of LGR4 that includes its N-terminal leucine-rich repeat (LRRNT) extracellular domain fused to the ß-galactosidase (Fig. 1BGoGo). The efficient translation of ß-galactosidase mRNA allowed convenient analysis of LGR4 expression by staining tissues with X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) in heterozygous and homozygous animals.

LGR4 Expression Based on ß-Galactosidase Transgene Expression in Mutant LGR4 Mice
Whole mount X-gal staining of heterozygous fetuses at embryonic d 19.5 (E19.5) to estimate the ß-galactosidase activity showed that the LGR4 gene was widely expressed in diverse tissues (Fig. 2Go and Table 1Go). X-gal staining was observed in brain (periventricular area, PV; olfactory bulb, OB), nasal cavity (NC), spinal column (S) (Fig. 2AGo, upper panel), rib cage (R), heart (H), and intestine (Fig. 2AGo, lower panel). In contrast, the wild-type fetus at E19.5 showed a background staining only in the intestine (Fig. 2BGo, arrows). In the heterozygous fetuses, kidney and adrenal gland (Fig. 2CGo), as well as stomach, bladder, bone, and cartilage (Table 1Go), also showed a strong ß-galactosidase expression. In contrast, liver, thymus, lung (Fig. 2AGo, lower panel), spleen, pancreas, and skeletal muscle did not show X-gal staining (Table 1Go). In adult heterozygous mice, whole-mount X-gal staining showed a similar expression pattern in most organs similar to that found in the fetuses (Table 1Go). However, in the adult, ß-galactosidase expression was weaker in the heart but stronger in the liver (Table 1Go).



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  		Fig. 2. Diverse Tissue Expression of the ß-Galactosidase Reporter Gene Driven by the Endogenous LGR4 Promoter in LGR4 Mutant Mice

A, Whole-mount staining of heterozygous (+/–) animals at embryonic d 19.5 (E19.5). Transverse sections of the embryo showed ß-galactosidase activity in different organs. Upper panel, Positive staining in the periventricular (PV) area, olfactory bulb (OB), nasal cavity (NC), and spinal column (S). Lower panel, Positive staining in the heart (H), intestine, and ribs (R), and minimal staining in the liver. No staining was apparent in the thymus (T) and lung. B, Whole-mount staining of wild-type animals at E19.5 served as a control. C, ß-Galactosidase activity in the kidney (K) and adrenal gland (Ad) at embryonic d 19.5 (E19.5) of heterozygous (+/–) but not wild-type embryos (+/+). D, ß-Galactosidase staining of kidney sections in null (–/–) newborns indicated strong staining in the parietal epithelium of Bowman’s capsule (arrowheads) surrounding the glomerulus (g) and in the epithelium of some tubules (t) (arrows). Sections were counterstained with Nuclear Fast Red.

 

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  		Table 1. Tissue Expression Pattern for LGR4 in Fetal/Newborn and Adult Mice

 
To determine the exact cell types expressing the ß-galactosidase transgene, X-gal staining was done on selective organ sections of heterozygous and homozygous newborns (day of birth, D0). In kidney sections, the transgene was found in the epithelial cells of some tubules (t) (Fig. 2DGo, arrows) and Bowman’s capsule of glomeruli (g) (Fig. 2DGo, arrowheads). The transgene was also expressed in the cardiac muscle of the heart, the epithelium, and the smooth muscle layer of the digestive tract of newborns (data not shown). Although no staining was apparent on the whole organ, low levels of staining were observed in liver, spleen, and medulla of thymus (Table 1Go).

LGR4 Expression Based on Immunohistochemical Analysis of Endogenous LGR4 in Wild-Type Mice
In addition to studies of transgene expression, we further analyzed endogenous LGR4 expression in wild-type animals by using a specific antibody generated against the ectodomain of LGR4. In the kidney, specific LGR4 staining was found in epithelial cells of selective tubules (t) (Fig. 3AGo, arrows). In contrast, no immunoreactivity was observed in the kidney stained with the preimmune serum (Fig. 3BGo). A strong immunoreactivity was also present in the zona fasciculata (f), but lower in the zona glomerulosa (g) of the adrenal gland (Fig. 3CGo). Furthermore, immunoreactive LGR4 was found in the epithelial cells (e) of the gut (Fig. 3DGo, arrow), together with lesser staining in the muscularis mucosae layer (m) (Fig. 3DGo). In addition, positive staining was found in the keratinized stratified squamous epithelium (e) of the nonglandular stomach (Fig. 3EGo, arrow). Only background immunoreactivity was observed in the stomach stained with the preimmune serum (Fig. 3FGo). These data confirmed the wide tissue distribution of the LGR4 receptor observed using the X-gal staining.



