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Eric H. Davidson - One of the best experts on this subject based on the ideXlab platform.
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A provisional regulatory Gene Network for specification of endomesoderm in the sea urchin embryo.
2018Co-Authors: Eric H. Davidson, Andrew Ransick, Takuya Minokawa, Paola Oliveri, Gabriele Amore, Jonathan P Rast, Cristina Calestani, Chiou-hwa Yuh, Veronica F. Hinman, César Arenas-menaAbstract:We present the current form of a provisional DNA sequence-based regulatory Gene Network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the Network is in a continuous process of revision and growth as new Genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The Network contains over 40 Genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the Network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between Genes in the Network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant Genes in the Network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory Genes in the Network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from Genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The Network model predicts the cis-regulatory inputs that link each Gene into the Network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the Genes in the Network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The Network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation Genes. Each of the temporal-spatial phases of specification is represented in a subelement of the Network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.
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a provisional regulatory Gene Network for specification of endomesoderm in the sea urchin embryo
Developmental Biology, 2002Co-Authors: Eric H. Davidson, Andrew Ransick, Takuya Minokawa, Paola Oliveri, Gabriele Amore, Jonathan P Rast, Cristina Calestani, Veronica F. Hinman, Cesar Arenasmena, Ochan OtimAbstract:We present the current form of a provisional DNA sequence-based regulatory Gene Network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the Network is in a continuous process of revision and growth as new Genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The Network contains over 40 Genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the Network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between Genes in the Network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant Genes in the Network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory Genes in the Network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from Genetic expression constructs and from injected mRNAs; (4) blockade of the β-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The Network model predicts the cis-regulatory inputs that link each Gene into the Network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the Genes in the Network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The Network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg_2 endomesodermal domain; the blastula-stage separation of the central veg_2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg_2 endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation Genes. Each of the temporal–spatial phases of specification is represented in a subelement of the Network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.
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a regulatory Gene Network that directs micromere specification in the sea urchin embryo
Developmental Biology, 2002Co-Authors: Paola Oliveri, Deanna M Carrick, Eric H. DavidsonAbstract:Micromeres and their immediate descendants have three known developmental functions in regularly developing sea urchins: immediately after their initial segregation, they are the source of an unidentified signal to the adjacent veg2 cells that is required for normal endomesodermal specification; a few cleavages later, they express Delta, a Notch ligand which triggers the conditional specification of the central mesodermal domain of the vegetal plate; and they exclusively give rise to the skeletogenic mesenchyme of the postgastrular embryo. We demonstrate the key components of the zygotic regulatory Gene Network that accounts for micromere specificity. This Network is a subelement of the overall endomesoderm specification Network of the Strongylocentrotus purpuratus embryo. A central role is played by a newly discovered Gene encoding a paired class homeodomain transcription factor which in micromeres acts as a repressor of a repressor: the Gene is named pmar1 (paired-class micromere anti-repressor). pmar1 is expressed only during cleavage and early blastula stages, and exclusively in micromeres. It is initially activated as soon as the micromeres are formed, in response to Otx and β-Catenin/Tcf inputs. The repressive nature of the interactions mediated by the pmar1 Gene product was shown by the identical effect of introducing mRNA encoding the Pmar1 factor, and mRNA encoding an Engrailed-Pmar1 (En-Pmar1) repressor domain fusion. In both cases, the effects are derepression: of the delta Gene; and of skeletogenic Genes, including several transcription factors normally expressed only in micromere descendants, and also a set of downstream skeletogenic differentiation Genes. The spatial phenotype of embryos bearing exogenous mRNA encoding Pmar1 factor or En-Pmar1 is expansion of the domains of expression of the downstream Genes over most or all of the embryo. This results in transformation of much of the embryo into skeletogenic mesenchyme cells that express skeletogenic markers. The normal role of pmarl is to prevent, exclusively in the micromeres, the expression of a repressor that is otherwise operative throughout the embryo. This function accounts for the localization of delta transcription in micromeres, and thereby for the conditional specification of the vegetal plate mesoderm. It also explains why skeletogenic differentiation Gene batteries normally function only in micromere descendants. More Generally, the regulatory Network subelement emerging from this work shows how the specificity of micromere function depends on continuing global regulatory interactions, as well as on early localized inputs.
