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Role of Rac1 in the regulation of axis specification and cell migration during early mouse development
ABSTRACTS / Developmental Biology 319 (2008) 576–586
inversion in both cases is inferred and has never been shown directly. We have used 3D reconstructions and cell tracing in chick embryos to show that the cardiogenic mesoderm is organized such that lateralmost cells are incorporated into the cardiac inflow (atria and left ventricle) while medially placed cells are incorporated into the cardiac outflow (right ventricle and outflow tract). This happens because the cardiogenic mesoderm is inverted concomitant with movement of the anterior intestinal portal caudomedially to form the foregut pocket. The bilateral cranial cardiogenic fields fold medially and ventrally and fuse. After folding the seam made by ventral fusion will become the greater curvature of the heart loop. The caudal border of the cardiogenic mesoderm which ends up dorsally coincides with the inner curvature. Physical ablation of selected areas of the cardiogenic mesoderm based on this new fate map confirmed these results and, in addition, showed that the right and left atria arise from the right and left heart fields. These findings provide a unified concept of heart fields and heart tube formation for avians and mammals. doi:10.1016/j.ydbio.2008.05.410
Program/Abstract # 387 The function of the mammalian Pumilio gene, Pum1, in early embryonic development of mice Henrike Siemen a,b, Eugene Xu c, Oliver Brüstle b, Renee A. Reijo Pera a a Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA b Institute of Reconstructive Neurobiology, University of Bonn Medical Center, Bonn, Germany c Department of Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA The mammalian Pumilio genes are members of a conserved family of RNA-binding proteins called the Puf protein family, which act as translational repressors. Puf proteins are characterized by their consensus RNA binding motif; the Pum-homology domain (Pum-HD). They are key regulators in stem cell and germ cell maintenance in diverse organisms from invertebrates to vertebrates. In this study we explore the role of mammalian Pumilio1, which has been identified in mice and humans. However, its function remains unresolved. Our initial characterization demonstrates that Pum1 is widely expressed in human and mice tissues and in ES cells. To further investigate the role of Pum1 in mammals, we used a genetrap strategy to generate mice that lack a functional Pum-HD and express lacZ under the endogenous control of Pum1. Loss of Pum1 leads to preimplantational embryonic lethality. However, heterozygous mutant mice are viable and fertile. Therefore, our results suggest that at least one copy of Pum1 is essential for early embryonic development in mice. Future directions will elucidate the mechanism behind the early embryonic lethality.
583
The primary germ layers of the mammalian embryo are specified from pluripotent epiblast cells during gastrulation. While considerable effort has been dedicated to elucidating the patterning signals mediating lineage commitment, the mechanisms regulating the pluripotency of epiblast cells remain poorly defined. We propose that cell intrinsic mechanisms exert tight control over transcriptional circuits of pluripotency to allow for rapid and appropriate differentiation during gastrulation. Recent discoveries have revealed a network of transcription factors (Nanog, Oct4, Sox2) required for self renewal of pluripotent embryonic stem cells (ESC) in vitro, and for expansion of the precursors of the epiblast. Tcf3, a transcription factor required for correct gastrulation, has been identified as a limiter of this pluripotency network that acts by repressing Nanog expression in ESC. To determine whether a similar network controls pluripotency within the epiblast, we examined the function of Tcf3 in gastrulating mouse embryos. We show that Tcf3 is required to restrict Nanog expression to the site of primitive streak formation. Furthermore, the ectopic expression of Nanog in Tcf3−/− embryos coincides with delayed or defective processes of lineage commitment as determined by altered expression patterns of Brachyury, Sox1 and Otx2. Using novel Tcf3ΔN knock-in mice, we show that Tcf3 is required to act as a transcriptional repressor independently of Beta-catenin. These findings provide new insight into the importance of negative regulation of pluripotency for lineage commitment during development and suggest new mechanisms for the patterning of the embryo during gastrulation. doi:10.1016/j.ydbio.2008.05.412
Program/Abstract # 389 Role of paracrine Furin activity during gastrulation Daniel Mesnard a, Martyn Donnison b, Peter L. Pfeffer b, Daniel B. Constam a a EPFL-ISREC, Epalinges, Switzerland b AgResearch, Hamilton, New Zealand Axis and germ layer formation in vertebrates are orchestrated by the secreted subtilisin-like proprotein convertases (SPC) Furin and PACE4. Genetic evidence in the mouse suggested that Furin and PACE4 are provided by the extraembryonic ectoderm (ExE) to activate the Nodal precursor in adjacent epiblast during gastrulation, but soluble forms of these proteases and their distribution have never been directly observed in vivo. In addition, we hypothesized that Nodal signaling may be stimulated already after implantation by an early wave of transient Furin expression in the visceral endoderm (Mesnard et al., 2005), possibly to achieve maximal Nodal signal duration (BenHaim et al., 2005). To visualize ExE-derived Furin, and to monitor its effect on Nodal signaling, we expressed a Furin-GFP transgene in the ExE of wild-type and Furin−/−;Pace4−/− double mutant embryos. We show that GFP-tagged Furin suppresses the precocious neural differentiation in Furin−/−;Pace4−/− double mutant embryos, and stimulates Nodal signaling.
