Lrp6 Mutant Mouse Lines in Comparison to β-Catenin Conditional Mutant Mouse Lines
In this study, we demonstrated that most types of the external organ defects in the Lrp6-floxdel homozygous mice were identical to those seen in the conventional Lrp6-βgeo homozygotes. In our conditional gene-targeting mice, the 394-bp exon 2 of Lrp6 gene was floxed by two loxP sites, which caused a frameshift by Cre deletion resulting in severe but identical defects. The mouse Lrp6 gene was located in chromosome 6. The known protein-coding transcript (NM_008514.3) consisted of 23 exons (that encoded a transcript of 9,368 base pairs with the translation of 1,613 amino acids). Thus, the genomic deletion of exon 2 only allowed translation of a short nonfunctional residue (18 amino acids) encoded by the exon 1 of Lrp6.
By contrast, the conventional Lrp6-βgeo mutant mouse line was generated by a gene-trap approach, in which the βgeo (fused β-galatosidase and neomycine) reporter cassette joined to the first 321 amino acids of the Lrp6 protein in-frame (Pinson et al.,2000). No Lrp6 mRNA was detected from the Lrp6-βgeo homozygous embryos and embryonic fibroblasts by Northern blot (Pinson et al.,2000). The lack of Lrp6 mRNA and severe defects in the conventional Lrp6 mutant mice suggest that the residual first 321 amino acids is not functional in vivo, and that no functional splice variants of Lrp6 may exist.
Of interest, two spontaneous point mutant mouse lines of Lrp6 were identified. The crooked tail mice with exencephaly (cranial but not spinal neural tube defects) were found to have a single nucleotide substitution in exon 7, which resulted in a G494D conversion in the second YWTD-EGF repeat domain of Lrp6 (Carter et al.,2005). In the ringelschwanz mice with spina bifida (the spinal neural tube defects) and skeletal defects, a missense mutation was found, resulting in a R886W transition in the Dkk-binding region of Lrp6 (Kokubu et al.,2004). In contrast to the severe and broad defects with embryonic lethality seen in Lrp6-βgeo and Lrp6-floxdel mice, these spontaneous Lrp6 mutant mice could survive partially or fully after birth with milder and more limited organ defects. The region- or organ-specific birth defects in the mouse models were linked with point mutation sites in the Lrp6 gene, suggesting that Lrp6 may also play similar roles in human genetic diseases.
Lrp6 plays a key role in the canonical Wnt/β-catenin signaling pathway. Three conditional mutant mouse lines of β-catenin have been generated for loss- and gain-of-function analyses (Harada et al.,1999; Brault et al.,2001; Huelsken et al.,2001; Grigoryan et al.,2008). Nevertheless, the use of the Lrp6 conditional mutant mouse line will provide us with significantly distinct implications in comparison to the β-catenin conditional mutant mouse lines. In general, the β-catenin conditional mutants will have even more severe and broader defects than seen in the Lrp6 conditional mutants. This is largely because β-catenin has dual roles in Wnt signaling and cell adhesion (Brembeck et al.,2006). β-Catenin null mice die during gastrulation (Haegel et al.,1995), while Lrp6-null mice can survive around birth with broad but unique organ defects. Thus, the Lrp6 conditional mutant mouse line can be used specifically for modeling and dissecting birth defects/congenital diseases.
Disease Connection of the Lrp6 Conditional Mutant Mouse Model and the Phenotypic Influence of the Genetic Background
Each year approximately 3% of babies are born with physical or mental abnormalities resulting from birth defects or developmental disorders (Canfield et al.,2006). The leading categories of birth defects are congenital cardiovascular defects (1 in 115 births), musculoskeletal defects (1 in 130 births), genital and urinary tract disorders (1 in 135 births), nervous system and eye defects (1 in 235 births), cleft lip/palate (1 in 930 births), and spina bifida (1 in 2,000 births; estimated incidences by March of Dimes Perinatal Data Center, 2000). Most formats of these common birth defects are seen in Lrp6 mutant mice.