Laurent Journot - One of the best experts on this subject based on the ideXlab platform.
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a systems level approach to parental genomic imprinting the imprinted Gene Network includes extracellular matrix Genes and regulates cell cycle exit and differentiation
Genome Research, 2015Co-Authors: Hala Al Adhami, Brendan Evano, Anne Le Digarcher, Charlotte Gueydan, Emeric Dubois, Hugues Parrinello, Tristan Bouschet, Annie Varrault, Christelle Dantec, Laurent JournotAbstract:Genomic imprinting is an epiGenetic mechanism that restrains the expression of ∼100 eutherian Genes in a parent-of-origin-specific manner. The reason for this selective targeting of Genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted Genes are coexpressed in a Network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipoGenesis in vitro, and muscle reGeneration in vivo. Imprinted Gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted Gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted Gene Network includes biallelically expressed, nonimprinted Genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor–activated signaling. These observations show that imprinted Genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.
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Modulation of imprinted Gene Network in placenta results in normal development of in vitro manipulated mouse embryos
Human Molecular Genetics, 2010Co-Authors: Patricia Fauque, Anne Le Digarcher, Anne Gabory, Laurent Journot, Anne Ripoche, Jorg Tost, Florence Busato, Françoise Mondon, Ivo Gut, Pierre JouannetAbstract:Genomic imprinting regulates the expression of a group of Genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these Genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting Genes from the recently described imprinted Gene Network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other Genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated Network of imprinted Genes results in a compensatory process capable of correcting potential dysfunction of placenta.
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h19 acts as a trans regulator of the imprinted Gene Network controlling growth in mice
Development, 2009Co-Authors: Anne Gabory, Azim M Surani, Anne Le Digarcher, Marieanne Ripoche, Francoise Watrin, Ahmed Ziyyat, Thierry Forne, Justin F X Ainscough, Helene Jammes, Laurent JournotAbstract:The imprinted H19 Gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 Gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other Genes from the newly described imprinted Gene Network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several Genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 Gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.
Antonio Miceli - One of the best experts on this subject based on the ideXlab platform.
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activation of a Gene Network in durum wheat roots exposed to cadmium
BMC Plant Biology, 2018Co-Authors: Alessio Aprile, Erika Sabella, Marzia Vergine, A Genga, M Siciliano, Eliana Nutricati, Patrizia Rampino, Mariarosaria De Pascali, Andrea Luvisi, Antonio MiceliAbstract:Among cereals, durum wheat (Triticum turgidum L. subsp. durum) accumulates cadmium (Cd) at higher concentration if grown in Cd-polluted soils. Since cadmium accumulation is a risk for human health, the international trade organizations have limited the acceptable concentration of Cd in edible crops. Therefore, durum wheat cultivars accumulating low cadmium in grains should be preferred by farmers and consumers. To identify the response of durum wheat to the presence of Cd, the transcriptomes of roots and shoots of Creso and Svevo cultivars were sequenced after a 50-day exposure to 0.5 μM Cd in hydroponic solution. No phytotoxic effects or biomass reduction was observed in Creso and Svevo plants at this Cd concentration. Despite this null effect, cadmium was accumulated in root tissues, in shoots and in grains suggesting a good cadmium translocation rate among tissues. The mRNA sequencing revealed a General transcriptome rearrangement after Cd treatment and more than 7000 Genes were found differentially expressed in root and shoot tissues. Among these, the up-regulated Genes in roots showed a clear correlation with cadmium uptake and detoxification. In particular, about three hundred Genes were commonly up-regulated in Creso and Svevo roots suggesting a well defined molecular strategy characterized by the transcriptomic activation of several transcription factors mainly belonging to bHLH and WRKY families. bHLHs are probably the activators of the strong up-regulation of three NAS Genes, responsible for the synthesis of the phytosiderophore nicotianamine (NA). Moreover, we found the overall up-regulation of the methionine salvage pathway that is tightly connected with NA synthesis and supply the S-adenosyl methionine necessary for NA biosynthesis. Finally, several vacuolar NA chelating heavy metal transporters were vigorously activated. In conclusion, the exposure of durum wheat to cadmium activates in roots a complex Gene Network involved in cadmium translocation and detoxification from heavy metals. These findings are confident with a role of nicotianamine and methionine salvage pathway in the accumulation of cadmium in durum wheat.