doi:10.1016/j.ydbio.2008.05.411 doi:10.1016/j.ydbio.2008.05.413
Program/Abstract # 388 Tcf3 regulation of pluripotency for lineage commitment during gastrulation Jackson A. Hoffman, Bradley J. Merrill Department of Biochemistry and Molecular Genetics, UIC, Chicago, IL 60607, USA
Program/Abstract # 390 Role of Rac1 in the regulation of axis specification and cell migration during early mouse development Isabelle M. Migeotte, Kathryn V. Anderson Developmental Biology Program, Sloan-Kettering Institute, NY, NY, USA
584
ABSTRACTS / Developmental Biology 319 (2008) 576–586
It was shown earlier that Rac1 null embryos die during gastrulation, but the basis of lethality was not clear. We found that Rac1 null embryos fail to specify the anterior–posterior (AP) body axis. The embryos express Brachyury, a primitive streak (PS) marker, as a ring at the embryonic–extraembryonic border. Migration of the anterior visceral endoderm (AVE), which is required to define the position of the PS, is virtually suppressed. We also found that many epiblastderived cells undergo apoptosis. We used a Rac1 conditional allele and the Sox2-Cre transgene to inactivate Rac1 in the e6.5 epiblast. The embryos survive to e8.5 and specify a normal AP axis. At e7.5, the PS of the mutants is enlarged. Nevertheless, the epithelial-to-mesenchymal transition seems to happen normally, suggesting a cell migration defect. At e8.5, the mutants display an open neural tube, an open gut, no defined somites, cardia bifida, and a big PS. These phenotypes resemble those of mutants that lack Nap1, a regulator of WAVEmediated cell migration, which suggests that Rac1 and Nap1 act together in vivo. However, unlike Nap1 mutants, apoptosis is increased in the Rac1 epiblast, PS and mesoderm. We then inactivated Rac1 using a TTR-Cre transgene (G. Kwon and A. K. Hadjantonakis, unpublished), which promotes deletion in most cells of the VE prior to the time of AVE migration. We observed a defect in AVE migration in a portion of the VE-deleted embryos, supporting the hypothesis that Rac1 is required in the AVE for proper migration. The VE-deleted embryos survive to e8, and analysis of axis specification in these embryos is in progress. doi:10.1016/j.ydbio.2008.05.414
Program/Abstract # 391 The role of Pten in anterior–posterior (AP) axis formation in the mouse embryo Joshua E. Bloomekatz a,b, Andrew Rakeman, Heather Alcorn b, Kathryn V. Anderson a,b a Weill-Cornell Graduate School, USA b Sloan-Kettering Institute, USA We are interested in understanding the morphogenetic mechanisms involved in the formation of the anterior–posterior (AP) axis in the early mouse embryo. We have identified an ENU-induced missense allele of Pten, M1, that displays defects in AP axis specification. The primitive streak, the site of gastrulation, marks the posterior side of the early embryo. Mutant embryos homozygous for either PtenM1 or a targeted null allele of Pten are defective in primitive streak specification: 50% of Pten mutant embryos display multiple primitive streaks, as visualized by the expression of Brachyury. The position of the primitive streak is controlled by signals from the anterior visceral endoderm (AVE). The AVE is an extra-embryonic structure which forms at the distal tip of the embryo and then migrates to the presumptive anterior side. Approximately 50% of Pten−/− mutant embryos display defects in the movement of AVE cells. Conditional removal of Pten in embryonic tissues only, using the Sox2-cre transgene, confirms Pten's role in AVE migration for proper AP specification. A missense mutation in Nap1, kahlo, has a similar phenotype to Pten−/− mutant embryos; 15% of Nap1khlo mutant embryos show primitive streak duplications (Rakeman, et al., 2006). Nap1 regulates actin polymerization in migrating cells. Nap1 and Pten display a genetic interaction in AP axis specification: 100% of Nap1khlo/khlo;Pten+/− mutant embryos display multiple primitive streaks. These results indicate that Nap1 and Pten work together to control AP axis specification. We are investigating the molecular nature of this genetic interaction. doi:10.1016/j.ydbio.2008.05.415