Particularly in this study, we observed the severe cleft palate and less dominant cleft lip in Lrp6-floxdel mice. Cleft lip with or without cleft palate (CLP) is a result of the failure of fusion in the lip and/or roof of the mouth during early embryonic development with complex but largely unknown etiology (Schutte and Murray,1999; Cobourne,2004). Although CLP is common in humans but is rare in mice, in which many mutant mouse lines exhibit only cleft lip and with incomplete penetrance. Thus, the Lrp6 mutant mouse will be a valuable genetic model for CLP study. We have recently found the severe CLP in the conventional Lrp6-βgeo mice with full penetrance (Song et al.,2009a). The cleft lip in Lrp6-floxdel mutants is less severe than seen in Lrp6-βgeo mice. Different strain backgrounds in these mutant mouse lines may cause the varied severity of cleft lip. We used FLP1 and CMV-Cre mouse lines to delete the FRT-flanked neo cassette and the loxP-flanked exon 2 in the Lrp6-loxP-Neo-FRT mice, respectively. The FLP1 transgenic construct was injected into fertilized eggs from B6SJLF2 mice, and then crossed with C57BL/6 mice (Rodriguez et al.,2000). The CMV-Cre mice were derived from BALB/c-1 background and backcrossed to the C57BL/6 background (Schwenk et al.,1995). The conventional Lrp6-βgeo mice were maintained in C57BL/6 background with early lethality of some homozygous embryos which died between E9.5 to E11.5 (Song et al.,2009b). Therefore, we backcrossed and maintained the Lrp6-floxdel mice in a compound background of C57BL/6 and CD1, which increased the survival rate of the Lrp6-floxdel homozygous embryos during the late gestation with expected Mendelian rations. However, all of the non-C57BL/6 backgrounds, particularly the CD1 background, may contribute to the reduced severity of cleft lip in Lrp6-floxdel mice.
In addition, it has been reported that exencephaly, the cranial neural tube defect, occurred in approximately half of the Lrp6-βgeo homozygous mice at C57BL/6 background (Pinson et al.,2000). We noted a low occurrence rate (approximately 1 in 10) of the exencephaly in Lrp6-floxdel homozygotes at the compound background of C57BL/6 mixed with CD1. By contrast, we observed full penetrant spina bifida (the caudal neural tube defects) in either Lrp6-βgeo or Lrp6-floxdel homozygotes regardless of their different strain backgrounds. These results suggest that the phenotypic influence of the mouse strain background is limited to particular, but not all fundamental defects existed in Lrp6-deficient mice.
We also detected full penetrant agenesis of external genitalia with no visible structures in either Lrp6-floxdel or Lrp6-βgeo homozygous embryos. Recently, β-catenin has been shown to play important roles in all three germ layer-derived endodermal, mesenchymal, and ectodermal lineage cells during early development of external genitalia (Lin et al.,2008). These results suggest that Lrp6 may act upstream of β-catenin in the canonical Wnt signaling pathway to regulate the external genitalia morphogenesis. Another possibility of the agenesis of external genitalia in Lrp6-deficience mice may be the indirect cause of the truncation of caudal body axis by loss-of-function of canonical Wnt signaling. The severe disruption of the caudal body axis in Lrp6 null embryos has been suggested to resemble the phenotypes observed in Wnt3a null or the hypomorphic vestigial tail mice which lack many posterior structures and can be affected by Wnt3a dosage and genetic background (Greco et al.,1996; Pinson et al.,2000). There are no previous reports of external genitalia defects in either Wnt3a or Lrp6 mutant mice. However, the external genitalia are likely still developed, by re-examining the published data of Wnt3a/vestigial tail mutant mice (Fig. 4B in Greco et al.,1996 and Fig. 3c in Pinson et al.,2000), or the compound mutant mice of vestigial tail and Lrp6-βgeo mice (Fig. 3b,d in Pinson et al.,2000). These data suggest that Lrp6 and its mediated canonical Wnt signaling may play a direct role in genitalia development, which is independent from their roles in caudal neural tube development.
The successful generation of the conditional Lrp6-flox mice in this study provides a significant opportunity to dissect the tissue- or cell lineage-specific roles of Lrp6-mediated signaling pathways during organogenesis and related birth defects. Many Cre transgenic mouse lines have been used to dissect the cell lineage-specific roles of β-catenin during development (Grigoryan et al.,2008). These Cre mouse lines can be crossed with our Lrp6-flox mice to investigate the distinct and common roles of Lrp6 and β-catenin in development and related congenital defects.