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Showing papers in "Genesis in 2003"


Journal ArticleDOI
01 Feb 2003-Genesis
TL;DR: Genome‐wide epigenetic reprogramming by demethylation occurs in early mouse embryos and primordial germ cells, which can result in the transgenerational inheritance of epigenetic states of IAPs, which could lead to heritable epimutations of neighbouring genes through influencing their transcriptional states.
Abstract: Genome-wide epigenetic reprogramming by demethylation occurs in early mouse embryos and primordial germ cells. In early embryos many single-copy sequences become demethylated both by active and passive demethylation, whereas imprinted gene methylation remains unaffected. In primordial germ cells single-copy and imprinted sequences are demethylated, presumably by active demethylation. Here we investigated systematically by bisulphite sequencing the methylation profiles of IAP and Line1 repeated sequence families during preimplantation and primordial germ cell development. Whereas Line1 elements were substantially demethylated during both developmental periods, IAP elements were largely resistant to demethylation, particularly during preimplantation development. This may be desirable in order to prevent IAP retrotransposition, which could cause mutations. In turn, this can result in the transgenerational inheritance of epigenetic states of IAPs, which could lead to heritable epimutations of neighbouring genes through influencing their transcriptional states.

640 citations


Journal ArticleDOI
01 Jan 2003-Genesis
TL;DR: This review focuses on what is known about the role of BMP signaling in gastrulation, mesoderm formation, left–right asymmetry, neural patterning, skeletal and limb development, organogenesis, and gametogenesis as revealed by BMP‐signaling mutants.
Abstract: During the past two decades, a significant amount of data has been accumulated revealing the intriguing functions of bone morphogenetic proteins (BMPs) in all aspects of embryonic development and organogenesis. Numerous genes encoding BMPs, BMP receptors, and their downstream signal transducers have been mutated in the mouse through targeted mutagenesis. This review focuses on what is known about the role of BMP signaling in gastrulation, mesoderm formation, left-right asymmetry, neural patterning, skeletal and limb development, organogenesis, and gametogenesis as revealed by BMP-signaling mutants.

387 citations


Journal ArticleDOI
01 Jan 2003-Genesis
TL;DR: A transgenic mouse line that expresses Cre recombinase exclusively in podocytes is reported, and Histological analysis of the kidneys showed that β‐gal expression was confined to podocytes.
Abstract: We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.

296 citations


Journal ArticleDOI
01 Nov 2003-Genesis
TL;DR: The data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the NotCh1 gene during early embryogenesis.
Abstract: The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.

254 citations


Journal ArticleDOI
01 Jan 2003-Genesis
TL;DR: The PLP/ CreERT animals should be very useful in elucidating and distinguishing a particular gene's function in the formation and maintenance of the myelin sheath and in analyzing mature oligodendrocyte function in pathological conditions.
Abstract: To explore the function of genes expressed by myelinating cells we have developed a model system that allows for the inducible ablation of predetermined genes in oligodendrocytes and Schwann cells. The Cre/loxP recombination system provides the opportunity to generate tissue-specific somatic mutations in mice. We have used a fusion protein between the Cre recombinase and a mutated ligand-binding domain of the human estrogen receptor (CreER(T)) to obtain inducible, site-specific recombination. CreER(T) expression was placed under the transcriptional control of the regulatory sequences of the myelin proteolipid protein (PLP) gene, which is abundantly expressed in oligodendrocytes and to a lesser extent in Schwann cells. The CreER(T) fusion protein translocated to the nucleus and mediated the recombination of a LacZ reporter transgene in myelinating cells of PLP/CreER(T) mice injected with the synthetic steroid tamoxifen. In untreated animals CreER(T) remained cytoplasmic, and there was no evidence of recombination. The PLP/ CreER(T) animals should be very useful in elucidating and distinguishing a particular gene's function in the formation and maintenance of the myelin sheath and in analyzing mature oligodendrocyte function in pathological conditions.

