Isabelle Migeotte

Université Libre de Bruxelles, Bruxelles, Brussels Capital, Belgium

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Publications (49)126.91 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: We have recently developed a microsystem to electroporate a few cells at the surface of early post-implantation mouse embryos. We could achieve the efficient, reproducible, and safe transfection of various genetic markers, which allowed single cell fate studies during morphogenesis. However, our single-use polymeric device necessitated to be fabricated in a clean room the day before each experiment. Thus, we here introduce an all-glass chip that any biologist can easily recycle in its laboratory. Most importantly, during the technological evolution process we could validate a comprehensive design strategy based on finite element model simulations. Indeed, both the embryo and the microsystem were represented as very simple electric objects and stationary computations enabled to properly predict the voltage pulse amplitude that would yield optimal device performances.
    No preview · Article · Aug 2014 · ECS Transactions
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    [Show abstract] [Hide abstract] ABSTRACT: The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.
    Preview · Article · May 2014 · Development
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    Full-text · Dataset · Jun 2013
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    [Show abstract] [Hide abstract] ABSTRACT: Harstfield syndrome is the rare and unique association of holoprosencephaly (HPE) and ectrodactyly, with or without cleft lip and palate, and variable additional features. All the reported cases occurred sporadically. Although several causal genes of HPE and ectrodactyly have been identified, the genetic cause of Hartsfield syndrome remains unknown. We hypothesised that a single key developmental gene may underlie the co-occurrence of HPE and ectrodactyly. We used whole exome sequencing in four isolated cases including one case-parents trio, and direct Sanger sequencing of three additional cases, to investigate the causative variants in Hartsfield syndrome. We identified a novel FGFR1 mutation in six out of seven patients. Affected residues are highly conserved and are located in the extracellular binding domain of the receptor (two homozygous mutations) or the intracellular tyrosine kinase domain (four heterozygous de novo variants). Strikingly, among the six novel mutations, three are located in close proximity to the ATP's phosphates or the coordinating magnesium, with one position required for kinase activity, and three are adjacent to known mutations involved in Kallmann syndrome plus other developmental anomalies. Dominant or recessive FGFR1 mutations are responsible for Hartsfield syndrome, consistent with the known roles of FGFR1 in vertebrate ontogeny and conditional Fgfr1-deficient mice. Our study shows that, in humans, lack of accurate FGFR1 activation can disrupt both brain and hand/foot midline development, and that FGFR1 loss-of-function mutations are responsible for a wider spectrum of clinical anomalies than previously thought, ranging in severity from seemingly isolated hypogonadotropic hypogonadism, through Kallmann syndrome with or without additional features, to Hartsfield syndrome at its most severe end.
    Full-text · Article · Jun 2013 · Journal of Medical Genetics
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    [Show abstract] [Hide abstract] ABSTRACT: Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.
    Full-text · Article · Feb 2012 · Developmental Biology
  • Isabelle Migeotte · Joaquim Grego-Bessa · Kathryn V Anderson
    [Show abstract] [Hide abstract] ABSTRACT: The establishment of the mammalian body plan depends on signal-regulated cell migration and adhesion, processes that are controlled by the Rho family of GTPases. Here we use a conditional allele of Rac1, the only Rac gene expressed early in development, to define its roles in the gastrulating mouse embryo. Embryos that lack Rac1 in the epiblast (Rac1Δepi) initiate development normally: the signaling pathways required for gastrulation are active, definitive endoderm and all classes of mesoderm are specified, and the neural plate is formed. After the initiation of gastrulation, Rac1Δepi embryos have an enlarged primitive streak, make only a small amount of paraxial mesoderm, and the lateral anlage of the heart do not fuse at the midline. Because these phenotypes are also seen in Nap1 mutants, we conclude that Rac1 acts upstream of the Nap1/WAVE complex to promote migration of the nascent mesoderm. In addition to migration phenotypes, Rac1Δepi cells fail to adhere to matrix, which leads to extensive cell death. Cell death is largely rescued in Rac1Δepi mutants that are heterozygous for a null mutation in Pten, providing evidence that Rac1 is required to link signals from the basement membrane to activation of the PI3K-Akt pathway in vivo. Surprisingly, the frequency of apoptosis is greater in the anterior half of the embryo, suggesting that cell survival can be promoted either by matrix adhesion or by signals from the posterior primitive streak. Rac1 also has essential roles in morphogenesis of the posterior notochordal plate (the node) and the midline.
    No preview · Article · Jul 2011 · Development
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    [Show abstract] [Hide abstract] ABSTRACT: The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.
    Preview · Article · Jun 2011 · The Journal of Immunology
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    [Show abstract] [Hide abstract] ABSTRACT: Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.
    Full-text · Article · Apr 2011 · Proceedings of the National Academy of Sciences
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    Jeffrey D Lee · Isabelle Migeotte · Kathryn V Anderson
    [Show abstract] [Hide abstract] ABSTRACT: The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.
