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ABSTRACT: We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.
Development 06/2001; 128(10):1911-21. · 6.60 Impact Factor
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ABSTRACT: Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.
Development 04/2001; 128(5):753-65. · 6.60 Impact Factor
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ABSTRACT: We have identified a novel mouse member of the Wnt family, Wnt13. Among mouse Wnt genes, Wnt13 is most closely related to Wnt2. Sequence comparisons and chromosomal localization strongly suggest that Wnt13, rather than Wnt2, is the mouse orthologue of both the human WNT13 and Xenopus XWnt2 genes. Wnt13 is expressed in the embryonic mesoderm during gastrulation. At later stages, transcripts are detected in the dorsal midline of the diencephalon and mesencephalon, the heart primordia, the periphery of the lung bud and the otic and optic vesicles. These data suggest that Wnt13 function might partially overlap with those of other Wnt genes in the cell signaling mechanisms controlling mesoderm specification during gastrulation and some aspects of brain, heart and lung formation.
Mechanisms of Development 05/1998; 73(1):107-16. · 2.83 Impact Factor
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D Acampora,
S Mazan,
F Tuorto,
V Avantaggiato,
J J Tremblay,
D Lazzaro,
A di Carlo,
A Mariano,
P E Macchia,
G Corte,
V Macchia,
J Drouin, P Brûlet,
A Simeone
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ABSTRACT: Genetic and molecular approaches have enabled the identification of regulatory genes critically involved in determining cell types in the pituitary gland and/or in the hypothalamus. Here we report that Otx1, a homeobox-containing gene of the Otx gene family, is postnatally transcribed and translated in the pituitary gland. Cell culture experiments indicate that Otx1 may activate transcription of the growth hormone (GH), follicle-stimulating hormone (betaFSH), luteinizing hormone (betaLH) and alpha-glycoprotein subunit (alphaGSU) genes. Analysis of Otx1 null mice indicates that, at the prepubescent stage, they exhibit transient dwarfism and hypogonadism due to low levels of pituitary GH, FSH and LH hormones which, in turn, dramatically affect downstream molecular and organ targets. Nevertheless, Otx1-/- mice gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with restored growth and gonadal function at 4 months of age. Expression patterns of related hypothalamic and pituitary cell type restricted genes, growth hormone releasing hormone (GRH), gonadotropin releasing hormone (GnRH) and their pituitary receptors (GRHR and GnRHR) suggest that, in Otx1-/- mice, hypothalamic and pituitary cells of the somatotropic and gonadotropic lineages appear unaltered and that the ability to synthesize GH, FSH and LH, rather than the number of cells producing these hormones, is affected. Our data indicate that Otx1 is a new pituitary transcription factor involved at the prepubescent stage in the control of GH, FSH and LH hormone levels and suggest that a complex regulatory mechanism might exist to control the physiological need for pituitary hormones at specific postnatal stages.
Development 05/1998; 125(7):1229-39. · 6.60 Impact Factor
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ABSTRACT: Mice deficient for the homeotic gene Hoxc-8 suffer from a congenital prehension deficiency of the forepaw. During embryogenesis, Hoxc-8 is highly expressed in motoneurons within spinal cord segments C7 to T1. These motoneurons innervate forelimb distal muscles that move the forepaw. In Hoxc-8 mutant embryos, formation of these muscles is normal, but their innervation is perturbed. From E13.5 onwards, distal muscles normally supplied by C(7-8) MNs also receive ectopic projections from C(5-6) and T1 motoneurons. Coordinates of motor pools are altered along the rostrocaudal and also the mediolateral axes. Following this aberrant connectivity pattern and during the time of naturally occurring cell death, apoptosis is specifically enhanced in C7-T1 motoneurons. Loss of Hox-encoded regional specifications subsequently leads to a numerical deficit of motoneurons and an irreversible disorganization of motor pools. In Hoxc-8 null mutants, C(7-8) motoneurons lose their selective advantage in growth cone pathfinding behavior and/or target recognition, two essential steps in the establishment and maintenance of a functional nervous system.