241 citations


Journal ArticleDOI
01 Mar 2003-Genesis
TL;DR: Direct evidence for the fate of Flk1+ cells in development is obtained using a knock‐in mouse line where Cre is expressed in Flk2+ cells, providing direct evidence that Flk 1+ cells are progenitors for muscles, in addition to hematopoietic and vascular endothelial cells.
Abstract: Flk1 is one of the specific cell surface receptors for vascular endothelial growth factor and one of the most specific markers highlighting the earliest stage of hematopoietic and vascular lineages. However, recent new evidence suggests that these Flk1(+) mesodermal progenitor cells also contribute to muscle lineages. All evidence is based on the experiments using in vitro differentiation and in vivo transplantation systems. Although this approach revealed a differentiation potential range of Flk1(+) cells that is wider than previously expected, it fails to determine whether Flk1(+) cells contribute to muscle lineage as part of the normal developmental process. To obtain direct evidence for the fate of Flk1(+) cells in development, we used a knock-in mouse line where Cre is expressed in Flk1(+) cells. Studies with these Cre lines provide direct evidence that Flk1(+) cells are progenitors for muscles, in addition to hematopoietic and vascular endothelial cells.

222 citations


Journal ArticleDOI
01 Apr 2003-Genesis
TL;DR: It is formally shown that striated muscle is the production site of functional myostatin and that this member of the TGFβ family of growth and differentiation factors regulates muscle mass not only during early embryogenesis but throughout development, indicating that myostarin antagonist could be used to treat muscle wasting and to promote muscle growth in man and animals.
Abstract: By using a conditional gene targeting approach exploiting the cre-lox system, we show that postnatal inactivation of the myostatin gene in striated muscle is sufficient to cause a generalized muscular hypertrophy of the same magnitude as that observed for constitutive myostatin knockout mice. This formally demonstrates that striated muscle is the production site of functional myostatin and that this member of the TGFbeta family of growth and differentiation factors regulates muscle mass not only during early embryogenesis but throughout development. It indicates that myostatin antagonist could be used to treat muscle wasting and to promote muscle growth in man and animals.

179 citations


Journal ArticleDOI
01 Nov 2003-Genesis
TL;DR: A Pbx1‐dependent pathway that regulates the expansion of SF‐1 positive cells essential for adrenal formation and gonadal differentiation is established and an early requirement for PbX1 in urogenital development is demonstrated.
Abstract: Pbx1 encodes a TALE (three amino acid loop extension) class homeodomain protein that participates in multimeric transcriptional complexes to regulate developmental gene expression. Previous studies demonstrate a critical role for Pbx1 as a developmental regulator whose absence results in embryonic lethality and multiple tissue and organ system abnormalities. Here we report a requirement for Pbx1 in the differentiation of urogenital organs, where Pbx1 is widely expressed in mesenchymal tissues. The complete lack of adrenal glands and formation of gonads displaying rudimentary sexual differentiation correlated with decreased cellular proliferation in Pbx1(-/-) genital ridges. Furthermore, expression of steroidogenic factor-1 (SF-1), a nuclear receptor essential for adrenal organogenesis, was reduced to minimal levels in Pbx1 mutants, indicating an upstream function for Pbx1 in adrenocortical development. Finally, loss of Pbx1 markedly reduces urogenital ridge outgrowth and results in impaired differentiation of the mesonephros and kidneys and the absence of Mullerian ducts. These findings establish a Pbx1-dependent pathway that regulates the expansion of SF-1 positive cells essential for adrenal formation and gonadal differentiation and demonstrate an early requirement for Pbx1 in urogenital development.

158 citations


Journal ArticleDOI
01 May 2003-Genesis
TL;DR: It is reported here that hsf2−/− mice exhibit multiple phenotypes, including an increased prenatal lethality occurring between mid‐gestation to birth, with fetal death probably due to central nervous system defects including collapse of the lateral ventricles and ventricular hemorrhages, and suggest that hSF2 has a major function in controlling expression of genes important for embryonic development and maintenance of sperm production.
Abstract: Summary: Heat shock transcription factors (Hsfs) are major transactivators of heat shock protein (Hsp) genes in the response to stress stimuli, but are also thought to be involved in embryonic development and spermatogenesis. Among the three known mammalian Hsfs, Hsf1 is recognized as the most effective transactivator of Hsps in response to thermal challenge, but the role of Hsf2 in regulation of genes under normal or increased stress conditions in vivo remains elusive. To study its physiological function in vivo, we generated mice deficient in hsf2 by gene targeting. We report here that hsf2−/− mice exhibit multiple phenotypes, including an increased prenatal lethality occurring between mid-gestation to birth, with fetal death probably due to central nervous system defects including collapse of the lateral ventricles and ventricular hemorrhages. Approximately 30% of hsf2−/− animals surviving to adulthood exhibited brain abnormalities characterized by marked dilation of the third and lateral ventricles. In addition, disruption of hsf2 resulted in reduced female fertility; however, despite ubiquitous expression in the testes and markedly reduced testis size and sperm count, only a small reduction in fertility was apparent in hsf2−/− male mice. Immunoblotting and gene expression microarray analysis of hsf2−/− embryos did not reveal reduced Hsp expression levels, indicating that the defects observed in hsf2−/− embryos may not result from disruption of Hsp expression. These findings suggest that hsf2 has a major function in controlling expression of genes important for embryonic development and maintenance of sperm production. genesis 36:48–61, 2003. © 2003 Wiley-Liss, Inc.