Program/Abstract # 392 Anterior axis duplication in mouse embryos caused by mutation in Porcn Steffen Biechele a,b, Brian J. Cox a, Owen J. Tamplin a,b, Mei Lu a, Janet Rossant a,b a Program in Developmental and Stem Cell Biology, Sick Kids Hospital, Toronto, Canada b Department of Molecular Genetics, University of Toronto, Canada Wnt signaling plays important roles in development and disease. In mammals, 19 Wnts activate several different pathways such as the canonical Wnt signaling pathway and the non-canonical planar cell polarity (PCP) pathway. Due to functional redundancy of Wnt ligands, compound mutants and deletion of downstream pathway components have been used to investigate the role of Wnt signaling. These efforts have focused on the cells that receive Wnt signal, whereas cells secreting Wnt have not been extensively studied. The conserved Xchromosomal gene Porcupine (Porcn) encodes an O-acyl transferase required for lipid modification, secretion and gradient formation of several, if not all Wnt ligands. Mutations in the human homolog of Porcn cause Goltz Syndrome, an X-linked dominant disorder that is usually observed in females. Rare cases of postzygotic mutations in males suggest that Porcn mutations could underlie some human cases of X-linked male lethality. This study investigates the role of Porcn in early mouse development. Porcn null embryos have been generated by aggregation of wildtype embryos with Porcn-deficient embryonic stem (ES) cells. ES cell derived embryos fail to complete gastrulation and show duplication of the anterior body axis. This observation is consistent with the model of a Wnt signal gradient being required for embryonic axis induction; high Wnt signal induces posterior tissues, whereas low Wnt signal promotes anterior structures. Finally, we are generating a conditional allele of Porcn to further examine its role in embryonic development. doi:10.1016/j.ydbio.2008.05.416
Program/Abstract # 393 Redundant function of Wnt5a and Wnt11 in somitogenesis and anteroposterior axis elongation Hai Song a, Andreas Kispert b, Yingzi Yang a a GDRB, National Human Genome Research Institute, Bethesda, MD, USA b Institute For Molecular Biology, Medizinische Hochschule Hannover, 30625 Hannover, Germany Wnts are a large family of secreted glycoproteins that play diverse roles during embryogenesis and transduce their signals through several different pathways. Mouse Wnt5a mutant embryos show shortened anteroposterior (A-P) axis. However, the morphology of Wnt11 mutant embryos is largely normal. Since Wnt5a and Wnt11 have overlapped expression pattern and mainly signal through noncanonical Wnt signaling during mouse development, they may play redundant roles during A-P axis elongation and somitogenesis. To test this, we generated Wnt5a and Wnt11 double mutant mouse embryos. The double mutant embryos exhibited much shorter A-P axis, irregular somites and heart-looping defect. The expression of Wnt3a and Fgf8 was normal in the tail bud. However, the expression of Notch1 and Notch2 was greatly reduced in the presomitic mesoderm (PSM) of double mutant embryos. Consistently, the expression of target genes of Notch signaling was also greatly reduced in the PSM of double mutant embryos. Bilateral symmetry of somite formation was disrupted in the double mutant embryos. Furthermore, the somite patterning of double mutant embryos was disrupted, due to the reduced expression of Shh. Myogenesis of double mutant embryos
inversion in both cases is inferred and has never been shown directly. We have used 3D reconstructions and cell tracing in chick embryos to show that the cardiogenic mesoderm is organized such that lateralmost cells are incorporated into the cardiac inflow (atria and left ventricle) while medially placed cells are incorporated into the cardiac outflow (right ventricle and outflow tract). This happens because the cardiogenic mesoderm is inverted concomitant with movement of the anterior intestinal portal caudomedially to form the foregut pocket. The bilateral cranial cardiogenic fields fold medially and ventrally and fuse. After folding the seam made by ventral fusion will become the greater curvature of the heart loop. The caudal border of the cardiogenic mesoderm which ends up dorsally coincides with the inner curvature. Physical ablation of selected areas of the cardiogenic mesoderm based on this new fate map confirmed these results and, in addition, showed that the right and left atria arise from the right and left heart fields. These findings provide a unified concept of heart fields and heart tube formation for avians and mammals. doi:10.1016/j.ydbio.2008.05.410