Development 02/1998; 125(2):279-91. · 6.60 Impact Factor
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ABSTRACT: The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ-TTC gene fusion retains the biological functions of both proteins in vivo-i.e. , retrograde transynaptic transport of the TTC fragment and beta-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.
Proceedings of the National Academy of Sciences 09/1997; 94(17):9400-5. · 9.68 Impact Factor
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ABSTRACT: Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferative, survival and differentiation activities on a wide range of cell types. A role for LIF in embryonic development is suggested by: i) its ability to stimulate the proliferation of embryonic stem (ES) cells in vitro, while maintaining their totipotency and ii) by both its maternal and embryonic expression at the time of blastocyst implantation. Functional studies of LIF and its receptor during mouse embryogenesis have been performed using the techniques of targeted gene replacement and transgene expression in ES cells to produce transgenic mice bearing either loss- or gain-of-function mutations for LIF activity. Whereas, the phenotype observed in the LIF gain-of-function mutant mice supports a role for LIF in early embryogenesis, the loss-of-function phenotypes point to more specialized functions for LIF in development and further reveal the redundant feature of the LIF cytokine/receptor family.
European cytokine network 01/1997; 7(4):699-712. · 1.73 Impact Factor
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ABSTRACT: The morphogenesis of the brain and the differentiation of the neural structures are highly complex processes. A series of temporally and spatially regulated morphogenetic events gives rise to smaller areas that are phylogenetically, functionally and often morphogenetically different. Candidate genes for positional information and differentiation during morphogenesis have been isolated. Both in vivo inactivation in mice and impairment in human diseases revealed, that they are required in regional specification and/or correct cell-type induction. We have previously cloned and characterized the murine Otx1 gene, which is related to orthodenticle (otd), a homeobox-containing gene required for Drosophila head development. Expression data during murine embryogenesis and postnatal brain development support the idea that Otx1 could be required for correct brain and sense organs development. To decipher its role in vivo we produced null mice by replacing Otx1 with the lacZ gene. Otx1-/- mice showed spontaneous epileptic behaviour and multiple abnormalities affecting mainly the telencephalic temporal and perirhinal areas, the hippocampus, the mesencephalon and the cerebellum, as well as the acoustic and visual sense organs. Our findings indicate that the Otx1 gene product is required for proper brain functions.
Nature Genetics 11/1996; 14(2):218-22. · 35.53 Impact Factor
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ABSTRACT: We have replaced part of the mouse homeogene Otx2 coding region with the E. coli lacZ coding sequence, thus creating a null allele of Otx2. By 9.5 dpc, homozygous mutant embryos are characterized by the absence of forebrain and midbrain regions. From the early to midstreak stages, endomesodermal cells expressing lacZ fail to be properly localized anteriorly. In the ectodermal layer, lacZ transcription is progressively extinguished, being barely detectable by the late streak stage. These data suggest that Otx2 expression in endomesoderm and ectoderm is required for anterior neuroectoderm specification. In gastrulating heterozygous embryos, a post-transcriptional repression acts on lacZ transcripts in the ectoderm, but not in the external layer, suggesting that different post-transcriptional mechanisms control Otx2 expression in both layers.
Development 11/1995; 121(10):3279-90. · 6.60 Impact Factor
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ABSTRACT: Nicotine affects many aspects of behaviour including learning and memory through its interaction with neuronal nicotinic acetylcholine receptors (nAChR). Functional nAChRs are pentameric proteins containing at least one type of alpha-subunit and one type of beta-subunit. The involvement of a particular neuronal nicotinic subunit in pharmacology and behaviour was examined using gene targeting to mutate beta 2, the most widely expressed nAChR subunit in the central nervous system. We report here that high-affinity binding sites for nicotine are absent from the brains of mice homozygous for the beta 2-subunit mutation. Further, electrophysiological recording from brain slices reveals that thalamic neurons from these mice do not respond to nicotine application. Finally, behavioural tests demonstrate that nicotine no longer augments the performance of beta 2-1- mice on passive avoidance, a test of associative memory. Paradoxically, mutant mice are able to perform better than their non-mutant siblings on this task.