154 citations


Journal ArticleDOI
01 Jan 2003-Genesis
TL;DR: A beginner's guide for analysing cardiovascular defects in mouse mutants is provided to provide a good indicator of both the type of the cardiovascular defect present and the possible underlying cause/s.
Abstract: Summary: Since the advent of mouse targeted mutations, gene traps, an escalating use of a variety of complex transgenic manipulations, and large-scale chemical mutagenesis projects yielding many mutants with cardiovascular defects, it has become increasingly evident that defects within the heart and vascular system are largely responsible for the observed in utero lethality of the embryo and early fetus. If a transgenically altered embryo survives implantation but fails to be born, it usually indicates that there is some form of lethal cardiovascular defect present. A number of embryonic organ and body systems, including the central nervous system, gut, lungs, urogenital system, and musculoskeletal system appear to have little or no survival value in utero (Copp, 1995). Cardiovascular abnormalities include the failure to establish an adequate yolk-sac vascular circulation, which results in early lethality (E8.5–10.5); poor cardiac function (E9.0–birth); failure to undergo correct looping and chamber formation of the primitive heart tube (E9.0–11.0); improper septation, including division of the common ventricle and atria and the establishment of a divided outflow tract (E11.0–13.0); inadequate establishment of the cardiac conduction system (E12.0–birth); and the failure of the in utero cardiovascular system to adapt to adult life (birth) and close the interatrial and aorta-pulmonary trunk shunts that are required for normal fetal life. Importantly, the developmental timing of lethality is usually a good indicator of both the type of the cardiovascular defect present and may also suggest the possible underlying cause/s. The purpose of this review is both to review the literature and to provide a beginner's guide for analysing cardiovascular defects in mouse mutants. genesis 35:1–21, 2003. © 2002 Wiley-Liss, Inc.

151 citations


Journal ArticleDOI
01 May 2003-Genesis
TL;DR: It is suggested that FGF4 is required to maintain trophectoderm and primitive endoderm identity at embryonic day 4.5 and that postimplantation lethality of Fgf4−/− embryos likely results from the failure of proper differentiation and function of extraembryonic cell types.
Abstract: FGF4, a member of the fibroblast growth factor (FGF) family, is absolutely required for periimplantation mouse development, although its precise role at this stage remains unknown. The nature of the defect leading to postimplantation lethality of embryos lacking zygotic FGF4 is unclear and little is known about downstream targets of FGF4-initiated signaling within the various cellular compartments of the blastocyst. Here we report that postimplantation lethality of Fgf4(-/-) embryos is unlikely to reflect strictly mitogenic requirements for FGF4. Rather, our results suggest that FGF4 is required to maintain trophectoderm and primitive endoderm identity at embryonic day 4.5. This result is consistent with the reported in vitro activity of FGF4 in maintaining trophoblast stem cells and with the requirement for receptor tyrosine kinase signaling in primitive endoderm formation. Thus, postimplantation lethality of Fgf4(-/-) embryos likely results from the failure of proper differentiation and function of extraembryonic cell types.

Journal ArticleDOI
01 Jun 2003-Genesis
TL;DR: Two lines of transgenic mice that express Cre recombinase in melanoblasts provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.
Abstract: Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.