Program/Abstract # 387 The function of the mammalian Pumilio gene, Pum1, in early embryonic development of mice Henrike Siemen a,b, Eugene Xu c, Oliver Brüstle b, Renee A. Reijo Pera a a Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA b Institute of Reconstructive Neurobiology, University of Bonn Medical Center, Bonn, Germany c Department of Obstetrics and Gynecology, Northwestern University, Chicago, IL, USA The mammalian Pumilio genes are members of a conserved family of RNA-binding proteins called the Puf protein family, which act as translational repressors. Puf proteins are characterized by their consensus RNA binding motif; the Pum-homology domain (Pum-HD). They are key regulators in stem cell and germ cell maintenance in diverse organisms from invertebrates to vertebrates. In this study we explore the role of mammalian Pumilio1, which has been identified in mice and humans. However, its function remains unresolved. Our initial characterization demonstrates that Pum1 is widely expressed in human and mice tissues and in ES cells. To further investigate the role of Pum1 in mammals, we used a genetrap strategy to generate mice that lack a functional Pum-HD and express lacZ under the endogenous control of Pum1. Loss of Pum1 leads to preimplantational embryonic lethality. However, heterozygous mutant mice are viable and fertile. Therefore, our results suggest that at least one copy of Pum1 is essential for early embryonic development in mice. Future directions will elucidate the mechanism behind the early embryonic lethality.
583
The primary germ layers of the mammalian embryo are specified from pluripotent epiblast cells during gastrulation. While considerable effort has been dedicated to elucidating the patterning signals mediating lineage commitment, the mechanisms regulating the pluripotency of epiblast cells remain poorly defined. We propose that cell intrinsic mechanisms exert tight control over transcriptional circuits of pluripotency to allow for rapid and appropriate differentiation during gastrulation. Recent discoveries have revealed a network of transcription factors (Nanog, Oct4, Sox2) required for self renewal of pluripotent embryonic stem cells (ESC) in vitro, and for expansion of the precursors of the epiblast. Tcf3, a transcription factor required for correct gastrulation, has been identified as a limiter of this pluripotency network that acts by repressing Nanog expression in ESC. To determine whether a similar network controls pluripotency within the epiblast, we examined the function of Tcf3 in gastrulating mouse embryos. We show that Tcf3 is required to restrict Nanog expression to the site of primitive streak formation. Furthermore, the ectopic expression of Nanog in Tcf3−/− embryos coincides with delayed or defective processes of lineage commitment as determined by altered expression patterns of Brachyury, Sox1 and Otx2. Using novel Tcf3ΔN knock-in mice, we show that Tcf3 is required to act as a transcriptional repressor independently of Beta-catenin. These findings provide new insight into the importance of negative regulation of pluripotency for lineage commitment during development and suggest new mechanisms for the patterning of the embryo during gastrulation. doi:10.1016/j.ydbio.2008.05.412
Program/Abstract # 389 Role of paracrine Furin activity during gastrulation Daniel Mesnard a, Martyn Donnison b, Peter L. Pfeffer b, Daniel B. Constam a a EPFL-ISREC, Epalinges, Switzerland b AgResearch, Hamilton, New Zealand Axis and germ layer formation in vertebrates are orchestrated by the secreted subtilisin-like proprotein convertases (SPC) Furin and PACE4. Genetic evidence in the mouse suggested that Furin and PACE4 are provided by the extraembryonic ectoderm (ExE) to activate the Nodal precursor in adjacent epiblast during gastrulation, but soluble forms of these proteases and their distribution have never been directly observed in vivo. In addition, we hypothesized that Nodal signaling may be stimulated already after implantation by an early wave of transient Furin expression in the visceral endoderm (Mesnard et al., 2005), possibly to achieve maximal Nodal signal duration (BenHaim et al., 2005). To visualize ExE-derived Furin, and to monitor its effect on Nodal signaling, we expressed a Furin-GFP transgene in the ExE of wild-type and Furin−/−;Pace4−/− double mutant embryos. We show that GFP-tagged Furin suppresses the precocious neural differentiation in Furin−/−;Pace4−/− double mutant embryos, and stimulates Nodal signaling.