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  		Fig. 3. Immunolocalization of LGR4 in Wild-Type Newborn Mice Using Antibodies against the LGR4 Ectodomain

A, Epithelial cells of selective cortical renal tubules (t) (arrows) in the kidney cortex showed positive staining. B, No staining was detected in the section of cortex incubated with nonimmune serum. C, The zona fasciculata (f) of the adrenal gland was immunoreactive in contrast to low staining in the zona glomerulosa (g). D, Intestinal epithelial cells (e) were stained positively (arrow), whereas the muscularis mucosae (m) showed minimal reactivity. E, The keratinized, stratified squamous epithelium of the nonglandular stomach (e) (arrow) showed positive staining. F, Background staining was detected in a stomach section incubated with nonimmune serum.

 
Perinatal Lethality of LGR4 Null Mice
We compared the litter sizes among seven intercrosses of wild-type (+/+) parents, 14 intercrosses of heterozygous (+/–) parents, and 14 intercrosses between heterozygous (+/–) and wild-type (+/+) parents. As shown in Fig. 4Go (D0), a 17% decrease in litter size was found in the heterozygous intercrosses (8.6 ± 0.5 pups per litter) as compared with those from wild-type parents (10.4 ± 0.5 pups per litter). At weaning (Fig. 4Go; D21), further decreases in offspring survival were evident in heterozygous intercrosses (+/– x +/–; 6.5 ± 0.6 pups per litter) as compared with wild-type intercrosses (+/+ x +/+; 10 ± 0.4 pups per litter) (P < 0.05).



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  		Fig. 4. Litter Size at Birth and d 21 of Age (D21) in Wild-Type and Mutant LGR4 Mice

The number of pups per litter was determined from wild-type (+/+) and heterozygous (+/–) breedings at birth [d 0 (D0) and weaning (D21). Numbers in parentheses indicate the number of litters studied. Results are presented as the mean ± SEM. *, P < 0.05, –/– vs. +/+.

 
We further genotyped individual pups from 27 heterozygous intercrosses to estimate the ratio of wild-type, heterozygous, and null mice in the offspring. Based on the assumption that all wild-type pups, constituting 25% of offspring, are viable, we observed a decrease in the expected number of heterozygous and homozygous mice at birth (Table 2Go, upper portion). These data suggested that fetal death occurred during embryonic development, and only 40% of the expected homozygous pups were born. These LGR4 null mice never showed any apparent signs of respiratory distress but were smaller in size and became progressively weaker after birth. Indeed, most (eight of 14) of the surviving LGR4 null newborns died postnatally at 1 d after birth (D1). In addition, the heterozygous mice also showed embryonic lethality with 90% of the expected heterozygous pups found at birth, and 19% (14 of 73) of them died postnatally (Table 2Go, upper portion). Only one female and two male LGR4 null mice survived until the adult stage (Table 2Go, upper portion). They did not present obvious differences in external phenotypes in comparison with their littermates. Breeding tests indicated that both male and female LGR4 null mice were infertile. After being killed, the two LGR4 null males showed swollen testes and kidneys whereas the one LGR4 null female exhibited enlarged uteri.


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  		Table 2. Neonatal and Embryonic Lethality of LGR4 Null Mice

 
To confirm the embryonic lethality of mutant mice, a group of seven pregnant heterozygous mice were killed for the genotyping of their fetuses at embryonic day 19.5. As shown in Table 2Go (lower panel), only 76% and 91% of the expected homozygous and heterozygous pups were present, respectively. Furthermore, five of 13 (38.5%) homozygous fetuses at E19.5 were not viable at the time of surgery. These data confirm the predicted embryonic lethality of the LGR4 null fetuses.