Journal ArticleDOI
01 Oct 2003-Genesis
TL;DR: The Sox2 gene is expressed in several undifferentiated cell types and a Sox2Cre transgene that mediates epiblast‐specific Cre‐mediated modification of gene activity in the embryo is reported that is active in the female germline.
Abstract: Summary: The Sox2 gene is expressed in several undifferentiated cell types. In an earlier study we described a Sox2Cre transgene that mediates epiblast-specific Cre-mediated modification of gene activity in the embryo. Here we report that this transgene is active in the female germline. Consequently, all offspring that arise from female mice heterozygous for the Sox2Cre transgene have demonstrable Cre activity irrespective of whether they inherit the transgene itself. Maternal inheritance of Cre activity allows the efficient modification of gene activity for functional analysis. genesis 37:51–53, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Sep 2003-Genesis
TL;DR: Findings support the critical role of Oct4 in regulating stem cell identity and suggest that future experiments using RNAi in ES cells can elucidate the roles of other genes affecting lineage specification during differentiation.
Abstract: Summary: We examined whether suppression of Oct4 via RNA interference (RNAi) would affect embryonic stem (ES) cell lineage choice. Cells were transfected with plasmids containing an independently expressed reporter gene and an RNA polymerase type III promoter to constitutively express small stem-loop RNA transcripts corresponding to Oct4 mRNA. Cells transfected with Oct4 RNAi constructs demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal differentiation. These findings support the critical role of Oct4 in regulating stem cell identity and suggest that future experiments using RNAi in ES cells can elucidate the roles of other genes affecting lineage specification during differentiation. genesis 37:18–24, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Jul 2003-Genesis
TL;DR: A conditional Ptc allele in mice is described which will have utility for the temporospatial ablation of Ptc function and is described as a conditional patched gene in mice.
Abstract: The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function.

Journal ArticleDOI
01 Apr 2003-Genesis
TL;DR: Two genes influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFβ pathway in C. elegans and homologs of these genes may be involved in tissue specificity and/or crosstalk of TGF β pathways in other animals.
Abstract: In the nematode Caenorhabditis elegans, a TGFβ-related signaling pathway regulates body size and male tail morphogenesis. We sought to identify genes encoding components or modifiers of this pathway in a large-scale genetic screen. Remarkably, this screen was able to identify essentially all core components of the TGFβ signaling pathway. Among 34 Small mutants, many mutations disrupt genes encoding recognizable components of the TGFβ pathway: DBL-1 ligand, DAF-4 type II receptor, SMA-6 type I receptor, and SMA-2, SMA-3, and SMA-4 Smads. Moreover, we find that at least 11 additional complementation groups can mutate to the Small phenotype. Four of these 11 genes, sma-9, sma-14, sma-16, and sma-20 affect male tail morphogenesis as well as body size. Two genes, sma-11 and sma-20, also influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFβ pathway in C. elegans. Other genes may represent tissue-specific factors or parallel pathways for body size control. Because of the conservation of TGFβ signaling pathways, homologs of these genes may be involved in tissue specificity and/or crosstalk of TGFβ pathways in other animals. genesis 35:239–247, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 May 2003-Genesis
TL;DR: The results imply that the underlying mechanism regulating early CNS patterning is conserved, despite several substantial differences in neurogenesis between mammals and amphibians.
Abstract: Summary: SOX3 is one of the earliest neural markers in vertebrates and is thought to play a role in specifying neuronal fate. To investigate the regulation of Sox3 expression we identified cis-regulatory regions in the Sox3 promoter that direct tissue-specific heterologous marker gene expression in transgenic mice. Our results show that an 8.3 kb fragment, comprising 3 kb upstream and 3 kb downstream of the Sox3 transcriptional unit, is sufficient in a lacZ reporter construct to reproduce most aspects of Sox3 expression during CNS development from headfold to midgestation stages. The apparently uniform expression of Sox3 in the neural tube depends, however, on the combined action of distinct regulatory modules within this 8.3 kb region. Each of these gives expression in a subdomain of the complete expression pattern. These are restricted along both the rostral-caudal and dorso-ventral axes and can be quite specific, one element giving expression largely confined to V2 interneuron precursors. We also find that at least some of the regulatory sequences are able to drive expression of the transgene in the CNS Xenopus laevis embryos in a manner that reflects the endogenous Sox3 expression pattern. These results imply that the underlying mechanism regulating early CNS patterning is conserved, despite several substantial differences in neurogenesis between mammals and amphibians. genesis 36:12–24, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Apr 2003-Genesis
TL;DR: It is shown that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm‐ isoform is absolutely required for cuticle biosynthesis and survival in Drosophila.
Abstract: Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.

