Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 4925-4932 ◽  
Author(s):  
W. Shawlot ◽  
M. Wakamiya ◽  
K.M. Kwan ◽  
A. Kania ◽  
T.M. Jessell ◽  
...  
Keyword(s):  
Marker Gene ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Primitive Streak ◽  
Mouse Embryos ◽  
Chimeric Mouse ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Targeted Mutation ◽  
Head Formation

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominantly wild-type cells, we found that Lim1(−)(/)(−) cells were able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that embryonic day 9.5 chimeric embryos displayed a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1(−)(/)(−) embryonic stem cells into wild-type tetraploid blastocysts lacked forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1(−)(/)(−) embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1(−)(/)(−) cells and the embryo proper of largely wild-type cells, also phenocopied the Lim1(−)(/)(−) headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse.

The transcription factor HNF3beta is required in visceral endoderm for normal primitive streak morphogenesis

Development ◽  
1998 ◽  
Vol 125 (16) ◽  
pp. 3015-3025 ◽  
Author(s):  
D. Dufort ◽  
L. Schwartz ◽  
K. Harpal ◽  
J. Rossant
Keyword(s):  
Transcription Factor ◽  
Primitive Streak ◽  
Chimeric Mouse ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Targeted Mutation ◽  
Notochord Cell ◽  
Cell Aggregations ◽  
Left Right Asymmetry ◽  
Embryonic Ectoderm

During early embryogenesis, the transcription factor HNF3beta is expressed in visceral and definitive endoderm, node, notochord and floorplate. A targeted mutation in the HNF3β gene results in the lack of a definitive node and notochord. Furthermore, lack of HNF3beta results in failure of proper primitive streak elongation. To address whether HNF3beta is required in visceral endoderm, we have used tetraploid embryo-ES cell aggregations to generate chimeric mouse embryos with wild-type visceral endoderm and homozygous mutant HNF3beta embryonic ectoderm or vice versa. Replacing the visceral endoderm of mutant HNF3beta embryos rescued proper primitive streak elongation and, conversely, mutant visceral endoderm imposed a severe embryonic-extraembryonic constriction on wild-type embryonic ectoderm. Restoration of normal streak morphogenesis was not sufficient to allow formation of the node and notochord in HNF3beta mutant embryos. Thus, our results demonstrate that HNF3beta has two separate roles in primitive streak formation. One is to act within the visceral endoderm to promote proper streak morphogenesis. The second is autonomous to the node and its precursors and involves specification of node and notochord cell fates. HNF3beta mutant embryos rescued for the embryonic-extraembryonic constriction developed further than mutant embryos, allowing examination of later roles for HNF3beta. We show that such mutant embryos lack foregut and midgut endoderm. In addition, left-right asymmetry is affected in the mutant embryos.

scholarly journals The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2433-2445 ◽  
Author(s):  
J.P. Martinez Barbera ◽  
M. Clements ◽  
P. Thomas ◽  
T. Rodriguez ◽  
D. Meloy ◽  
...  
Keyword(s):  
Normal Development ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Definitive Endoderm ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Zona Limitans Intrathalamica ◽  
Ventral Thalamus ◽  
Septum Transversum ◽  
Early Mouse

The homeobox gene Hex is expressed in the anterior visceral endoderm (AVE) and rostral definitive endoderm of early mouse embryos. Later, Hex transcripts are detected in liver, thyroid and endothelial precursor cells. A null mutation was introduced into the Hex locus by homologous recombination in embryonic stem cells. Hex mutant embryos exhibit varying degrees of anterior truncation as well as liver and thyroid dysplasia. The liver diverticulum is formed but migration of hepatocytes into the septum transversum fails to occur. Development of the thyroid is arrested at the thyroid bud stage at 9.5 dpc. Brain defects are restricted to the rostral forebrain and have a caudal limit at the zona limitans intrathalamica, the boundary between dorsal and ventral thalamus. Analysis of Hex(−/−) mutants at early stages shows that the prospective forebrain ectoderm is correctly induced and patterned at 7.5 days post coitum (dpc), but subsequently fails to develop. AVE markers are expressed and correctly positioned but development of rostral definitive endoderm is greatly disturbed in Hex(−/−) embryos. Chimeric embryos composed of Hex(−/−) cells developing within a wild-type visceral endoderm show forebrain defects indicating that Hex is required in the definitive endoderm. All together, these results demonstrate that Hex function is essential in definitive endoderm for normal development of the forebrain, liver and thyroid gland.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1881-1892 ◽  
Author(s):  
Annette Damert ◽  
Lucile Miquerol ◽  
Marina Gertsenstein ◽  
Werner Risau ◽  
Andras Nagy
Keyword(s):  
Yolk Sac ◽  
Endothelial Differentiation ◽  
Vascular Endothelial ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Hypomorphic Allele ◽  
Targeted Mutation ◽  
Blood Formation ◽  
Opposite Situation ◽  
Endothelial Growth Factor

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegflo. Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing ‘Vegf wild-type’ visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the ‘Vegf wild-type’ yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.

