SummarySkeletal defects in ringelschwanz mutant mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis Research articleDevelopment and disease 5470 of somitogenesis (Aulehla et al., 2003; Hamblet et al., 2002) and of bone mass in adults in humans and mice (Boyden et al., 2002; Gong et al., 2001;Kato et al., 2002;Little et al., 2002). In the context of somite development, Wnt signaling mediated by Wnt3a has been implicated in the specification and propagation of progenitor cells of the paraxial mesoderm in the primitive streak or in the tail bud (Takada et al., 1994;Yoshikawa et al., 1997), and this Wnt signaling is transduced through the canonical β-catenin signaling pathway (Galceran et al., 1999; Galceran et al., 2001). As late functions, Wnt signaling is also known to play essential roles in the dorsoventral patterning of formed somites, which is required for proper development of the dermomyotome and the myotome (Capdevila et al., 1998; Fan et al., 1997;Münsterberg et al., 1995;Wagner et al., 2000). However, whether Wnt signaling plays any significant role in the periodic morphogenetic movement of somitogenesis that takes place in the presomitic mesoderm (PSM) had not been clear until recently. Mouse dishevelled 2 (Dvl2), together with its paralog dishevelled 1 (Dvl1), has recently been shown to be required for somite segmentation, through the analysis of Dvl2-single and Dvl1;Dvl2-double knockout mice (Hamblet et al., 2002). Furthermore, it has recently been demonstrated that a paralog of Axin, Axin2 (also called conductin) exhibits a dynamic and cyclic expression profile in the PSM. This finding, together with the detailed analysis of the notch-delta signaling activity in Wnt3a mutants, has provided clear evidence for the involvement of Wnt signaling in the process of somitogenesis in the PSM, functioning upstream of notch-delta signaling (Aulehla et al., 2003). On the other hand, recent studies have also elucidated another, unexpected functional aspect of Wnt signaling in the postnatal life, with the identification of Lrp5 as one of the key genetic factors that control bone mass. Positional cloning of the gene responsible for osteoporosispseudoglioma syndrome (OPPG), an autosomal recessive disorder in humans, revealed that loss-of-function mutations in LRP5 lead to a low bone mass phenotype (osteoporosis) (Gong et al., 2001). Despite the availability of Lrp6-null mouse mutants (Pinson et al., 2000), whether Lrp6 plays any roles in somitogenesis during development and in the control of bone mass during adult life has not been known, because of strong pleiotropic effects of Lrp6 deficiency that leads to neonatal lethality (Pinson et al., 2000). In the present study, we demonstrate that Lrp6 is required for somitogenesis and osteogenesis, through the analysis of a novel spontaneous mouse mutation ringelschwanz (rs), identified in this study as a viable hypomorphic allele of Lrp6. Materials and methods MiceThe rs mutant strain is maintained on the BALB/c background. For a backcross m...
Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect of these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic mutations in GATA1. To better understand functional interplay between trisomy 21 and GATA1 mutations in hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs) and genome-editing technologies. Comparative analysis of these engineered iPSCs demonstrated that trisomy 21 perturbed hematopoietic development through the enhanced production of early hematopoietic progenitors and the upregulation of mutated GATA1, resulting in the accelerated production of aberrantly differentiated cells. These effects were mediated by dosage alterations of RUNX1, ETS2, and ERG, which are located in a critical 4-Mb region of chromosome 21. Our study provides insight into the genetic synergy that contributes to multi-step leukemogenesis.
Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.
Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.
Low-density lipoprotein receptor-related protein 5 (LRP5) regulates bone acquisition by controlling bone formation. Because roles of LRP6, another co-receptor for Wnts, in postnatal bone metabolism have not been fully elucidated, we studied bone phenotype in mice harboring an Lrp6 hypomorphic mutation, ringelschwanz (rs), and characterized the mutant protein.
A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.
The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4,6-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.Transposable elements, constituting a large part of the mammalian genome, may have a major impact on the evolution and integrity of the genome (30). One of the most active transposases in mammalian cells, Sleeping Beauty (SB), was reconstructed from the salmonid genome and it is categorized within the Tc1/mariner superfamily of transposons (20). SB-based gene mobilization has been utilized as a tool for insertional mutagenesis (3,4,7,14,19,24) and several gene therapy paradigms (32, 39). The SB transposon system has been developed as a nonautonomous system consisting of two independent components: transposon and transposase. The SB transposon is a DNA fragment flanked by the terminal inverted repeats (IRs) and is mobilized by SB transposase. Each IR contains two copies of a short (15-to 20-bp) direct repeat (DR), and the resultant structures are named IR/DRs. SB transposase can initiate a transposition event by binding to IR/DRs. The two IR/DRs are then probably paired through interactions of the transposase subunits (21), thereby forming a synaptic complex (Fig. 1A), followed by excision and reinsertion of the SB transposon into another locus. The excision events result in a "footprint" containing several additional base pairs at the excised site.The epigenetic modification for transposable-element mobility is an important issue for understanding the relationship between host and exogenous elements, such as transposons. When the copy number of the retrotransposon is increased in Arabidopsis, the transposed retrotransposon is inactivated by DNA methylation. These retrotranspo...
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