Nature 04/1995; 374(6517):65-7. · 36.28 Impact Factor
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ABSTRACT: Developmental genetics in Drosophila led to the isolation of the homeotic genes which are involved in the cellular positional information. In vertebrates, homologous genes have been characterized and play similar roles in the spatial determination. However, the subtle mechanisms by which positional identity is specified by the Hox genes co-expression are little known. The Hoxc-8 null mutation can induce homeosis at the anterior margin of Hoxc-8 expression, where the mosaicism observed suggests that, at least in the segmented paraxial mesoderm, the number of cells expressing a Hox gene is a determinant parameter. A combinatorial model is also suggested by several genetic experiments where the level of Hox genes expression is modified. Expression of some related Hox genes in Hoxc-8 homozygous mutants is not altered, suggesting the absence of genetic interregulations between these genes.
Comptes Rendus de l Académie des Sciences - Series III - Sciences de la Vie 10/1993; 316(9):1009-24.
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ABSTRACT: Leukaemia inhibitory factor (LIF) has a variety of effects on different cell types in vitro, inhibiting the differentiation of embryonic stem cells and promoting the survival and/or proliferation of primitive haematopoietic precursors and primordial germ cells. Here we show that LIF-deficient mice derived by gene targeting techniques have dramatically decreased numbers of stem cells in spleen and bone marrow. Injection of spleen and marrow cells from these mice promotes long-term survival of lethally irradiated wild-type animals, however, showing that the LIF- stem cells remain pluripotent. The numbers of committed progenitors are also reduced in the spleen but not the bone marrow, suggesting that stem cells interact differently with the splenic and medullary microenvironment. Heterozygous animals are intermediate in phenotype, implying that LIF has a dosage effect, and defects in stem cell number can be compensated by exogenous LIF. LIF thus appears to be required for the survival of the normal pool of stem cells, but not their terminal differentiation.
Nature 06/1993; 363(6427):361-4. · 36.28 Impact Factor
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ABSTRACT: Leukemia inhibitory factor (LIF) is a cytokine active in vitro on different target cells. It is detected in vivo during mouse gestation in both extraembryonic membranes and maternal tissues. Two isoforms have been described maintaining embryonic stem cells in culture in a pluripotent state. However, overexpression of their cDNAs in chimeric mouse embryos observed between 6.5 and 9.5 days postcoitus gave strikingly different phenotypes. Embryos overexpressing the diffusible form of LIF cDNA looked essentially normal. Chimerae expressing LIF associated with the extracellular matrix cDNA showed an abnormal proliferation of tissues and the absence of differentiated mesoderm. They have not undertaken the normal pathway of gastrulation.
Proceedings of the National Academy of Sciences 10/1992; 89(17):8195-9. · 9.68 Impact Factor
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ABSTRACT: We have replaced the Hox-3.1 coding sequence with the E. coli lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Homozygous mice were born alive, but most of them died within a few days. In the trunk region of homozygotes, several skeletal segments were transformed into the likeness of more anterior ones, as observed in Drosophila with loss-of-function homeotic mutations. The most obvious transformations were the attachment of the 8th pair of ribs to the sternum and the appearance of a 14th pair of ribs on the 1st lumbar vertebra. The pattern of beta-galactosidase activity was identical in heterozygotes and homozygotes and reflected faithfully the Hox-3.1 expression pattern. Thus, the mutation modified the identity, rather than the position, of embryonic cells that would normally express Hox-3.1.
Cell 05/1992; 69(2):251-64. · 32.40 Impact Factor
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ABSTRACT: An embryonic stem (ES) cell line stably expressing lacZ under the control of an endogenous promoter has been isolated and used as a marker to follow the fate of ES cells injected into blastocysts and morulae, before midgestation. The results show a multisite pattern of blastocyst colonization by ES cells deposited into the blastocoel cavity and a low degree of mingling between ES cells and ICM cells. Furthermore, analysis of dispersal of ES cell descendants in postimplantation chimaeric embryos showed that colonization can be highly variable from one region of the embryo to another. In contrast, a high and reproducible degree of chimaerism was obtained when the ES cells were injected at the morula stage prior to ICM formation.