Decreases in Body Weight and Lower Kidney and Liver Weights in Newborn LGR4 Null Mice
Although no gross morphological abnormalities were found for the LGR4 null mice, 14% and 20% decreases (P < 0.05) in body weight were observed at birth and on day 1, respectively, as compared with their wild-type littermates (Fig. 5AGoGo). In contrast, there were no differences between the body weight of heterozygous and wild-type mice. We further analyzed individual organ weights of these animals. The absolute organ weight of the kidney, liver, heart, lung, thymus, and spleen showed significant reduction in the homozygous newborns compared with the heterozygous and wild-type newborns (Fig. 5BGoGo). However, when the ratio of organ to body weight was compared, a major reduction of the ratio was found only for kidney and liver in the LGR4 null newborns as compared with the heterozygous and wild-type mice (Fig. 5CGoGo).



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  		Fig. 5. Body and Organ Weights of Wild-Type and Mutant LGR4 Mice

A, Body weight of newborns [d 0 (D0) and 1-d-old pups (D1)]. B, Absolute organ weights and (C) ratio of organ weight to body weight in wild-type and mutant newborns. Numbers inside the bars indicate the number of animals studied. Results are presented as the mean ± SEM; *, P < 0.05, –/– vs. +/+ and +/–.

 


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  		Fig. 5A. Continued

 
Histological Analysis of Organs of LGR4 Null Newborns
The observed decreases in kidney weight in LGR4 null newborns was confirmed by histological analysis, showing a size reduction (Fig. 6Go, A and B). However, the kidneys from LGR4 null mice showed similar histoarchitecture as compared with their wild-type counterparts (Fig. 6Go, A–D). No apparent difference in the cortico (co)-medullary (m) proportions and no obvious structural abnormality of the glomeruli (arrows) or tubules (arrowheads) were observed in the LGR4 null mice (Fig. 6DGo) as compared with wild-type animals (Fig. 6CGo).



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  		Fig. 6. Histological Analysis of Kidney in Wild-Type and LGR4 Null Newborn Mice

Hematoxylin-eosin-stained sagittal section of the kidney from the LGR4 null newborn (B) demonstrates the smaller compared with its wild-type littermate (A). Higher magnification of a kidney section shows the cortex (co) and the medulla (m), which contain glomeruli (arrows) and tubules (arrowheads). Other than size, no obvious differences were observed between LGR4 null (D) and wild-type (C) animals.

 
In addition to the kidney, the liver of LGR4 null newborns also showed a major weight reduction. However, histological analysis did not reveal any deviations from normal structure (data not shown). Similarly, no gross or histological abnormalities were noted in the stomach, intestine, urinary bladder, heart, lung, spleen, thymus, or pancreas (data not shown).

Lack of Changes in the Expression of IGF-I, IGF-II, and IGF Receptor Type-I (IGFR-I) in LGR4 Null Mice
Earlier experiments have demonstrated the importance of the IGF signaling system for embryonic and neonatal body and organ growth (13, 14, 15, 16). In particular, liver-derived IGF-I has been shown to be essential for in utero body growth (17), and kidney IGF-II is involved in renal development (18, 19). We tested possible changes in the expression levels of IGF-I, IGF-II, and IGFR-I transcripts in liver and kidney between wild-type and null newborn mice by RT-PCR. As shown in Fig. 7Go, no differences in the relative expression level of IGF-I, IGF-II, and IGFR-I in the liver (upper panel) and the kidney (lower panel) could be found between wild-type and null newborn mice.



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  		Fig. 7. IGF-I, IGF-II, and IGFR-I Transcript Levels in Liver and Kidney of Wild-Type and LGR4 Null Newborn Mice

Real-time PCR was performed to estimate the mRNA levels for IGF-I and related genes in liver (top panel) and kidney (lower panel). ß-Actin transcript levels were also estimated for all samples to derive the target gene/ß-actin ratios. Numbers inside the bars indicate the number of animals studied. Results are presented as the mean ± SEM.

 
Assays of Serum and Urine Markers
Serum chemistry analyses for blood urea nitrogen (BUN) and creatinine are routinely used to evaluate renal functions (20), and the liver function during perinatal period was assessed by the measurement of aspartate aminotransferase (AST) (21) and serum glucose levels (22). Serum and urine samples were collected at birth from wild-type and LGR4 mutant mice to perform marker tests. As shown in Table 3Go, no significant differences in serum glucose, AST, and BUN or urine BUN and creatinine levels were found between wild-type, heterozygous, and homozygous newborn mice.


