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ABSTRACT: Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.
Developmental Biology 08/2012; 371(1):77-85. · 4.07 Impact Factor
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Jaime A Rivera-Pérez
Journal of Cellular Physiology 03/2011; 226(7):1713. · 3.87 Impact Factor
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ABSTRACT: Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail) transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.
PLoS ONE 01/2011; 6(5):e18778. · 4.09 Impact Factor
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ABSTRACT: In vitro culture of whole mouse embryos enables the maintenance of growth and morphogenesis of postimplantation embryos outside the uterine environment. This technological advent facilitates the observation of the development of embryos in real time whereby cell lineage and tissue morphogenesis can be traced with appropriate vital cell labels and molecular markers. Embryos in culture are also amenable to direct experimental manipulations for elucidating developmental mechanisms of embryogenesis, germ layer formation, and embryonic patterning. This chapter outlines protocols for culturing mouse embryos at the immediate postimplantation period. We also present a system of developmental staging so that the outcome of different embryo culture studies may be assessed properly with reference to the precise developmental stage of the embryos used for the specific experiments.
Methods in enzymology 01/2010; 476:185-203. · 1.90 Impact Factor
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ABSTRACT: Aurora-A is a conserved kinase implicated in mitotic regulation and carcinogenesis. Aurora-A was previously implicated in mitotic entry and spindle assembly, although contradictory results prevented a clear understanding of the roles of Aurora-A in mammals. We developed a conditional null mutation in the mouse Aurora-A gene to investigate Aurora-A functions in primary cells ex vivo and in vivo. We show here that conditional Aurora-A ablation in cultured embryonic fibroblasts causes impaired mitotic entry and mitotic arrest with a profound defect in bipolar spindle formation. Germ line Aurora-A deficiency causes embryonic death at the blastocyst stage with pronounced cell proliferation failure, mitotic arrest, and monopolar spindle formation. Aurora-A deletion in mid-gestation embryos causes an increase in mitotic and apoptotic cells. These results indicate that murine Aurora-A facilitates, but is not absolutely required for, mitotic entry in murine embryonic fibroblasts and is essential for centrosome separation and bipolar spindle formation in vitro and in vivo. Aurora-A deletion increases apoptosis, suggesting that molecular therapies targeting Aurora-A may be effective in inducing tumor cell apoptosis. Aurora-A conditional mutant mice provide a valuable system for further defining Aurora-A functions and for predicting effects of Aurora-A therapeutic intervention.
Molecular and cellular biology 01/2009; 29(4):1059-71. · 6.06 Impact Factor
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ABSTRACT: Crucial aspects of axial development in mice occur at early postimplantation stages from the time of implantation to the appearance of the primitive streak. However, this period of development is notoriously refractory to experimental approaches due to the small size of the conceptus and to the presence of the parietal yolk sac, a protective tripartite membrane that surrounds the developing egg cylinder. Here, we describe a method that combines enzymatic digestion and mechanical manipulation to remove the parietal yolk sac of conceptuses at stages between 5.5 and 6.5 days post coitum. This method, which is compatible with whole-mount in situ hybridization and immunostaining techniques, offers a significant improvement over conventional dissection techniques, and it will greatly facilitate research in early mammalian development.
Developmental Dynamics 03/2007; 236(2):489-93. · 2.54 Impact Factor
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ABSTRACT: The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.
Developmental Biology 01/2006; 288(2):363-71. · 4.07 Impact Factor
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ABSTRACT: Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.
Developmental Biology 10/2003; 261(2):470-87. · 4.07 Impact Factor
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ABSTRACT: In vitro culture of whole mouse embryos enables the maintenance of growth and morphogenesis of postimplantation embryos outside the uterine environment. This technological advent facilitates the observation of the development of embryos in real time whereby cell lineage and tissue morphogenesis can be traced with appropriate vital cell lables and molecular markers. Embryos in culture are also amenable to direct experimental manipulations for elucidating developmental mechanisms of embryogenesis, germ layer formation, and embryonic patterning. This chapter outlines protocols for culturing mouse embryos at the immediate postimplantation period. We also present a system of developmental staging so that the outcome of different embryo culture studies may be assessed properly with reference to the precise developmental stage of the embryos used for the specific experiments.
Methods in Enzymology.
