Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension

Abstract

The physiological role of an orphan G protein-coupled receptor, LGR5, was investigated by targeted deletion of this seven-transmembrane protein containing a large N-terminal extracellular domain with leucine-rich repeats. LGR5 null mice exhibited 100% neonatal lethality characterized by gastrointestinal tract dilation with air and an absence of milk in the stomach. Gross and histological examination revealed fusion of the tongue to the floor of oral cavity in the mutant newborns and immunostaining of LGR5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos. The observed ankyloglossia phenotype provides a model for understanding the genetic basis of this craniofacial defect in humans and an opportunity to elucidate the physiological role of the LGR5 signaling system during embryonic development.

FIG. 1.

Targeted disruption of the LGR5 genes and genotyping of LGR5 null mice. (A) Schematic representation of LGR5 genomic DNA, the targeting vector, and the disrupted gene. The LGR5 gene contains 18 exons, with exon 1 encoding the signal peptide for secretion (SP) and the N-terminal leucine-rich repeat domain. Exons 2 to 17 encode the 17 leucine-rich repeat (LRR) domains, and exon 18 encodes the seven-transmembrane region plus the C-terminal tail. In the targeting vector, the transgene containing the LacZ-MC1 neo cassette replaced exon 18 of the LGR5 gene, leading to the loss of LGR5 expression in the targeted allele. (B) PCR genotyping of wild-type (+/+), heterozygous (+/−), and LGR5 null (−/−) mice. Triplex PCR was performed with genomic DNA as the template together with three primers. Primers A and C allowed the amplification of an LGR5 gene fragment (673 bp) in the wild-type allele, whereas primers B and C amplified a chimeric gene fragment (467 bp) in the mutant allele. In heterozygous animals, both PCR products were generated. (C) Lack of expression of the LGR5 ectodomain-LacZ transgene in LGR5 null mice. (Top panel) Location of two sets of primers used to amplify the ectodomain of LGR5. (Lower panel) Amplification of LGR5 transcripts (a and b primers, 524 bp; c and d primers, 540 bp) in wild type (+/+) but not LGR5 null (−/−) mice. The levels of β-actin serve as positive controls.

FIG. 2.

Tissue expression pattern of LGR5 in mice. Real-time RT-PCR analyses were performed to estimate LGR5 transcript levels in diverse tissues from neonatal (left panel) and adult (right panel) mice. Results are expressed as the ratios between LGR5 and β-actin transcripts for normalization.

FIG. 3.

Characterization of LGR5 antibodies and immunostaining of LGR5 in adrenal gland. (A) Immunoblot analyses of affinity-purified 5BP corresponding to the ectodomain of LGR5. Some samples were pretreated with N-glycosidase to remove N-linked carbohydrate side chains. (B) Immunostaining of LGR5 in the adrenal glands of adult mice. Ab, antibody; C, cortex; M, medulla; R, zona reticularis.

FIG. 4.

Dilated gastrointestinal tract and abnormal craniofacial development in LGR5 null mice. (A) The wild-type LGR5 (+/+) neonates had milk in their stomachs (S), whereas the LGR5 null (−/−) neonates had dilated stomachs without milk. (B) The entire gastrointestinal tract of LGR5 null mice was dilated without milk, whereas the wild-type mice had a normal appearance. SI, small intestine. (C) In sagittal sections of the craniofacial region, the LGR5 null mice showed fusion of the tongue (T) to the mandible (M), whereas these two regions are separated in the wild-type mice. Identification of similar structures in the upper and lower jaws indicates the sections were taken at the same level. I, developing upper incisor; AB, developing alveolar bone of lower incisor. (D) In transverse serial sections of the mandible region, the tongue of LGR5 null mice was attached to the mandible, whereas the tongue of the wild-type animals in the same region was connected only in a posterior section. Similar sections are reflected by the morphology of the molar teeth (mt), and more anterior sections are shown on the right. (E) Immunostaining of LGR5 antigen in the epithelium of the tongue and the epithelium and mesenchyme of the mandible at E14.5. Ab, antibody. The boxed area in the upper panel is enlarged in the lower panels.

FIG. 4.

Dilated gastrointestinal tract and abnormal craniofacial development in LGR5 null mice. (A) The wild-type LGR5 (+/+) neonates had milk in their stomachs (S), whereas the LGR5 null (−/−) neonates had dilated stomachs without milk. (B) The entire gastrointestinal tract of LGR5 null mice was dilated without milk, whereas the wild-type mice had a normal appearance. SI, small intestine. (C) In sagittal sections of the craniofacial region, the LGR5 null mice showed fusion of the tongue (T) to the mandible (M), whereas these two regions are separated in the wild-type mice. Identification of similar structures in the upper and lower jaws indicates the sections were taken at the same level. I, developing upper incisor; AB, developing alveolar bone of lower incisor. (D) In transverse serial sections of the mandible region, the tongue of LGR5 null mice was attached to the mandible, whereas the tongue of the wild-type animals in the same region was connected only in a posterior section. Similar sections are reflected by the morphology of the molar teeth (mt), and more anterior sections are shown on the right. (E) Immunostaining of LGR5 antigen in the epithelium of the tongue and the epithelium and mesenchyme of the mandible at E14.5. Ab, antibody. The boxed area in the upper panel is enlarged in the lower panels.