Gene-trapped Embryonic Stem Cell Research Articles

Mutant non-coding RNA resource in mouse embryonic stem cells.

ABSTRACTGene trapping is a high-throughput approach that has been used to introduce insertional mutations into the genome of mouse embryonic stem (ES) cells. It is performed with generic gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA sequence tag for the rapid identification of the disrupted gene. Large-scale international efforts assembled a gene trap library of 566,554 ES cell lines with single gene trap integrations distributed throughout the genome. Here, we re-investigated this unique library and identified mutations in 2202 non-coding RNA (ncRNA) genes, in addition to mutations in 12,078 distinct protein-coding genes. Moreover, we found certain types of gene trap vectors preferentially integrating into genes expressing specific long non-coding RNA (lncRNA) biotypes. Together with all other gene-trapped ES cell lines, lncRNA gene-trapped ES cell lines are readily available for functional in vitro and in vivo studies.

Inactivation of the microRNA-183/96/182cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain.

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.

Initial characterization of mice null for Lphn3, a gene implicated in ADHD and addiction

The LPHN3 gene has been associated with both attention deficit-hyperactivity disorder (ADHD) and addiction, suggesting that it may play a role in the etiology of these disorders. Unfortunately, almost nothing is known about the normal functions of this gene, which has hampered understanding of its potential pathogenic role. To begin to characterize such normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression assays. Most notably, we found changes in dopamine and serotonin receptors and transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th, Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were examined at 4–6weeks of age, null mutants showed increased levels of dopamine and serotonin in the dorsal striatum. Finally, null mutant mice had a hyperactive phenotype in the open field test, independent of sex, and were more sensitive to the locomotor stimulant effects of cocaine. Considered together, these results suggest that Lphn3 plays a role in development and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is mediated through alterations in monoamine signaling.

Enhanced Hippocampal Long-Term Potentiation and Fear Memory in Btbd9 Mutant Mice

Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

Growth Defects and Impaired Cognitive–Behavioral Abilities in Mice with Knockout for Eif4h, a Gene Located in the Mouse Homolog of the Williams-Beuren Syndrome Critical Region

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1−/− animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated Aaligned (Aal) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1−/− gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1−/− testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

294 Generation of endoplasmic reticulum protein 44 (ERP44) knockout/knockin mice to investigate the role of ERP44 in calcium signaling and ER stress in cardiomyocytes

BACKGROUND: ERp44 is a novel unfolded protein response (UPR)-induced endoplasmic reticulum (ER) protein of the thioredoxin family. ERp44 transcripts are widely expressed in a variety of tissues and cell types and has been shown to be up regulated upon ER stress. ERp44 may be involved in multiple cellular functions including the inhibition of Ca release by inactivating inositol 1,4,5-trisphosphate receptor1 (IP3R1), and by supporting disulfide bond formation through reinforcing the Ero1 /oxidoreductase system. The role of ER stress in various cardiomyopathies is becoming clearer. Here, we hypothesize that ERp44 plays important roles in ER stress involved in heart disease by regulation of Ca signaling and activation of the ER stress response signaling. Dysregulation of Ca cycling in myocytes will activate ER stress pathway and lead to apoptotic cell death in cardiomyocytes. To study the role of ERp44 in Ca signaling and ER stress in vivo, we have generated ERp44 knockout/knockin mouse model systems. METHODS/RESULTS: We utilized the gene-trapped embryonic stem (ES) cell lines RST431 and XB599 (IGTC) to generate ERp44 (Txnd4) mutant mice through blastocyst injection. Analysis of the ERp44 (Txnd4) gene trapped lines RST431 and XB599 by RT-PCR and sequencing both mutant mice strains have the LacZ knockin allele inserted into intron 8 (RST431) and intron 1 (XB599). LacZ staining of tissue sections and immunoblot analysis of WT / ERp44-LacZ lines has confirmed LacZ protein expression in the heart, brain and other various tissues of these knockin ERp44 mutant mice. Mortality, in vivo echocardiography, isolated cardiomyocyte Ca2 transient assays, along with biochemical analysis of ER stress response pathways are reported. Ongoing work in our lab is focused on obtaining and analyzing ERp44-LacZ /ERp44LacZ double allele strains. CONCLUSION: We generated two ERp44 mutant mouse strains by utilizing gene trapped ES cell lines RST431 and XB599. RST431 mice have LacZ gene inserted in to downstream of ERp44 intron 8 and XB599 mice have LacZ inserted upstream of ERp44 intron 1. RST431 mouse strain represents a useful LacZ knockin ERp44 allele mouse model to study ER stress in the development of heart disease, as a reporter line, while the XB599 mouse strain represents the ERp44 knockout mouse model to study the role of ERp44 in development of ER stress involved heart disease. Heart and Stroke Foundation of Ontario, Canada Research Chair Program

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

Can controversies be put to REST?