Development 01/1991; 110(4):1241-8. · 6.60 Impact Factor
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ABSTRACT: Murine leukemia inhibitory factor (LIF) protein is a growth factor which has the ability to maintain the developmental potential of pluripotent embryonic stem cells through a specific receptor. We have examined the expression pattern of the LIF gene from the preimplantation stage (3.5 days post coitum) to the midgestation stage (12.5 days post coitum) of the mouse embryo. LIF transcripts were detected at the preimplantation blastocyst stage, whereas no transcripts were detectable in embryonic stem cells. LIF gene transcription continued in the extraembryonic tissue of the 7.5-day and in the placenta of 9.5-, 10.5-, and 12.5-day post coitum embryos. No transcripts were detected in the embryo proper of the corresponding stages. Our results suggest that this growth factor is synthesized in the extraembryonic part of the embryo and acts on the embryonic tissues during early mouse development.
Molecular and Cellular Biology 08/1990; 10(7):3801-5. · 5.53 Impact Factor
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ABSTRACT: Through gene targeting based upon homologous recombination in embryonic stem cells, a chosen gene can be inactivated and eventually a strain of mutant mice created. We have devised a procedure to specifically replace a targeted gene by another gene. A murine homeobox gene was disrupted at high frequency in embryonic stem cells by its replacement with Escherichia coli lacZ. Injection of such stem cells into blastocysts yielded chimeric embryos in which beta-galactosidase activity was driven by the Hox-3.1 promoter. This technique will allow the visual assessment at the cellular level of gene inactivation effects in transgenic mice.
Proceedings of the National Academy of Sciences 07/1990; 87(12):4712-6. · 9.68 Impact Factor
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ABSTRACT: Keratin 8 (Endo A) is expressed in simple epithelia, together with keratin 18 (Endo B). Filaments formed by this keratin pair are the first cytoskeletal elements induced during mouse embryogenesis. We have isolated Endo A cDNA clones from lambda gt11 libraries prepared with mRNA isolated from PCC4 embryonal carcinoma (EC) cells. Sequencing of three overlapping cDNAs and of a genomic clone allowed us to determine the complete sequence of the Endo A message. Analysis of the protein sequence deduced showed that the Endo A protein presented all the characteristics of intermediate filaments, including an alpha-helical central rod domain and nonhelical N- and C-termini. In the rod domain, the degree of similarity to the other members of the basic keratin family was high. A high degree of homology to keratin 8 of other species was observed, even in the non-helical domains. During these analyses, we found clones extending upstream of the normal 5' end of the mRNA. Sequence comparison between these cDNAs and the 5' upstream region of the Endo A gene suggested that they corresponded to transcripts initiated at an upstream alternative promoter. These observations supported previous results showing the presence of Endo A transcripts initiated upstream of the normal 5' end in mouse morulae and blastocysts.
Differentiation 02/1988; 37(1):40-6. · 2.81 Impact Factor
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ABSTRACT: A cDNA from the Hox-3.1 locus, isolated from a 10.5-day postcoitum (p.c.) mouse embryo cDNA library, and the putative encoded protein are described. The spatial distribution of Hox-3.1 gene transcripts from late gastrulation to embryonic day 14.5 p.c. was monitored by in situ hybridization, using a cDNA probe. When first detectable in 8.5-day p.c. embryos, the transcripts are distributed in all the tissues of the posterior end. At later stages, the distribution becomes progressively spatially restricted and tissue specific. By 12.5 days p.c., transcription is localized most intensely in the neural tube region lying above the heart. The early transcription pattern thus appears to be compatible with a regionalizing role for the Hox-3.1 gene.
Genes & Development 02/1988; 2(1):125-35. · 11.66 Impact Factor
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ABSTRACT: Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion.
Proceedings of the National Academy of Sciences 01/1986; 82(24):8535-9. · 9.68 Impact Factor