doi:10.1016/j.ydbio.2008.05.411 doi:10.1016/j.ydbio.2008.05.413
Program/Abstract # 388 Tcf3 regulation of pluripotency for lineage commitment during gastrulation Jackson A. Hoffman, Bradley J. Merrill Department of Biochemistry and Molecular Genetics, UIC, Chicago, IL 60607, USA
Program/Abstract # 390 Role of Rac1 in the regulation of axis specification and cell migration during early mouse development Isabelle M. Migeotte, Kathryn V. Anderson Developmental Biology Program, Sloan-Kettering Institute, NY, NY, USA
584
ABSTRACTS / Developmental Biology 319 (2008) 576–586
It was shown earlier that Rac1 null embryos die during gastrulation, but the basis of lethality was not clear. We found that Rac1 null embryos fail to specify the anterior–posterior (AP) body axis. The embryos express Brachyury, a primitive streak (PS) marker, as a ring at the embryonic–extraembryonic border. Migration of the anterior visceral endoderm (AVE), which is required to define the position of the PS, is virtually suppressed. We also found that many epiblastderived cells undergo apoptosis. We used a Rac1 conditional allele and the Sox2-Cre transgene to inactivate Rac1 in the e6.5 epiblast. The embryos survive to e8.5 and specify a normal AP axis. At e7.5, the PS of the mutants is enlarged. Nevertheless, the epithelial-to-mesenchymal transition seems to happen normally, suggesting a cell migration defect. At e8.5, the mutants display an open neural tube, an open gut, no defined somites, cardia bifida, and a big PS. These phenotypes resemble those of mutants that lack Nap1, a regulator of WAVEmediated cell migration, which suggests that Rac1 and Nap1 act together in vivo. However, unlike Nap1 mutants, apoptosis is increased in the Rac1 epiblast, PS and mesoderm. We then inactivated Rac1 using a TTR-Cre transgene (G. Kwon and A. K. Hadjantonakis, unpublished), which promotes deletion in most cells of the VE prior to the time of AVE migration. We observed a defect in AVE migration in a portion of the VE-deleted embryos, supporting the hypothesis that Rac1 is required in the AVE for proper migration. The VE-deleted embryos survive to e8, and analysis of axis specification in these embryos is in progress. doi:10.1016/j.ydbio.2008.05.414
Program/Abstract # 391 The role of Pten in anterior–posterior (AP) axis formation in the mouse embryo Joshua E. Bloomekatz a,b, Andrew Rakeman, Heather Alcorn b, Kathryn V. Anderson a,b a Weill-Cornell Graduate School, USA b Sloan-Kettering Institute, USA We are interested in understanding the morphogenetic mechanisms involved in the formation of the anterior–posterior (AP) axis in the early mouse embryo. We have identified an ENU-induced missense allele of Pten, M1, that displays defects in AP axis specification. The primitive streak, the site of gastrulation, marks the posterior side of the early embryo. Mutant embryos homozygous for either PtenM1 or a targeted null allele of Pten are defective in primitive streak specification: 50% of Pten mutant embryos display multiple primitive streaks, as visualized by the expression of Brachyury. The position of the primitive streak is controlled by signals from the anterior visceral endoderm (AVE). The AVE is an extra-embryonic structure which forms at the distal tip of the embryo and then migrates to the presumptive anterior side. Approximately 50% of Pten−/− mutant embryos display defects in the movement of AVE cells. Conditional removal of Pten in embryonic tissues only, using the Sox2-cre transgene, confirms Pten's role in AVE migration for proper AP specification. A missense mutation in Nap1, kahlo, has a similar phenotype to Pten−/− mutant embryos; 15% of Nap1khlo mutant embryos show primitive streak duplications (Rakeman, et al., 2006). Nap1 regulates actin polymerization in migrating cells. Nap1 and Pten display a genetic interaction in AP axis specification: 100% of Nap1khlo/khlo;Pten+/− mutant embryos display multiple primitive streaks. These results indicate that Nap1 and Pten work together to control AP axis specification. We are investigating the molecular nature of this genetic interaction. doi:10.1016/j.ydbio.2008.05.415