The contribution of REST to embryonic stem (ES) cell pluripotency has been uncertain. Two years ago, Singh et al. claimed that Rest(+/-) and REST knock-down ES cells expressed reduced levels of pluripotency markers, in contrast to a prior and subsequent reports. To understand the basis of this difference, we analysed the YHC334 (YHC) and RRC160 (RRC) gene-trap ES cell lines used by Singh et al., obtained directly from BayGenomics. Both REST mutant lines generated REST-betaGeo fusion proteins, but expressed pluripotency genes at levels similar to appropriately matched parental wild ES cells, consistent with expression being REST-independent.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.

Unintentional miRNA Ablation Is a Risk Factor in Gene Knockout Studies: A Short Report

One of the most powerful techniques for studying the function of a gene is to disrupt the expression of that gene using genetic engineering strategies such as targeted recombination or viral integration of gene trap cassettes. The tremendous utility of these tools was recognized this year with the awarding of the Nobel Prize in Physiology or Medicine to Capecchi, Evans, and Smithies for their pioneering work in targeted recombination mutagenesis in mammals. Another noteworthy discovery made nearly a decade ago was the identification of a novel class of non-coding genes called microRNAs. MicroRNAs are among the largest known classes of regulatory elements with more than 1000 predicted to exist in the mouse genome. Over 50% of known microRNAs are located within introns of coding genes. Given that currently about half of the genes in mouse have been knocked out, we investigated the possibility that intronic microRNAs may have been coincidentally deleted or disrupted in some of these mouse models. We searched published murine knockout studies and gene trap embryonic stem cell line databases for cases where a microRNA was located within or near the manipulated genomic loci, finding almost 200 cases where microRNA expression may have been disrupted along with another gene. Our results draw attention to the need for careful planning in future knockout studies to minimize the unintentional disruption of microRNAs. These data also raise the possibility that many knockout studies may need to be reexamined to determine if loss of a microRNA contributes to the phenotypic consequences attributed to loss of a protein-encoding gene.

Reduced Body Size and Decreased Intestinal Tumor Rates in HDAC2-Mutant Mice

Histone deacetylases (HDAC) reverse the acetylation of histone and nonhistone proteins and thereby modulate chromatin structure and function of nonhistone proteins. Many tumor cell lines and experimental tumors respond to HDAC inhibition. To assess the role of an individual HDAC isoenzyme in physiology and tumor development, HDAC2-mutant mice were generated from a gene trap embryonic stem cell clone. These mice express a catalytically inactive fusion protein of the NH(2)-terminal part of HDAC2 and beta-galactosidase, which fails to integrate into corepressor complexes with mSin3B. They are the first class 1 HDAC mutant mice that are viable although they are approximately 25% smaller than their littermates. Cell number and thickness of intestinal mucosa are reduced. Mutant embryonic fibroblasts fail to respond to insulin-like growth factor I (IGF) by the IGF-I-induced increase in cell number observed in wild-type cells. These data suggest a novel link between HDACs and IGF-I-dependent responses. Crossing of HDAC2-mutant with tumor-prone APC(min) mice revealed tumor rates that are lower in HDAC2-deficient mice by 10% to 100% depending on segment of the gut and sex of the mice. These mice provide evidence that the key functions of HDAC2, although not essential for survival of the organism, play a rate-limiting role for tumor development in vivo.