Journal ArticleDOI
01 May 2003-Genesis
TL;DR: Four ragged mutants represent an allelic series that reveal SOX18 structure–function relationships and implicate related SOX proteins in cardiovascular and hair follicle development and suggest that the ragged mutant SoX18 proteins act in a dominant‐negative fashion.
Abstract: The ragged (Ra) spontaneous mouse mutant is characterised by abnormalities in its coat and cardiovascular system. Four alleles are known and we have previously described mutations in the transcription factor gene Sox18 in the Ra and Ra(J) alleles. We report here Sox18 mutations in the remaining two ragged alleles, opossum (Ra(op)) and ragged-like (Ragl). The single-base deletions cause a C-terminal frameshift, abolishing transcriptional trans-activation and impairing interaction with the partner protein MEF2C. The nature of these mutations, together with the near-normal phenotype of Sox18-null mice, suggests that the ragged mutant SOX18 proteins act in a dominant-negative fashion. The four ragged mutants represent an allelic series that reveal SOX18 structure-function relationships and implicate related SOX proteins in cardiovascular and hair follicle development.

Journal ArticleDOI
01 Aug 2003-Genesis
TL;DR: The Cprlox strain will be valuable for conditional Cpr gene deletion and subsequent determination of the impact of CPR loss on the metabolism of endogenous and xenobiotic compounds, as well as on postnatal development and other biological functions.
Abstract: Summary: NADPH-cytochrome P450 reductase (CPR or POR) is the obligatory electron donor for all microsomal cytochrome P450 (CYP or P450)-catalyzed monooxygenase reactions. Disruption of the mouse Cpr gene has been reported to cause prenatal developmental defects and embryonic lethality. In this study, we generated a mouse model with a floxed Cpr allele (termed Cprlox). Homozygous Cprlox mice are fertile and without any histological abnormality or any change in CPR expression. The floxed Cpr allele was subsequently deleted efficiently by crossing Cprlox mice with transgenic mice having liver-specific Cre expression (Alb-Cre); the result was a decrease in the level of CPR protein in liver microsomes. The Cprlox strain will be valuable for conditional Cpr gene deletion and subsequent determination of the impact of CPR loss on the metabolism of endogenous and xenobiotic compounds, as well as on postnatal development and other biological functions. genesis 36:177–181, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Feb 2003-Genesis
TL;DR: The data indicate that oil body biogenesis can occur outside of the embryo and that oleosin‐GFP can be used to monitor early events in oil bodyBiogenesis in real‐time.
Abstract: We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.

Journal ArticleDOI
01 Jun 2003-Genesis
TL;DR: Two mouse lines for making Cre recombinase‐mediated gene disruptions largely confined to adult cerebellar granule cells are generated, each expressing the GABAA receptor α6 subunit gene, whose expression marks this cell type.
Abstract: Summary: The cerebellum maintains balance and orien-tation, refines motor action, stores motor memories, andcontributes to the timing aspects of cognition. We gen-erated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cer-ebellar granule cells. For this purpose we chose theGABA A receptor 6 subunit gene, whose expressionmarks this cell type. Here we describe mouse lines ex-pressing Cre recombinase generated by 1) Cre knockedinto the native 6 subunit gene by homologous recom-bination in embryonic stem cells; and 2) Cre recombinedinto an 6 subunit gene carried on a bacterial artificialchromosome (BAC) genomic clone. The fidelity of Creexpression was tested by crossing the mouse lines withthe ROSA26 reporter mice. The particular 6BAC clonewe identified will be valuable for delivering othergene products to cerebellar granule cells. genesis 36:97–103, 2003. © 2003 Wiley-Liss, Inc. Key words: Cre recombinase; BAC transgene; cerebellum;granule cell; GABA

Journal ArticleDOI
01 Oct 2003-Genesis
TL;DR: A new gene transduction technique is reported using microbubble‐enhanced sonoporation to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes and most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonopation.
Abstract: Summary: The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed. genesis 37:91–101, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Jun 2003-Genesis
TL;DR: Examination of embryonic and postnatal tissues revealed that esophageal skeletal muscle does not arise from transdifferentiation of committed smooth muscle cells.
Abstract: Summary: To determine the developmental history of murine esophageal skeletal muscle, smooth muscle cells were fate mapped by lineage-specific recombination and phenotypically marked by eGFP. Examination of embryonic and postnatal tissues revealed that esophageal skeletal muscle does not arise from transdifferentiation of committed smooth muscle cells. genesis 36:81–82, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Apr 2003-Genesis
TL;DR: A generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis‐Brachyury‐promoter/Green Fluorescent Protein‐reporter (Ci‐Bra‐GFP) construct is reported, indicating integration into the genome.
Abstract: Summary: We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F0 adults tested, were found to transmit the transgene to their offspring (F1s) at frequencies of 42% and 23%. The transgene was further inherited by the F2 in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F3 and F4 generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos. genesis 35:248–259, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Aug 2003-Genesis
TL;DR: To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter are generated, indicating that transgenic Cre is functional.
Abstract: Summary: To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We intercrossed TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of “floxed” genes in catecholaminergic neurons. genesis 36:196–202, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Jun 2003-Genesis
TL;DR: It is shown that the trapped laminin γ1 fusion protein “co‐trapped” endogenous β1 intracellularly and the co‐trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.
Abstract: Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.

