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1.
Development ; 146(7)2019 04 05.
Article in English | MEDLINE | ID: mdl-30890572

ABSTRACT

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.


Subject(s)
Cell Differentiation/physiology , Ectoderm/cytology , Germ Layers/cytology , Animals , Cell Differentiation/genetics , Cells, Cultured , Ectoderm/metabolism , Gastrulation/genetics , Gastrulation/physiology , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Germ Layers/metabolism , Mice , Signal Transduction/genetics , Signal Transduction/physiology
2.
Genesis ; 56(9): e23246, 2018 09.
Article in English | MEDLINE | ID: mdl-30114334

ABSTRACT

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.


Subject(s)
Gene Regulatory Networks , Genes, Homeobox , Head/embryology , LIM-Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Germ Cells/physiology , Transcription, Genetic , Xenopus laevis/embryology
3.
Development ; 142(11): 2069-79, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25977363

ABSTRACT

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.


Subject(s)
Embryo, Mammalian/metabolism , Head/embryology , LIM-Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Cadherins/metabolism , Endoderm/cytology , Endoderm/metabolism , Gene Deletion , Gene Expression Regulation, Developmental , Germ Layers/cytology , Germ Layers/metabolism , LIM-Homeodomain Proteins/genetics , Mice, Knockout , Models, Biological , Mutation , Phenotype , Signal Transduction , Transcription Factors/genetics , Wnt Proteins/metabolism
4.
Int J Dev Biol ; 55(1): 45-58, 2011.
Article in English | MEDLINE | ID: mdl-21305474

ABSTRACT

Sox17 is a transcription factor that is required for maintenance of the definitive endoderm in mouse embryos. By expression profiling of wild-type and mutant embryos and Sox17-overexpressing hepatoma cells, we identified genes with Sox17-dependent expression. Among the genes that were up-regulated in Sox17-null embryos and down-regulated by Sox17 expressing HepG2 cells is a set of genes that are expressed in the developing liver, suggesting that one function of Sox17 is the repression of liver gene expression, which is compatible with a role for Sox17 in maintaining the definitive endoderm in a progenitor state. Consistent with these findings, Sox17(-/-) cells display a diminished capacity to contribute to the definitive endoderm when transplanted into wild-type hosts. Analysis of gene ontology further revealed that many genes related to heart development were downregulated in Sox17-null embryos. This is associated with the defective development of the heart in the mutant embryos, which is accompanied by localised loss of Myocd-expressing cardiogenic progenitors and the malformation of the anterior intestinal portal.


Subject(s)
Embryo, Mammalian/metabolism , Gastrointestinal Tract/metabolism , Gene Expression Regulation, Developmental , HMGB Proteins/genetics , Myocardium/metabolism , SOXF Transcription Factors/genetics , Animals , Cell Transplantation/methods , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Endoderm/embryology , Endoderm/metabolism , Female , Gastrointestinal Tract/embryology , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HMGB Proteins/deficiency , Heart/embryology , Hep G2 Cells , Humans , In Situ Hybridization , Male , Mice , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , SOXF Transcription Factors/deficiency , Somites/embryology , Somites/metabolism
5.
Development ; 138(4): 667-76, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21228006

ABSTRACT

In mouse embryos, loss of Dickkopf-1 (DKK1) activity is associated with an ectopic activation of WNT signalling responses in the precursors of the craniofacial structures and leads to a complete truncation of the head at early organogenesis. Here, we show that ENU-induced mutations of genes coding for two WNT canonical pathway factors, the co-receptor LRP6 and the transcriptional co-activator ß-catenin, also elicit an ectopic signalling response and result in loss of the rostral tissues of the forebrain. Compound mutant embryos harbouring combinations of mutant alleles of Lrp6, Ctnnb1 and Dkk1 recapitulate the partial to complete head truncation phenotype of individual homozygous mutants. The demonstration of a synergistic interaction of Dkk1, Lrp6 and Ctnnb1 provides compelling evidence supporting the concepts that (1) stringent regulation of the level of canonical WNT signalling is necessary for head formation, (2) activity of the canonical pathway is sufficient to account for the phenotypic effects of mutations in three different components of the signal cascade and (3) rostral parts of the brain and the head are differentially more sensitive to canonical WNT signalling and their development is contingent on negative modulation of WNT signalling activity.


