Jane Brennan - Academia.edu (original) (raw)

Papers by Jane Brennan

Gene trapping in murine embryonic stem (ES) cells provides a method to create random insertional ... more Gene trapping in murine embryonic stem (ES) cells provides a method to create random insertional mutations in mice which are easily accessible to molecular characterisation. This approach involves electroporating a reporter gene construct into ES cells which is transcribed following integration into an endogenous transcription unit. In an effort to understand the molecular mechanisms that mediate cellular interactions during development I have used a modified gene trap vector, the secretory trap vector (pGT1. 8TM), to enrich for insertions in genes encoding secreted and membrane-spanning molecules. The selective property of this vector depends on the presence of a 680 bp fragment containing the transmembrane domain of CD4 fused to the bgeo reporter gene. Secretory trap lines were generated by electroporating pGTI . 8TM into CGR8 ES cells. The expression patterns of trapped genes were determined by monitoring reporter gene activity in chimeric embryos between 8.5d and 10. 5d of devel...

Nature, 2000

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and ti... more Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.

Methods in Molecular Biology, 2012

The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression a... more The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.

Nature Precedings, 2009

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is a consortium of laboratories ... more The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is a consortium of laboratories working to provide the scientific and medical community with gene expression data and tools to facilitate research (see "www.gudmap.org":http://www.gudmap.org). The data provided by GUDMAP includes large in situ hybridization screens (wholemount and section) and expression microarray analysis of components of the developing mouse urogenital system (including laser-captured material and FACS-isolated cells from transgenic reporter mice). In addition, a high-resolution anatomy ontology has been developed by members of the GUDMAP consortium to describe the subcompartments of the developing murine genitourinary tract. The GUDMAP Database Development Team and Editorial Office - both based in Edinburgh - function to ensure submission, curation, storage and presentation of the data submitted by the GUDMAP consortium. Our collective aim is twofold: 1) to simplify the process of submiss...

Mechanisms of Development, 2009

We have previously shown that lentiviral vectors can be used to generate transgenic chickens effi... more We have previously shown that lentiviral vectors can be used to generate transgenic chickens efficiently and that expression of introduced transgenes is not silenced on transmission through the germline. Transgene expression can be targeted in the predicted tissue-restricted manner, for example: muscle-specific expression is restricted to skeletal muscle using the rat myosin light chain 3 gene enhancer/promoter and oviduct-specific expression is demonstrated using regulatory elements of the chicken ovalbumin gene. We also produced transgenic chicken lines expressing GFP ubiquitously using the CMV IE enhancer fused to the chicken b-actin promoter/intron. High levels of GFP protein are visible at all stages of developing transgenic embryos. GFP embryos have been wildly used in cell-fate experiments where the grafted cells can be visualised and followed during embryo development. Here we are extending the repertoire of reporter genes with the development of transgenic lines that ubiquitously express membrane-localized GFP or monomericCherry (red), which will make invivo tracing cell shape changes and cell interactions possible. Founder males chimeric for transgenes coding for membrane-localized fluorescent proteins have been raised to sexual maturity and crossed to screen for transgenic offspring. In addition, we are generating transgenic lines carry photo-activatable GFP or photo-switchable CFP that should allow marking of cells insitu for lineage tracing studies without the need for grafting.

Gene Expression Patterns, 2007

Cataloguing gene expression during development of the genitourinary tract will increase our under... more Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17 to 27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the

Development, 2011

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium w... more The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

Cancer Research, Apr 15, 2006

Oncogene

Members of the protein superfamily of transmembrane receptor tyrosine kinases are key components ... more Members of the protein superfamily of transmembrane receptor tyrosine kinases are key components of intercellular signal transduction pathways that elicit appropriate cellular responses to environmental cues during development of multicellular organisms. In a search for additional receptor tyrosine kinases expressed during mouse embryogenesis we cloned the murine homolog of Eck, a member of the Eph subfamily, that maps to the distal region of mouse chromosome 4. Specific antisera defined Eck in murine embryonic cells as a glycoprotein of 130 kDa with an intrinsic autophosphorylation activity. Immunohistochemical staining and laser scanning microscopy revealed a dynamic and tightly regulated distribution of Eck receptor protein in the developing mouse embryo. During gastrulation, a high transient distribution of Eck was seen in mesodermal cells aggregating in the midline as notochordal plate. A similar restriction of Eck receptor protein was apparent along the rostrocaudal axis of th...