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Activation of a Gene Network in durum wheat roots exposed to cadmium
'Springer Science and Business Media LLC', 2018Co-Authors: Alessio Aprile, Erika Sabella, Marzia Vergine, A Genga, M Siciliano, Eliana Nutricati, Patrizia Rampino, Mariarosaria De Pascali, Andrea Luvisi, Antonio MiceliAbstract:Abstract Background Among cereals, durum wheat (Triticum turgidum L. subsp. durum) accumulates cadmium (Cd) at higher concentration if grown in Cd-polluted soils. Since cadmium accumulation is a risk for human health, the international trade organizations have limited the acceptable concentration of Cd in edible crops. Therefore, durum wheat cultivars accumulating low cadmium in grains should be preferred by farmers and consumers. To identify the response of durum wheat to the presence of Cd, the transcriptomes of roots and shoots of Creso and Svevo cultivars were sequenced after a 50-day exposure to 0.5 μM Cd in hydroponic solution. Results No phytotoxic effects or biomass reduction was observed in Creso and Svevo plants at this Cd concentration. Despite this null effect, cadmium was accumulated in root tissues, in shoots and in grains suggesting a good cadmium translocation rate among tissues. The mRNA sequencing revealed a General transcriptome rearrangement after Cd treatment and more than 7000 Genes were found differentially expressed in root and shoot tissues. Among these, the up-regulated Genes in roots showed a clear correlation with cadmium uptake and detoxification. In particular, about three hundred Genes were commonly up-regulated in Creso and Svevo roots suggesting a well defined molecular strategy characterized by the transcriptomic activation of several transcription factors mainly belonging to bHLH and WRKY families. bHLHs are probably the activators of the strong up-regulation of three NAS Genes, responsible for the synthesis of the phytosiderophore nicotianamine (NA). Moreover, we found the overall up-regulation of the methionine salvage pathway that is tightly connected with NA synthesis and supply the S-adenosyl methionine necessary for NA biosynthesis. Finally, several vacuolar NA chelating heavy metal transporters were vigorously activated. Conclusions In conclusion, the exposure of durum wheat to cadmium activates in roots a complex Gene Network involved in cadmium translocation and detoxification from heavy metals. These findings are confident with a role of nicotianamine and methionine salvage pathway in the accumulation of cadmium in durum wheat
Paola Oliveri - One of the best experts on this subject based on the ideXlab platform.
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A provisional regulatory Gene Network for specification of endomesoderm in the sea urchin embryo.
2018Co-Authors: Eric H. Davidson, Andrew Ransick, Takuya Minokawa, Paola Oliveri, Gabriele Amore, Jonathan P Rast, Cristina Calestani, Chiou-hwa Yuh, Veronica F. Hinman, César Arenas-menaAbstract:We present the current form of a provisional DNA sequence-based regulatory Gene Network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the Network is in a continuous process of revision and growth as new Genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The Network contains over 40 Genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the Network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between Genes in the Network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant Genes in the Network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory Genes in the Network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from Genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The Network model predicts the cis-regulatory inputs that link each Gene into the Network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the Genes in the Network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The Network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation Genes. Each of the temporal-spatial phases of specification is represented in a subelement of the Network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.