Program/Abstract # 392 Anterior axis duplication in mouse embryos caused by mutation in Porcn Steffen Biechele a,b, Brian J. Cox a, Owen J. Tamplin a,b, Mei Lu a, Janet Rossant a,b a Program in Developmental and Stem Cell Biology, Sick Kids Hospital, Toronto, Canada b Department of Molecular Genetics, University of Toronto, Canada Wnt signaling plays important roles in development and disease. In mammals, 19 Wnts activate several different pathways such as the canonical Wnt signaling pathway and the non-canonical planar cell polarity (PCP) pathway. Due to functional redundancy of Wnt ligands, compound mutants and deletion of downstream pathway components have been used to investigate the role of Wnt signaling. These efforts have focused on the cells that receive Wnt signal, whereas cells secreting Wnt have not been extensively studied. The conserved Xchromosomal gene Porcupine (Porcn) encodes an O-acyl transferase required for lipid modification, secretion and gradient formation of several, if not all Wnt ligands. Mutations in the human homolog of Porcn cause Goltz Syndrome, an X-linked dominant disorder that is usually observed in females. Rare cases of postzygotic mutations in males suggest that Porcn mutations could underlie some human cases of X-linked male lethality. This study investigates the role of Porcn in early mouse development. Porcn null embryos have been generated by aggregation of wildtype embryos with Porcn-deficient embryonic stem (ES) cells. ES cell derived embryos fail to complete gastrulation and show duplication of the anterior body axis. This observation is consistent with the model of a Wnt signal gradient being required for embryonic axis induction; high Wnt signal induces posterior tissues, whereas low Wnt signal promotes anterior structures. Finally, we are generating a conditional allele of Porcn to further examine its role in embryonic development. doi:10.1016/j.ydbio.2008.05.416
Program/Abstract # 393 Redundant function of Wnt5a and Wnt11 in somitogenesis and anteroposterior axis elongation Hai Song a, Andreas Kispert b, Yingzi Yang a a GDRB, National Human Genome Research Institute, Bethesda, MD, USA b Institute For Molecular Biology, Medizinische Hochschule Hannover, 30625 Hannover, Germany Wnts are a large family of secreted glycoproteins that play diverse roles during embryogenesis and transduce their signals through several different pathways. Mouse Wnt5a mutant embryos show shortened anteroposterior (A-P) axis. However, the morphology of Wnt11 mutant embryos is largely normal. Since Wnt5a and Wnt11 have overlapped expression pattern and mainly signal through noncanonical Wnt signaling during mouse development, they may play redundant roles during A-P axis elongation and somitogenesis. To test this, we generated Wnt5a and Wnt11 double mutant mouse embryos. The double mutant embryos exhibited much shorter A-P axis, irregular somites and heart-looping defect. The expression of Wnt3a and Fgf8 was normal in the tail bud. However, the expression of Notch1 and Notch2 was greatly reduced in the presomitic mesoderm (PSM) of double mutant embryos. Consistently, the expression of target genes of Notch signaling was also greatly reduced in the PSM of double mutant embryos. Bilateral symmetry of somite formation was disrupted in the double mutant embryos. Furthermore, the somite patterning of double mutant embryos was disrupted, due to the reduced expression of Shh. Myogenesis of double mutant embryos