Oncogene

The protein superfamily of transmembrane receptor tyrosine kinases (RTKs) are essential component... more The protein superfamily of transmembrane receptor tyrosine kinases (RTKs) are essential components of intercellular signalling pathways necessary for normal cellular regulation. We report the cloning and developmental expression pattern of Nsk2, a novel, structurally distinct mammalian RTK characterised by a putative extracellular region bearing four immunoglobulin-like domains. The Nsk2 locus was mapped to the distal region of mouse chromosome 13 and was found to be expressed preferentially in skeletal muscle amongst adult mouse tissues. Moreover, increased steady-state levels of Nsk2 transcripts were apparent on terminal differentiation of committed skeletal myoblast cell lines in vitro and multiple isoforms of the Nsk2 RTK were identified in skeletal myotube cultures. RNA in situ hybridisation studies of mouse embryos confirmed skeletal myogenesis to be a major site of Nsk2 expression during normal embryogenesis, and identified other likely sites of Nsk2 function in ganglia of th...

Development (Cambridge, England), Jan 15, 2015

Malformation of the urogenital tract represents a considerable paediatric burden, with many defec... more Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external g...

Developmental biology, 2007

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serin... more Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serine protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodeling and tumorigenesis, such as HGFA, matriptase, prostasin and hepsin. We generated HAI-1 deficient mice, which died in utero due to placental defects. The HAI-1(-/-) placental labyrinth exhibited a complete failure of vascularization and a compact morphology of the trophoblast layer. Immunofluorescent staining of collagen IV and laminin and electron microscopy analysis revealed that this aberrant labyrinth architecture was associated with disrupted basement membranes located at the interface of chorionic trophoblasts and allantoic mesoderm. Unlike the placental labyrinth, basement membranes and vasculogenesis were normal in embryo and yolk sac. Therefore, basement membrane defects appear to be the underlying cause for the greatly impaired vascularization and trophoblast branchin...

Development (Cambridge, England), 2002

The TGFbeta-related growth factor Nodal governs anteroposterior (AP) and left-right (LR) axis for... more The TGFbeta-related growth factor Nodal governs anteroposterior (AP) and left-right (LR) axis formation in the vertebrate embryo. A conserved intronic enhancer (ASE), containing binding sites for the fork head transcription factor Foxh1, modulates dynamic patterns of Nodal expression during early mouse development. This enhancer is responsible for early activation of Nodal expression in the epiblast and visceral endoderm, and at later stages governs asymmetric expression during LR axis formation. We demonstrate ASE activity is strictly Foxh1 dependent. Loss of this autoregulatory enhancer eliminates transcription in the visceral endoderm and decreases Nodal expression in the epiblast, but causes surprisingly discrete developmental abnormalities. Thus lowering the level of Nodal signaling in the epiblast disrupts both orientation of the AP axis and specification of the definitive endoderm. Targeted removal of the ASE also dramatically reduces left-sided Nodal expression, but the earl...

Molecular Embryology, 1999

ABSTRACT

Nature, 2001

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of ... more Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.

Philosophical Transactions of the Royal Society B: Biological Sciences, 2003

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissu... more Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior–posterior axis of the embryo occurs through coordinated cell movements that rotate a pre–existing proximal–distal (P–D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12–24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the ‘organizer’ region and include the precursors of the so–called ‘axial mesendoderm’, namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor–β ligand nodal are involved in orchestrating the proce...

Mechanisms of Development, 1999

Site specific recombinases have provided the experimental strategy necessary to modulate the expr... more Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.

Genes & Development, 2002

Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" t... more Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" tissue remains unknown due to the early lethality of Nodal mutant embryos. Here we used a genetic strategy to selectively remove Nodal activity from the node. Embryos lacking Nodal in the node fail to initiate molecular asymmetry in the left lateral plate mesoderm and exhibit multiple left-right patterning defects. Nodal may also act as a short-range signal to establish a functional midline barrier. Our findings confirm that the mouse node is instrumental in initiating left-right axis specification and identify Nodal as the key morphogen regulating this process.

Gene trapping in murine embryonic stem (ES) cells provides a method to create random insertional ... more Gene trapping in murine embryonic stem (ES) cells provides a method to create random insertional mutations in mice which are easily accessible to molecular characterisation. This approach involves electroporating a reporter gene construct into ES cells which is transcribed following integration into an endogenous transcription unit. In an effort to understand the molecular mechanisms that mediate cellular interactions during development I have used a modified gene trap vector, the secretory trap vector (pGT1. 8TM), to enrich for insertions in genes encoding secreted and membrane-spanning molecules. The selective property of this vector depends on the presence of a 680 bp fragment containing the transmembrane domain of CD4 fused to the bgeo reporter gene. Secretory trap lines were generated by electroporating pGTI . 8TM into CGR8 ES cells. The expression patterns of trapped genes were determined by monitoring reporter gene activity in chimeric embryos between 8.5d and 10. 5d of devel...