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a provisional regulatory Gene Network for specification of endomesoderm in the sea urchin embryo
Developmental Biology, 2002Co-Authors: Eric H. Davidson, Andrew Ransick, Takuya Minokawa, Paola Oliveri, Gabriele Amore, Jonathan P Rast, Cristina Calestani, Veronica F. Hinman, Cesar Arenasmena, Ochan OtimAbstract:We present the current form of a provisional DNA sequence-based regulatory Gene Network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the Network is in a continuous process of revision and growth as new Genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The Network contains over 40 Genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the Network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between Genes in the Network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant Genes in the Network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory Genes in the Network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from Genetic expression constructs and from injected mRNAs; (4) blockade of the β-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The Network model predicts the cis-regulatory inputs that link each Gene into the Network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the Genes in the Network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The Network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg_2 endomesodermal domain; the blastula-stage separation of the central veg_2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg_2 endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation Genes. Each of the temporal–spatial phases of specification is represented in a subelement of the Network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.
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a regulatory Gene Network that directs micromere specification in the sea urchin embryo
Developmental Biology, 2002Co-Authors: Paola Oliveri, Deanna M Carrick, Eric H. DavidsonAbstract:Micromeres and their immediate descendants have three known developmental functions in regularly developing sea urchins: immediately after their initial segregation, they are the source of an unidentified signal to the adjacent veg2 cells that is required for normal endomesodermal specification; a few cleavages later, they express Delta, a Notch ligand which triggers the conditional specification of the central mesodermal domain of the vegetal plate; and they exclusively give rise to the skeletogenic mesenchyme of the postgastrular embryo. We demonstrate the key components of the zygotic regulatory Gene Network that accounts for micromere specificity. This Network is a subelement of the overall endomesoderm specification Network of the Strongylocentrotus purpuratus embryo. A central role is played by a newly discovered Gene encoding a paired class homeodomain transcription factor which in micromeres acts as a repressor of a repressor: the Gene is named pmar1 (paired-class micromere anti-repressor). pmar1 is expressed only during cleavage and early blastula stages, and exclusively in micromeres. It is initially activated as soon as the micromeres are formed, in response to Otx and β-Catenin/Tcf inputs. The repressive nature of the interactions mediated by the pmar1 Gene product was shown by the identical effect of introducing mRNA encoding the Pmar1 factor, and mRNA encoding an Engrailed-Pmar1 (En-Pmar1) repressor domain fusion. In both cases, the effects are derepression: of the delta Gene; and of skeletogenic Genes, including several transcription factors normally expressed only in micromere descendants, and also a set of downstream skeletogenic differentiation Genes. The spatial phenotype of embryos bearing exogenous mRNA encoding Pmar1 factor or En-Pmar1 is expansion of the domains of expression of the downstream Genes over most or all of the embryo. This results in transformation of much of the embryo into skeletogenic mesenchyme cells that express skeletogenic markers. The normal role of pmarl is to prevent, exclusively in the micromeres, the expression of a repressor that is otherwise operative throughout the embryo. This function accounts for the localization of delta transcription in micromeres, and thereby for the conditional specification of the vegetal plate mesoderm. It also explains why skeletogenic differentiation Gene batteries normally function only in micromere descendants. More Generally, the regulatory Network subelement emerging from this work shows how the specificity of micromere function depends on continuing global regulatory interactions, as well as on early localized inputs.
Ochan Otim - One of the best experts on this subject based on the ideXlab platform.
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a provisional regulatory Gene Network for specification of endomesoderm in the sea urchin embryo
Developmental Biology, 2002Co-Authors: Eric H. Davidson, Andrew Ransick, Takuya Minokawa, Paola Oliveri, Gabriele Amore, Jonathan P Rast, Cristina Calestani, Veronica F. Hinman, Cesar Arenasmena, Ochan OtimAbstract:We present the current form of a provisional DNA sequence-based regulatory Gene Network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the Network is in a continuous process of revision and growth as new Genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The Network contains over 40 Genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the Network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between Genes in the Network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant Genes in the Network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory Genes in the Network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from Genetic expression constructs and from injected mRNAs; (4) blockade of the β-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The Network model predicts the cis-regulatory inputs that link each Gene into the Network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the Genes in the Network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The Network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg_2 endomesodermal domain; the blastula-stage separation of the central veg_2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg_2 endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation Genes. Each of the temporal–spatial phases of specification is represented in a subelement of the Network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.