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  		Table 3. Serum and Urine Markers in Wild-Type and Mutant LGR4 Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
LGR4 is an orphan receptor belonging to the LGR superfamily. Here, we characterized the expression pattern of LGR4 and investigated its physiological roles in null mice generated by a gene trap approach. Histochemical staining of endogenous LGR4 protein and ß-galactosidase transgene driven by the LGR4 gene promoter showed that LGR4 is highly expressed in kidney, adrenal gland, stomach, intestine, cartilage, bone, and heart of newborn mice. Genotyping and animal survival analysis of heterozygous intercrosses revealed embryonic lethality of the LGR4 homozygous fetuses. For the LGR4 null mice that survived at birth, most of them died within the first 2 d of life. Furthermore, they showed a 14% decrease in body weight at birth, together with decreases in kidney and liver weights of 30% and 40%, respectively. Histological analyses, however, did not reveal any kidney or liver abnormalities. The observed pre- and postnatal lethality, as well as the IUGR of LGR4 null mice, underlies the importance of the LGR4 signaling system for the survival and the growth of animals during the perinatal stage.

LGR4 null mice were generated using the secretory trap approach (9, 11, 12). The trapping of the LGR4 gene generated a fusion protein consisting of a portion of the extracellular domain of the LGR4 receptor fused to an intracellular membrane-tethered ß-galactosidase enzyme. The fusion proteins were sequestrated in the endoplasmic reticulum, minimizing any effect they may have at the cell surface (8). Creation of the LGR4 null mice was demonstrated by the PCR amplification of a fusion product between the LGR4 intron 1 and the trap vector, the absence of LGR4 wild-type transcripts, and the detection of the ß-galactosidase activity.

Taking advantage of the expression of the ß-galactosidase reporter gene driven by the endogenous LGR4 promoter, the expression of LGR4 was studied in heterozygous and homozygous mice (11). X-gal staining and PCR analysis (data not shown) of tissues from transgenic fetuses and newborns showed that the LGR4 gene was highly expressed in diverse tissues. Immunohistochemical staining of LGR4 of wild-type tissues further confirmed the LGR4 gene expression pattern, with high levels in epithelial cells of the kidney tubules and the gut, in the cardiac muscle, and in the zona fasciculata of the adrenal gland. A similar tissue expression profile was found in the adult, with the exception of higher expression of LGR4 in the liver and lower expression in the heart. These data are consistent with earlier analysis of LGR4 mRNA expression based on Northern blotting (7). Using the transgene expression analysis, we did not confirm the high expression of LGR4 mRNA in muscle observed by Loh et al. (7). The nature of this apparent discrepancy is not clear. Although similar patterns of LGR4 mRNA expression were found for most human tissues, human pancreas showed the highest transcript level (1, 7), perhaps due to species variations. Nevertheless, overall results suggest a wide tissue expression of LGR4. In addition to its expression in adults, LGR4 is also expressed in embryos (7) and could be involved in embryonic development.

LGR4 null mice showed a 14% reduction in body weight at birth, associated with pronounced decreases of kidney and liver weights. Similar weight reductions were observed in newborns of humans, rodents, and pigs exhibiting the syndrome of IUGR (23, 24, 25, 26, 27). Although IUGR has been associated with intrauterine infections, genetic factors, uteroplacental deficiency, and diseases of the mother, the causes for 40% of these cases are still unknown (28). Defects in the IGF and insulin pathways constitute examples of hormonal signaling abnormalities associated with IUGR (29, 30). The present finding of severe IUGR phenotypes in LGR4 null mice suggested that the LGR4 receptor pathway could be important in fetal growth.

Similar to LGR4 null mice, low body weight at birth has been found in mice with the mutated IGF-I, IGF-II, and IGFR-I gene (14, 15, 31, 32). Tissue-specific deletion of IGF-I in the liver further revealed that hepatic-derived IGF-I was necessary for intrauterine growth (17). However, in LGR4 null mice, hepatic IGF-I, IGF-II, and IGFR-I transcript levels were not changed, suggesting circulating levels of IGFs were maintained in LGR4 null newborns. Furthermore, LGR4 null mice did not show impaired development of the diaphragm and intercostal muscles found in IGF-I null mice (15). Moreover, an increase in liver weight was found in IGF-I null mice (14), whereas a decrease in liver weight was observed in LGR4 null mice. Apart from the common growth retardation found in LGR4 and IGF-II null newborns, IGF-II null mice did not exhibit perinatal lethality (31). These data suggest that the severe defects of LGR4 deficiency cannot be explained by decreases in IGF-II expression during embryonic development.