Nature, 2000

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and ti... more Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.

Methods in Molecular Biology, 2012

The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression a... more The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.

Nature Precedings, 2009

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is a consortium of laboratories ... more The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is a consortium of laboratories working to provide the scientific and medical community with gene expression data and tools to facilitate research (see "www.gudmap.org":http://www.gudmap.org). The data provided by GUDMAP includes large in situ hybridization screens (wholemount and section) and expression microarray analysis of components of the developing mouse urogenital system (including laser-captured material and FACS-isolated cells from transgenic reporter mice). In addition, a high-resolution anatomy ontology has been developed by members of the GUDMAP consortium to describe the subcompartments of the developing murine genitourinary tract. The GUDMAP Database Development Team and Editorial Office - both based in Edinburgh - function to ensure submission, curation, storage and presentation of the data submitted by the GUDMAP consortium. Our collective aim is twofold: 1) to simplify the process of submiss...

Mechanisms of Development, 2009

We have previously shown that lentiviral vectors can be used to generate transgenic chickens effi... more We have previously shown that lentiviral vectors can be used to generate transgenic chickens efficiently and that expression of introduced transgenes is not silenced on transmission through the germline. Transgene expression can be targeted in the predicted tissue-restricted manner, for example: muscle-specific expression is restricted to skeletal muscle using the rat myosin light chain 3 gene enhancer/promoter and oviduct-specific expression is demonstrated using regulatory elements of the chicken ovalbumin gene. We also produced transgenic chicken lines expressing GFP ubiquitously using the CMV IE enhancer fused to the chicken b-actin promoter/intron. High levels of GFP protein are visible at all stages of developing transgenic embryos. GFP embryos have been wildly used in cell-fate experiments where the grafted cells can be visualised and followed during embryo development. Here we are extending the repertoire of reporter genes with the development of transgenic lines that ubiquitously express membrane-localized GFP or monomericCherry (red), which will make invivo tracing cell shape changes and cell interactions possible. Founder males chimeric for transgenes coding for membrane-localized fluorescent proteins have been raised to sexual maturity and crossed to screen for transgenic offspring. In addition, we are generating transgenic lines carry photo-activatable GFP or photo-switchable CFP that should allow marking of cells insitu for lineage tracing studies without the need for grafting.

Gene Expression Patterns, 2007

Cataloguing gene expression during development of the genitourinary tract will increase our under... more Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17 to 27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the

Development, 2011

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium w... more The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

Cancer Research, Apr 15, 2006

Oncogene

Members of the protein superfamily of transmembrane receptor tyrosine kinases are key components ... more Members of the protein superfamily of transmembrane receptor tyrosine kinases are key components of intercellular signal transduction pathways that elicit appropriate cellular responses to environmental cues during development of multicellular organisms. In a search for additional receptor tyrosine kinases expressed during mouse embryogenesis we cloned the murine homolog of Eck, a member of the Eph subfamily, that maps to the distal region of mouse chromosome 4. Specific antisera defined Eck in murine embryonic cells as a glycoprotein of 130 kDa with an intrinsic autophosphorylation activity. Immunohistochemical staining and laser scanning microscopy revealed a dynamic and tightly regulated distribution of Eck receptor protein in the developing mouse embryo. During gastrulation, a high transient distribution of Eck was seen in mesodermal cells aggregating in the midline as notochordal plate. A similar restriction of Eck receptor protein was apparent along the rostrocaudal axis of th...

Oncogene

The protein superfamily of transmembrane receptor tyrosine kinases (RTKs) are essential component... more The protein superfamily of transmembrane receptor tyrosine kinases (RTKs) are essential components of intercellular signalling pathways necessary for normal cellular regulation. We report the cloning and developmental expression pattern of Nsk2, a novel, structurally distinct mammalian RTK characterised by a putative extracellular region bearing four immunoglobulin-like domains. The Nsk2 locus was mapped to the distal region of mouse chromosome 13 and was found to be expressed preferentially in skeletal muscle amongst adult mouse tissues. Moreover, increased steady-state levels of Nsk2 transcripts were apparent on terminal differentiation of committed skeletal myoblast cell lines in vitro and multiple isoforms of the Nsk2 RTK were identified in skeletal myotube cultures. RNA in situ hybridisation studies of mouse embryos confirmed skeletal myogenesis to be a major site of Nsk2 expression during normal embryogenesis, and identified other likely sites of Nsk2 function in ganglia of th...