The nuclear envelope protein MAN1 regulates TGFβ signaling and vasculogenesis in the embryonic yolk sac

MAN1 is an integral protein of the inner nuclear membrane of the nuclear envelope (NE). MAN1 interacts with SMAD transcription factors, which in turn are regulated by the Transforming growth factor beta (TGFbeta) superfamily of signaling molecules. To determine the role of MAN1 in mouse development, we used a gene-trap embryonic stem cell clone to derive mice with a functional mutation in MAN1 (Man1(GT/GT)). Expression of Man1 during early development is initially low but increases at embryonic day 9.5 (E9.5). Coincident with this increase, homozygous gene-trapped Man1 (Man1(GT/GT)) embryos die by E10.5. Examination of mutant embryos and tetraploid rescue experiments reveals that abnormal yolk-sac vascularization is the probable cause of lethality. We also established embryonic stem cell lines and their differentiated derivatives that are homozygous for the Man1(GT) allele. Using these lines, we show that the Man1(GT) allele results in increased phosphorylation, nuclear localization and elevated levels of SMAD transcriptional activity, predominantly of SMAD2/3, which are regulated by the ALK5 signaling pathway. Our studies identify a previously uncharacterized role for an integral nuclear envelope protein in the regulation of yolk-sac angiogenesis by TGFbeta signaling and reveal that the NE has an essential role in regulating transcription factor activity during mouse development.

Absence of hearing loss in a mouse model for DFNA17 and MYH9-related disease: The use of public gene-targeted ES cell resources

Multiple mouse embryonic stem (ES) cell banks expand the capability to characterize functions of genes implicated in human disease and to develop mouse models for the further understanding of disease pathology. Genetic diseases that result in hearing loss can provide insight into causative molecular mechanisms for deafness. We utilized BayGenomics, the public mouse ES cell bank, to identify gene-trapped ES cell lines associated with hearing loss. We identified two gene-trapped ES cell lines specific for the non-muscle myosin heavy chain class IIA or myosin heavy chain IX (Myh9). Inherited mutations in the Myh9 gene have been linked to non-syndromic hereditary hearing impairment DFNA17 as well as ‘MYH9-related disease’ characterized by macrothrombocytopenia, leukocyte inclusions, and in some patients deafness. Mutant Myh9 mice were derived from one of these ES cell lines that underwent germline transmission for in-depth otological examination. No homozygous mice however were identified at birth, consistent with recently published data describing the embryonic lethality of homozygous mutations in Myh9. We provide evidence that adult heterozygous Myh9 mouse inner ears contain half wild-type levels of Myh9 mRNA. Hearing loss however was not observed in heterozygous Myh9 mice in contrast to human Myh9-related diseases. Aged heterozygous Myh9 mice also did not show signs of cochleosaccular degeneration common in DFNA17. Although inheritance of Myh9 mutations in humans is dominant, we conclude that heterozygous loss of Myh9 is not critical to hearing function in mice by itself.

ADP-Ribosylation Factor-Like 3 Is Involved in Kidney and Photoreceptor Development

ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (-/-) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (-/-) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.

The N-terminal half of talin2 is sufficient for mouse development and survival

Using a talin2 gene-trapped embryonic stem cell clone, we have developed a talin2 mutant mouse line that expresses the N-terminal half (1–1295) of talin2 fused with β-galactosidase. The homozygous mutant mice appear to be normal and healthy. In the testis, talin2 expresses as a shorter form with a unique 30 residues at N-terminus linking to a common C-terminus from 1122 to 2453 of the long form. The resulting talin2 in the mutant testis only contains 204 residues of the wild-type testis talin2. However, it did not seem to affect the morphology of testis or reproduction of male mice. In fact, male and female mutant mice are fertile. Utilizing the expression of talin2(1–1295)/β-galactosidase fusion protein, we have examined the distribution of talin2 in tissues. In contrast to talin1, talin2 expression is more restricted in tissues and cell types.

Gene trap screening as an effective approach for identification of Wnt‐responsive genes in the mouse embryo

In this study, we examined whether gene trap methodology, which would be available for systematic identification and functional analysis of genes, is effective for screening of Wnt-responsive genes during mouse development. We screened out two individual clones among 794 gene-trapped embryonic stem cell lines by their in vitro response to WNT-3A proteins. One gene was mainly expressed in the ductal epithelium of several developing organs, including the kidney and the salivary glands, and the other gene was expressed in neural crest cells and the telencephalic flexure. The spatial and temporal expression of these two genes coincided well with that of several Wnt genes. Furthermore, the expression of these two genes was significantly decreased in embryos deficient for Wnts or in cultures of embryonic tissues treated with a Wnt signal inhibitor. These results indicate that the gene trap is an effective method for systematic identification of Wnt-responsive genes during embryogenesis.