Although IUGR is usually associated with increases in perinatal mortality, the LGR4 null mice showed a more severe phenotype. Most of the LGR4 null newborn mice (eight of 14) and some heterozygotes (11 of 73) died on the first day after birth. However, these pups breathed normally, and milk was found in their stomachs. They became progressively weaker and eventually showed postnatal lethality due to unknown causes. The observed lethality of heterozygous LGR4 pups suggests the importance of the LGR4 gene in newborn survival. The essential role of LGR4 in overall development was underscored by intrauterine death, evidenced by a reduced number of homozygous and heterozygous pups at birth and by the presence of nonviable fetuses in utero.

In LGR4 null mice, the kidney showed a 30% weight reduction compared with wild-type animals. This phenotype could be the consequence of defects in renal morphogenesis, which is dependent upon the IGF signaling system (33). IGF-I, IGF-II, and IGFR-I are expressed in the mouse fetal kidney and IGF-II is the major fetal form essential for kidney differentiation (19) and metanephros growth (18). However, in the kidneys of LGR4 null newborns, we did not observe a decrease in IGF-I, IGF-II, and IGFR-I mRNA levels, suggesting that the IGF paracrine system was not disrupted at this stage of development. Indeed, histological analysis of kidney sections did not show any structural abnormalities. Using platelet-endothelial cell adhesion molecule-1 immunostaining of the endothelial cells of the glomeruli capillaries (34), we did not observe obvious abnormalities in the vascularity of the glomeruli in LGR4 null newborn kidneys (data not shown). Likewise, serum BUN levels were similar in wild-type, heterozygous, and homozygous newborns. These data suggest that the kidney filtration ability was not altered in LGR4 null mice, and kidney malfunction does not likely account for the perinatal death of the newborns.

LGR4 gene disruption affected liver growth with a 40% weight reduction. However, we did not observe hepatic structural abnormalities in LGR4 null mice. Disproportionate alteration of the fetal liver mass has been described in disorders that cause augmented or diminished somatic growth. In the rat, IUGR induced by artery ligation or maternal fasting is associated with a 30% decrease in liver weight (23, 35). However, our data showed that LGR4 expression was minimal in newborn liver, suggesting that the observed liver abnormality could be due to LGR4 action in extrahepatic sites. Despite the observed reduction in liver size in LGR4 null mice, we did not observe impaired liver function. Serum levels of the hepatic enzyme, AST (21), were similar between wild-type and null newborns. We also did not detect significant changes in serum glucose levels. Glucose is a major metabolic fuel during perinatal development, and failure to correct postnatal hypoglycemia is lethal in neonatal rats (22). Normal serum glucose levels observed in LGR4 null newborn suggest that liver glycogenolysis and gluconeogenesis were not altered (22, 36).

The present results demonstrate the wide tissue expression of LGR4 and the importance of this orphan receptor in embryonic and neonatal development. The observed decreases in body growth and organ development suggest an IUGR phenotype. Both fetal and perinatal mortality occurred, not allowing the identification of a single cause of death. Future development of tissue-specific LGR4 null mice is needed to elucidate the essential role of LGR4 in kidney and diverse other tissues expressing this orphan receptor. In addition, the eventual identification of the cognate ligand for this receptor could further advance our understanding of the physiological roles of this ligand/receptor system during fetal and neonatal development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of Transgenic Mice by the Gene-Trap Strategy
LGR4 null mice were generated based on the secretory-trap approach as previously described (8, 11, 12) by disrupting the endogenous LGR4 gene (Fig. 1BGoGo). The secretory-trap vector (pGT0,1,2tm-pfs, 11.98 kb) includes the mouse En-2 SA sequence, a fragment of CD4 containing the transmembrane domain inserted in frame with ßgeo (the LacZ-neomycin-phosphotransferase fusion gene) together with a downstream IRES, the PLAP gene, and the simian virus 40 polyadenylation signal in the pGT1tm vector (Fig. 1BGoGo) (8, 11, 12). PCR analyses indicated that transcript splicing occurred between the splice donor site in exon 1 of the LGR4 gene and the SA site of the trap vector. This insertion resulted in the generation of two proteins. The first chimeric protein composed of the first 151 amino acids of LGR4 corresponding to the LRRNT type domain fused to the transmembrane region of CD4, together with the ß-galactosidase enzyme encoded by the trap vector. Due to the presence of the IRES, the PLAP protein was also generated.