Development (Cambridge, England), Jan 15, 2015

Malformation of the urogenital tract represents a considerable paediatric burden, with many defec... more Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external g...

Developmental biology, 2007

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serin... more Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serine protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodeling and tumorigenesis, such as HGFA, matriptase, prostasin and hepsin. We generated HAI-1 deficient mice, which died in utero due to placental defects. The HAI-1(-/-) placental labyrinth exhibited a complete failure of vascularization and a compact morphology of the trophoblast layer. Immunofluorescent staining of collagen IV and laminin and electron microscopy analysis revealed that this aberrant labyrinth architecture was associated with disrupted basement membranes located at the interface of chorionic trophoblasts and allantoic mesoderm. Unlike the placental labyrinth, basement membranes and vasculogenesis were normal in embryo and yolk sac. Therefore, basement membrane defects appear to be the underlying cause for the greatly impaired vascularization and trophoblast branchin...

Development (Cambridge, England), 2002

The TGFbeta-related growth factor Nodal governs anteroposterior (AP) and left-right (LR) axis for... more The TGFbeta-related growth factor Nodal governs anteroposterior (AP) and left-right (LR) axis formation in the vertebrate embryo. A conserved intronic enhancer (ASE), containing binding sites for the fork head transcription factor Foxh1, modulates dynamic patterns of Nodal expression during early mouse development. This enhancer is responsible for early activation of Nodal expression in the epiblast and visceral endoderm, and at later stages governs asymmetric expression during LR axis formation. We demonstrate ASE activity is strictly Foxh1 dependent. Loss of this autoregulatory enhancer eliminates transcription in the visceral endoderm and decreases Nodal expression in the epiblast, but causes surprisingly discrete developmental abnormalities. Thus lowering the level of Nodal signaling in the epiblast disrupts both orientation of the AP axis and specification of the definitive endoderm. Targeted removal of the ASE also dramatically reduces left-sided Nodal expression, but the earl...

Molecular Embryology, 1999

ABSTRACT

Nature, 2001

Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of ... more Shortly after implantation the mouse embryo comprises three tissue layers. The founder tissue of the embryo proper, the epiblast, forms a radially symmetric cup of epithelial cells that grows in close apposition to the extra-embryonic ectoderm and the visceral endoderm. This simple cylindrical structure exhibits a distinct molecular pattern along its proximal-distal axis. The anterior-posterior axis of the embryo is positioned later by coordinated cell movements that rotate the pre-existing proximal-distal axis. The transforming growth factor-beta family member Nodal is known to be required for formation of the anterior-posterior axis. Here we show that signals from the epiblast are responsible for the initiation of proximal-distal polarity. Nodal acts to promote posterior cell fates in the epiblast and to maintain molecular pattern in the adjacent extra-embryonic ectoderm. Both of these functions are independent of Smad2. Moreover, Nodal signals from the epiblast also pattern the visceral endoderm by activating the Smad2-dependent pathway required for specification of anterior identity in overlying epiblast cells. Our experiments show that proximal-distal and subsequent anterior-posterior polarity of the pregastrulation embryo result from reciprocal cell-cell interactions between the epiblast and the two extra-embryonic tissues.

Philosophical Transactions of the Royal Society B: Biological Sciences, 2003

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissu... more Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior–posterior axis of the embryo occurs through coordinated cell movements that rotate a pre–existing proximal–distal (P–D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12–24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the ‘organizer’ region and include the precursors of the so–called ‘axial mesendoderm’, namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor–β ligand nodal are involved in orchestrating the proce...

Mechanisms of Development, 1999

Site specific recombinases have provided the experimental strategy necessary to modulate the expr... more Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.

Genes & Development, 2002

Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" t... more Nodal is expressed at the lateral edges of the mouse node, but its function in this "organizer" tissue remains unknown due to the early lethality of Nodal mutant embryos. Here we used a genetic strategy to selectively remove Nodal activity from the node. Embryos lacking Nodal in the node fail to initiate molecular asymmetry in the left lateral plate mesoderm and exhibit multiple left-right patterning defects. Nodal may also act as a short-range signal to establish a functional midline barrier. Our findings confirm that the mouse node is instrumental in initiating left-right axis specification and identify Nodal as the key morphogen regulating this process.