Journal ArticleDOI
01 Apr 2003-Genesis
TL;DR: The absence of a phenotype in the Bmp4‐deficient telencephalon along with recent genetic studies establishing a role for a BMP4 receptor, BMPRIA, in telENCEphalic midline development, demonstrate that loss of Bmp 4 function in the telencephon can be compensated for by at least one other Bmp gene, the identity of which has not yet been determined.
Abstract: The embryonic telencephalon is patterned into several areas that give rise to functionally distinct structures in the adult forebrain. Previous studies have shown that BMP4 and BMP2 can induce features characteristic of the telencephalic midline in cultured explants, suggesting that the normal role of BMP4 in the forebrain is to pattern the medial lateral axis of the telencephalon by promoting midline cell fates. To test this hypothesis directly in vivo, the Bmp4 gene was efficiently disrupted in the telencephalon using a CRE/loxP approach. Analysis of Bmp4-deficient telencephalons fails to reveal a defect in patterning, cell proliferation, differentiation, or apoptosis. The absence of a phenotype in the Bmp4-deficient telencephalon along with recent genetic studies establishing a role for a BMP4 receptor, BMPRIA, in telencephalic midline development, demonstrate that loss of Bmp4 function in the telencephalon can be compensated for by at least one other Bmp gene, the identity of which has not yet been determined.

Journal ArticleDOI
01 Aug 2003-Genesis
TL;DR: It is demonstrated that transient transfection of siRNA to primary cells can have substantial functional consequences and may be applicable to a variety of primary cell types.
Abstract: Summary: Short interfering (si) RNAs have now been shown to inhibit gene expression in several species, including mammals (Elbashir et al.: Nature 411:494–498, 2001; Fire et al.: Nature 391:806–811, 1998). RNA inhibition in primary cells such as stem cells would facilitate rapid gene discovery in a postgenome era. While retroviruses can deliver siRNA expression cassettes for stable expression (Barton and Medzhitov: Proc Natl Acad Sci USA 99:14943–14945, 2002; Paddison et al.: Proc Natl Acad Sci USA 99:1443–1448, 2002; Rubinson et al.: Nat Genet 33:401–406, 2003), an efficient method for direct transfer of siRNA to stem cells is still lacking. Here, we established electroporation to deliver siRNA to hematopoietic progenitors. On average, at least 80% of cells take up the RNA, and these display nearly 100% knockout of marker gene expression at both the RNA and protein level. Moreover, knockdown of the hematopoietic regulator, CD45, results in 3-fold more hematopoietic colonies in a progenitor assay. These results demonstrate that transient transfection of siRNA to primary cells can have substantial functional consequences. This technology may be applicable to a variety of primary cell types. genesis 36:203–208, 2003. © 2003 Wiley-Liss, Inc.

Journal ArticleDOI
01 Mar 2003-Genesis
TL;DR: The jewel wasp Nasonia vitripennis is considered the “Drosophila melanogaster of the Hymenoptera” and offers insect geneticists a means for applying haplo‐diploid genetics to the analysis of developmental processes.
Abstract: The jewel wasp Nasonia vitripennis is considered the "Drosophila melanogaster of the Hymenoptera." This diminutive wasp offers insect geneticists a means for applying haplo-diploid genetics to the analysis of developmental processes. As in bees, haploid males develop from unfertilized eggs, while diploid females develop from fertilized eggs. Nasonia's advantageous combination of haplo-diploid genetics and ease of handling in the laboratory facilitates screening the entire genome for recessive mutations affecting a developmental process of interest. This approach is currently directed toward understanding the evolution of embryonic pattern formation by comparing Nasonia embryogenesis to that of Drosophila. Haplo-diploid genetics also facilitates developing molecular maps and mapping polygenic traits. Moreover, Nasonia embryos are also proving amenable to cell biological analysis. These capabilities are being exploited to understand a variety of behavioral, developmental, and evolutionary processes, ranging from cytoplasmic incompatibility to the evolution of wing morphology.