Mouse Husbandry and Genotyping
Mice were housed under controlled humidity, temperature, and light regimen and fed ad libitum. Animal care was consistent with institutional and NIH guidelines. Heterozygous C57Bl/6J mice were intercrossed with Swiss Webster mice; 35 intercross litters were obtained by pairing male and female animals overnight. The day when the copulation plug was observed was considered as embryonic d 0.5 (E0.5) whereas the day of birth was designated as d 0 (D0). Approximately 250 mice (27 litters) from heterozygous intercrosses were genotyped to perform the present study. For genotyping, genomic DNA was isolated from tail biopsy specimens using the genomic DNA extraction kit (Promega Corp., Madison, WI). PCR was carried out using three primers (Fig. 1CGoGo): upstream primer A, 5'-CCAGTCACCACTCTTACACAATGGCTAAC-3' (nucleotide 9585044 to 9585072 in the mouse chromosome 2 contig) located in intron 1 of LGR4; downstream primer B, 5'-ATTCCCGTAGGAGATAGCGTCCTAG-3' (nucleotide 9586038 to 9586062) in intron 1 of LGR4; and the second downstream primer C, 5'-GGTCTTTGAGCACCAGAGGAC-3' (5'-end of the trap vector). Two PCR products were expected: the wild-type PCR product A/B (805 kb) and the transgene PCR product A/C (650 kb). Due to the insertion of the large 11.98-kb trap vector between primer A and primer B, the A/B PCR product could not be amplified in the transgene allele under the present PCR conditions (Fig. 1C).

X-gal Staining and Histological Analyses
To perform X-gal staining in heterozygous and homozygous newborns, whole embryos (E19.5) or organs were dissected in PBS and incubated with the ß-galactosidase fixative (0.2% glutaraldehyde, 1.5% formaldehyde, 2 mM MgCl2, 5 mM EGTA, 0.1 M sodium phosphate buffer, pH 8) for 15 min to 5 h. Skin was removed from embryos before fixation. Tissues were washed three times for 30 min in the washing buffer [0.1 M sodium phosphate buffer (pH 8), 2 mM MgCl2, 5 mM EGTA, 0.01% (wt/vol) sodium deoxycholate, 0.02% (vol/vol) Nonidet P40]. Subsequently, tissues were incubated in the staining solution [washing buffer containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6·6 H2O, and 1 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Invitrogen, Carlsbad, CA)] for 3 h at room temperature. The tissues were then rinsed in PBS before fixation in 3.7% formaldehyde overnight at 4 C. Photography was performed with transillumination using a dissecting microscope (Leica Microsystem, Bannockburn, IL). Wild-type newborns were used as negative controls. To perform ß-galactosidase staining of tissue sections, organs were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA), frozen on dry ice, and stored at –80 C. Cryosections (8 µm thickness) were dried for 1 h at room temperature. Fixation and staining were performed as described for whole organ processing. Counterstaining with Nuclear Fast Red (Sigma-Aldrich, St. Louis, MO) was performed before dehydration, clearing in Neo-clear (EM Science, Gibbstown, NJ), and coverslipping with Permount (Fisher Scientific, Santa Clara, CA).

For histological analysis, different organs from newborns were weighed and fixed by immersion in Bouin’s fixative for 16 h. After dehydration and embedding, paraffin blocks were sectioned at 4 µm thickness and stained with hematoxylin and eosin using standard procedures.

LGR4 immunohistochemical analysis was performed using rabbit polyclonal antibodies against the ectodomain of the receptor. For LGR4 antibody production, cDNA corresponding to the ectodomain of human LGR4 was subcloned into the pcDNA3.1Zeo vector (Invitrogen, San Diego, CA) and stably transfected in 293T cells. The LGR4 ectodomain protein was purified using Nickel-affinity columns and emulsified in Freud’s adjuvant before injection into rabbits to generate polyclonal antibodies. IgG was purified from bleeds using the Protein G Sepharose 4 Fast Flow column (Amersham Biosciences Corp., Piscataway, NJ). This specific LGR4 antiserum (dilution 1:1000) was used to localize LGR4 expression in wild-type newborn mice. Substitution of the primary antibody with the rabbit preimmune serum served as the negative control. Staining was performed using the Histostain-SPAEC kit following manufacturer’s instructions (Zymed Laboratories, Inc., South San Francisco, CA).