Correction of β Thalassemia by Homologous Recombination in Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 373-373 ◽  
Author(s):  
Thomas M. Ryan ◽  
Chiao-Wang Sun ◽  
Li-Chen Wu ◽  
Jin-Xiang Ren ◽  
Tim M. Townes
Keyword(s):  
Homologous Recombination ◽  
Es Cells ◽  
Globin Gene ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Microcytic Anemia ◽  
Globin Genes ◽  
Wild Type ◽  
Distribution Width ◽  
Erythrocyte Morphology

Abstract Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.

The homeoprotein Hex is required for hemangioblast differentiation

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2428-2435 ◽  
Author(s):  
Ying Guo ◽  
Rebecca Chan ◽  
Heather Ramsey ◽  
Weiming Li ◽  
Xiaodong Xie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Chimeric Mouse ◽  
Es Cell ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Temporal And Spatial

Abstract The first hematopoietic and endothelial progenitors are derived from a common embryonic precursor termed the hemangioblast. The genetic cascades that regulate the differentiation of the hemangioblast to hematopoietic and endothelial cells are largely unknown. In general, much of embryonic development is coordinately regulated by temporal and spatial expression of transcription factors, such as the Homeobox (Hox) gene family. We and others isolated a divergent homeobox gene termed Hex (or Prh) that is preferentially expressed in hematopoietic and endothelial cells. Using in vitro Hex-/- embryonic stem (ES) cell differentiation, in vivo yolk sac hematopoietic progenitor assays, and chimeric mouse analysis, we found that Hex is required for differentiation of the hemangioblast to definitive embryonic hematopoietic progenitors and to a lesser extent endothelial cells. Therefore, Hex is a novel regulator of hemangioblast differentiation to hematopoietic and endothelial cells. (Blood. 2003;102:2428-2435)

scholarly journals Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells

2005 ◽  
Vol 25 (24) ◽  
pp. 10791-10802 ◽  
Author(s):  
Claudia R. Amura ◽  
Lindsay Marek ◽  
Robert A. Winn ◽  
Lynn E. Heasley
Keyword(s):  
Pc12 Cell ◽  
Neural Differentiation ◽  
Es Cells ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Signal Pathways ◽  
Es Cell ◽  
Wild Type ◽  
Base Pairs ◽  
Promoter Sequences

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

scholarly journals Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

1992 ◽  
Vol 12 (8) ◽  
pp. 3636-3643 ◽  
Author(s):  
D G Kim ◽  
H M Kang ◽  
S K Jang ◽  
H S Shin
Keyword(s):  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Encephalomyocarditis Virus ◽  
Vector System ◽  
Chimeric Mouse ◽  
Entry Site ◽  
Cell Extracts ◽  
Ribosomal Entry

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

Cell autonomous and non-cell autonomous functions of Otx2 in patterning the rostral brain

Development ◽  
1999 ◽  
Vol 126 (19) ◽  
pp. 4295-4304 ◽  
Author(s):  
M. Rhinn ◽  
A. Dierich ◽  
M. Le Meur ◽  
S. Ang
Keyword(s):  
Cell Adhesion ◽  
Target Genes ◽  
Homeobox Gene ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Candidate Target ◽  
Important Regulator ◽  
Mutant Cells ◽  
The Brain ◽  
Brain Patterning

Previous studies have shown that the homeobox gene Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Here, we demonstrate that Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type cells in the brain, we observe a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 in mutant cells, whereas expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2.

Head induction in the chick by primitive endoderm of mammalian, but not avian origin

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 815-825 ◽  
Author(s):  
H. Knoetgen ◽  
C. Viebahn ◽  
M. Kessel
Keyword(s):  
Lower Layer ◽  
Homeobox Gene ◽  
Primitive Streak ◽  
Neural Plate ◽  
Visceral Endoderm ◽  
Avian Origin ◽  
Bmp Antagonist ◽  
Rabbit Embryos ◽  
Inductive Potential ◽  
Anterior Visceral Endoderm

Different types of endoderm, including primitive, definitive and mesendoderm, play a role in the induction and patterning of the vertebrate head. We have studied the formation of the anterior neural plate in chick embryos using the homeobox gene GANF as a marker. GANF is first expressed after mesendoderm ingression from Hensen's node. We found that, after transplantation, neither the avian hypoblast nor the anterior definitive endoderm is capable of GANF induction, whereas the mesendoderm (young head process, prechordal plate) exhibits a strong inductive potential. GANF induction cannot be separated from the formation of a proper neural plate, which requires an intact lower layer and the presence of the prechordal mesendoderm. It is inhibited by BMP4 and promoted by the presence of the BMP antagonist Noggin. In order to investigate the inductive potential of the mammalian visceral endoderm, we used rabbit embryos which, in contrast to mouse embryos, allow the morphological recognition of the prospective anterior pole in the living, pre-primitive-streak embryo. The anterior visceral endoderm from such rabbit embryos induced neuralization and independent, ectopic GANF expression domains in the area pellucida or the area opaca of chick hosts. Thus, the signals for head induction reside in the anterior visceral endoderm of mammals whereas, in birds and amphibia, they reside in the prechordal mesendoderm, indicating a heterochronic shift of the head inductive capacity during the evolution of mammalia.

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