Subject(s)
Head/embryology , Signal Transduction , Alleles , Animals , Base Sequence , Gene Expression Regulation, Developmental , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/metabolism , LDL-Receptor Related Proteins/genetics , LDL-Receptor Related Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , Mice, Inbred BALB C , Mutation , Phenotype , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolism
6.
Eur J Cell Biol ; 89(7): 489-98, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20223554

ABSTRACT

Previous studies have shown that the overexpression of tropomyosins leads to isoform-specific alterations in the morphology of subcellular compartments in neuronal cells. Here we have examined the role of the most abundant set of isoforms from the gamma-Tm gene by knocking out the alternatively spliced C-terminal exon 9d. Despite the widespread location of exon 9d-containing isoforms, mice were healthy and viable. Compensation by products containing the C-terminal exon 9c was seen in the adult brain. While neurons from these mice show a mild phenotype at one day in culture, neurons revealed a significant morphological alteration with an increase in the branching of dendrites and axons after four days in culture. Our data suggest that this effect is mediated via altered stability of actin filaments in the growth cones. We conclude that exon 9d-containing isoforms are not essential for survival of neuronal cells and that isoform choice from the gamma-Tm gene is flexible in the brain. Although functional redundancy does not exist between tropomyosin genes, these results suggest that significant redundancy exists between products from the same gene.


Subject(s)
Neurogenesis/physiology , Tropomyosin/metabolism , Alternative Splicing/genetics , Alternative Splicing/physiology , Animals , Axons/metabolism , Brain/cytology , Brain/metabolism , Cell Line , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Immunohistochemistry , Mice , Neurogenesis/genetics , Neurons/cytology , Neurons/metabolism , Phenotype , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tropomyosin/genetics
8.
Mech Dev ; 125(7): 587-600, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18486455

ABSTRACT

This fate-mapping study reveals that the progenitors of all major parts of the embryonic gut are already present in endoderm of the early-head-fold to early-somite stage (1-9 somites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal portal of the early-organogenesis stage (16-19 somites) embryo. Endoderm cells around and lateral to the node are allocated to the open "midgut" region of the embryonic gut. The posterior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also the open "midgut". Descendants of the posterior endoderm span a length of the gut from the level of the 3rd-5th somites to the posterior end of the embryonic gut. The formation of the anterior and posterior intestinal portals is accompanied by similar repertoires of morphogenetic tissue movement. We also discovered that cells on contralateral sides of the anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.


Subject(s)
Endoderm/cytology , Gastrointestinal Tract/anatomy & histology , Gastrointestinal Tract/embryology , Morphogenesis/physiology , Stem Cells/cytology , Animals , Cell Movement/physiology , Endoderm/physiology , Female , Liver/anatomy & histology , Liver/embryology , Mice , Mice, Inbred Strains , Stem Cells/physiology
9.
Development ; 135(10): 1791-801, 2008 May.
Article in English | MEDLINE | ID: mdl-18403408

ABSTRACT

Loss of Dkk1 results in ectopic WNT/beta-catenin signalling activity in the anterior germ layer tissues and impairs cell movement in the endoderm of the mouse gastrula. The juxtaposition of the expression domains of Dkk1 and Wnt3 is suggestive of an antagonist-agonist interaction. The downregulation of Dkk1 when Wnt3 activity is reduced reveals a feedback mechanism for regulating WNT signalling. Compound Dkk1;Wnt3 heterozygous mutant embryos display head truncation and trunk malformation, which are not found in either Dkk1(+/-) or Wnt3(+/-) embryos. Reducing the dose of Wnt3 gene in Dkk1(-/-) embryos partially rescues the truncated head phenotype. These findings highlight that head development is sensitive to the level of WNT3 signalling and that DKK1 is the key antagonist that modulates WNT3 activity during anterior morphogenesis.


Subject(s)
Head/embryology , Intercellular Signaling Peptides and Proteins/physiology , Morphogenesis/physiology , Wnt Proteins/physiology , Animals , Body Patterning/physiology , Down-Regulation , Gastrula/cytology , Gastrula/embryology , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Mutant Strains , Mutation , Signal Transduction , Wnt Proteins/genetics , Wnt3 Protein
10.
Development ; 134(2): 251-60, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17151016

ABSTRACT

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.


Subject(s)
Endoderm/cytology , Gastrula/cytology , Animals , Body Patterning/genetics , Cell Movement , Cell Transplantation , Digestive System/cytology , Digestive System/embryology , Female , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Pregnancy
11.
Mech Dev ; 124(2): 157-165, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17127040

ABSTRACT

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.