Quantification of IGF-I, IGF-II, and IGFR-I Transcript Levels Based on Real-Time PCR Analysis
mRNAs were extracted from different organs using the RNAeasy kit (QIAGEN, Valencia, CA). cDNA was synthesized with the Omniscript reverse transcriptase (QIAGEN) using the oligo-dT primer (Invitrogen). PCR was performed using SmartCycler (Cepheid, Inc., Sunnyvale, CA) according to the manufacturer’s protocol. The primers and probes for real-time PCR were as follows. IGF-I: sense, 5'-GATACACATCATGTCGTCTTCACAC-3'; antisense, 5'-GAACTGAAGAGCATCCACCAG-3'; probe, 5'-6-carboxy-fluorescein (FAM)-CTCTTCTACCTGGCGCTCTGCTTG-6-carboxy-tetramethyl-rhodamine (TAMRA)-3'; IGF-II: sense, 5'-GACCGCGGCTTCTACTTCAGCA-3'; antisense, 5'-GGAAGCAGCACTCTTCCACGATG-3'; probe, 5'-FAM-CTTCAAGCCGTGCCAACCGT-TAMRA-3'; IGFR-I: sense, 5'-GACATCTACGAGAC-GGACTACTACC-3', antisense, 5'-AGAATGAGTAGTGAAGACACCATCC-3'; probe, 5'-FAM-TGCCTGTGCGCTGGATGTCT-TAMRA-3'; and ß-actin: sense, 5'-GGACCTGACGGACTACCTCATG-3'; antisense, 5'-TCTTTGATGTCACGCACGATTT-3'; probe, 5'-FAM-CCTGACCGAGCGTGGCTACAGCTTC-TAMRA-3'. To determine the copy number of target transcripts, IGF-I, IGF-II, IGFR-I, and ß-actin cDNAs were used to generate calibration curves by plotting the threshold cycle (Ct) vs. the known copy number for each plasmid template. The copy numbers for target samples were determined according to the calibration curve. To correct for differences in RNA extraction, data were normalized by dividing the copy number of the target cDNA by that of ß-actin.

Blood and Urine Analysis
Blood was collected from the aorta of newborn mice in 50-µl nonheparinized capillary tubes. After clotting of blood, serum samples were analyzed for AST, glucose and BUN content after 1:5–1:20 dilutions. Urine was collected in 50-µl capillary tubes after gentle massage of the bladder. Urine samples were also analyzed for their BUN and creatinine content. All tests were performed by Stanford’s Veterinary Service Center’s diagnostic laboratory using standard procedures.

Data Analysis
All experimental data are presented as the mean ± SEM. Statistical significance was determined by ANOVA for multiple-group comparisons and by Tukey’s post-test. Significance was accepted at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Forbes (Department of Pediatrics, University of Virginia, Charlottesville, VA) for helpful advice on kidney structure and immunohistochemistry and Pauline L. Chu for technical advice on histology processing. We thank the personnel of the Research Animal Facility (Stanford University School of Medicine) for care and monitoring of animals and are grateful to Dr. G. S. Barsh’s laboratory (Departments of Pediatrics and Genetics, Stanford University) for the use of their stereoscope and digital camera system. We thank C. Spencer for editorial assistance.


   FOOTNOTES
 
This work was supported by the National Institutes of Health Grant HD23273 and by the National Institute of Mental Health Grant RO1 MH60612 (to M.T.-L.).

Abbreviations: AST, Aspartate aminotransferase; BUN, blood urea nitrogen; FAM, 5'-6-carboxy-fluorescein; IGFR-I, IGF receptor type I; IRES, internal ribosome entry sequence; IUGR, intrauterine growth retardation; LGR, leucine-rich repeat-containing, G protein-coupled receptor; LRR, leucine-rich repeat; LRRNT, N-terminal LRR; PLAP, placental alkaline phosphatase; SA, splice acceptor; SD, splice donor; TAMRA, 6-carboxy-tetramethyl-rhodamine; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.

Received for publication March 31, 2004. Accepted for publication May 28, 2004.


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