12.
Dev Biol ; 274(1): 171-87, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15355796

ABSTRACT

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anterior-posterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm was sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos.


Subject(s)
Cell Differentiation/physiology , Cell Movement/physiology , Endoderm/physiology , Gastrula/physiology , Homeodomain Proteins/physiology , Mice/embryology , Animals , DNA-Binding Proteins/metabolism , Electroporation , Galactosides , Gastrointestinal Tract/embryology , Gene Expression Regulation, Developmental/physiology , Green Fluorescent Proteins , Hepatocyte Nuclear Factor 3-beta , High Mobility Group Proteins/metabolism , Histological Techniques , In Situ Hybridization , Indoles , LIM-Homeodomain Proteins , Luminescent Proteins , Mice, Mutant Strains , Morphogenesis , Nuclear Proteins/metabolism , Transcription Factors/metabolism
13.
Dev Cell ; 4(1): 83-94, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12530965

ABSTRACT

To investigate Lim1 function during gastrulation, we used transcript depletion through DEED antisense oligonucleotides in Xenopus and cell transplantation in mice. Xenopus embryos depleted of Lim1 lack anterior head structures and fail to form a proper axis as a result of a failure of gastrulation movements, even though mesodermal cell identities are specified. Similar disruption of cell movements in the mesoderm is also observed in Lim1(-/-) mice. Paraxial protocadherin (PAPC) expression is lost in the nascent mesoderm of Lim1(-/-) mouse embryos and in the organizer of Lim1-depleted Xenopus embryos; the latter can be rescued to a considerable extent by supplying PAPC exogenously. We conclude that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation.


Subject(s)
Cell Movement , Gastrula/cytology , Gastrula/metabolism , Homeodomain Proteins/metabolism , Xenopus/embryology , Animals , Biological Evolution , Body Patterning , Cadherins/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , In Situ Hybridization , LIM-Homeodomain Proteins , Mesoderm/cytology , Mesoderm/metabolism , Mice , Phenotype , Protocadherins , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors , Xenopus/genetics , Xenopus/metabolism , Xenopus Proteins
14.
Genesis ; 35(1): 57-62, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12481299

ABSTRACT

We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2-3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8-9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism.


Subject(s)
Electroporation , Gastrula/physiology , Germ Layers/physiology , Alkaline Phosphatase/metabolism , Animals , Biomarkers , Cell Culture Techniques , Cell Lineage , Embryo, Mammalian/anatomy & histology , Genes, Reporter , Genetic Techniques , Green Fluorescent Proteins , Integrases/metabolism , Lac Operon/physiology , Luminescent Proteins/metabolism , Mice , Viral Proteins/metabolism
15.
Dev Biol ; 247(2): 251-70, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12086465

ABSTRACT

Loss of Twist function in the cranial mesenchyme of the mouse embryo causes failure of closure of the cephalic neural tube and malformation of the branchial arches. In the Twist(-/-) embryo, the expression of molecular markers that signify dorsal forebrain tissues is either absent or reduced, but those associated with ventral tissues display expanded domains of expression. Dorsoventral organization of the mid- and hindbrain and the anterior-posterior pattern of the neural tube are not affected. In the Twist(-/-) embryo, neural crest cells stray from the subectodermal migratory path and the late-migrating subpopulation invades the cell-free zone separating streams of cells going to the first and second branchial arches. Cell transplantation studies reveal that Twist activity is required in the cranial mesenchyme for directing the migration of the neural crest cells, as well as in the neural crest cells within the first branchial arch to achieve correct localization. Twist is also required for the proper differentiation of the first arch tissues into bone, muscle, and teeth.


Subject(s)
Neural Crest/embryology , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Prosencephalon/embryology , Transcription Factors , Animals , Cell Differentiation , Cell Movement , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , Genotype , Green Fluorescent Proteins , Heterozygote , High Mobility Group Proteins/metabolism , In Situ Hybridization , Luminescent Proteins/metabolism , Mice , Mice, Transgenic , Microscopy, Fluorescence , Morphogenesis , Mutation , Neural Crest/anatomy & histology , Nuclear Proteins/genetics , RNA, Messenger/metabolism , SOXE Transcription Factors , Time Factors , Twist-Related Protein 1 , beta-Galactosidase/analysis
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