    Preview · Article · Oct 2010 · Developmental Biology
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    Dataset: Video S11
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Absence of cell movement and long cellular projections in Rac1 VE-deleted embryos. The Rac1 VE-deleted embryo was dissected on e5.6 and imaged for 4 h 20 min. Frames are 10 min apart. A stack of 22 slices was taken, at 2 µm per slice. The embryo had developed overnight to a degree similar to wild-type embryos. Individual cells were pseudocolored in order to visualize their trajectories. The cell tracking was made using individual confocal images of each stack in order to increase the accuracy of the cell contours. (9.57 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Video S8
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Wild-type AVE cells are elongated. 3D reconstruction (Hex-GFP, green) from stacks of 150–200 optical sections (step size: 0.5 µm) was performed using Amira ResolveRT 4.1. The embryo outline (dotted line) was determined in Metamorph (Region outline tool) using bright field images. A single orthogonal plane of the embryo is shown in gray scale. The bounding box (orange lines) represents the volume analyzed in all the sections. The movie (from 0 to 130 degrees) includes 60 individual planes, rotated at 6 frames per second. The movie illustrates the elongated shape and the anterior protrusion of leading AVE cells. (8.92 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Video S1
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: AVE cells send long lamellipodia that are spatially and temporally coordinated. A wild-type embryo was dissected on e5.6 at mid-afternoon, and imaged for 6 h. Images were taken every 10 min. At each time point, a stack of 39 slices was taken, at 2 µm per slice. Extended focus images have been aligned to correct for the shift of the embryo, using the distal border as a reference point. Individual cells were pseudocolored to visualize their trajectories. Cells were tracked using individual confocal images of each stack to increase the accuracy of the cell contours. (9.84 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Figure S2
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: AVE cells that fail to migrate form a multilayered epithelium. Single confocal sections of e6.5 embryos stained for E-cadherin (magenta). At e6.5, AVE cells remained at the distal tip of the embryo in Rac1, FoxA2, and Nap1RRQ mutants expressing Hex-GFP (staining with anti-GFP antibody for wild-type, FoxA2, and RRQ embryos and native GFP for Rac1 null embryos). AVE cells formed several rows (arrows) and were linked by adherens junctions. In FoxA2 mutants (C), cells failed to express Hex-GFP but could be recognized through their columnar morphology (*). Nap1RRQ embryos (D) displayed a more severe phenotype than the Nap1khlo allele described in [10]. Scale bars = 50 µm. (7.97 MB TIF)
    Preview · Dataset · Aug 2010
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    Dataset: Video S9
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Lack of projections in a Rac1 null embryo. The embryo depicted in Video S9 is from a litter dissected on e5.5 at mid-afternoon and was selected because the Hex-GFP cells appeared to be round and to not have initiated migration. The embryo was smaller than its wild-type littermates at dissection but developed overnight to a degree similar to what we have observed in vivo. It was imaged from 6 pm to 10:30 am. Frames are 10 min apart. A stack of 23 slices was taken, at 2 µm per slice. The movie represents the first 5 h of imaging. It was genotyped the following day and confirmed to be Rac1 null. (5.95 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Figure S5
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Cell shape is irregular in the embryonic VE during AVE migration. 3D reconstructions of Z-stacks of early (lateral view) and late (anterior view) embryos expressing Hex-GFP (green, native GFP), stained for F-actin (red). The embryos presented as examples were cultured for 1 h prior to fixation. A stable epithelium has a majority of pentagonal or hexagonal cells. The number of sides per cell in the extra-embryonic and embryonic regions of early and late e5.5 embryos was quantified on 3D reconstructions of Z-stacks, considering all faces of the embryos. All cells from the embryonic portion were considered, regardless of Hex-GFP expression, as all cells are likely to change shape either actively or passively. In both groups, most cells had 3 or 4 sides in the embryonic region, and 5 or 6 sides in the extra-embryonic region, and this trend was stronger in younger embryos in which the epithelium is expected to be less stable. (6.10 MB TIF)
    Preview · Dataset · Aug 2010
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    Dataset: Figure S8
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Basal projection of migrating AVE cell. Individual confocal images (stills from Video S5) of a trailing wild-type Hex-GFP cell. The cell showed basal protrusive activity while maintaining its columnar structure. The protrusion was directed towards the embryonic/extra-embryonic border (top right) and the cell translocated over time. Scale bar = 30 µm. (6.88 MB TIF)
    Preview · Dataset · Aug 2010
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    Dataset: Figure S12
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Defects in AVE migration and axis specification in Nap1RRQ embryos. (A) 3D reconstructions of Z-stacks of e6.5 embryos expressing Hex-GFP (green, staining with anti-GFP antibody), stained for F-actin (red) or E-cadherin (magenta). In Nap1RRQ embryos, the distribution of Hex-GFP is abnormal, and many cells fail to initiate migration. Cells are round in the VE of mutant embryos, reminiscent of the Rac1 mutant embryos. (B) Expression of Brachyury (T) at e7.5. In Nap1RRQ mutants, there appear to be multiple sites of streak initiation or, in the most severe cases, a ring of T-expressing cells in the proximal epiblast. (10.18 MB TIF)
    Preview · Dataset · Aug 2010
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    Dataset: Video S5
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Projections on trailing cells are located basally. Video S5 shows a detail from Video S1. A cell initially located at the distal end of the embryo was tracked for 3 h 50 min in individual confocal sections from each time point. Frames were aligned using the center of the cell body as a reference point. The cell was pseudocolored to highlight the dynamics of projections. (1.00 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Video S10
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Formation of a distal cluster of AVE cells in a Rac1 null embryo. Video S10 figures a cell from Video S9 (Rac1 null embryo). It was tracked for 2.5 h in individual confocal sections from each time point. Frames were aligned using the center of the cell body as a reference point. The cell contour was pseudocolored to highlight cell shape changes. Some cells seem to have lost contact with the basement membrane. (0.86 MB AVI)
    Preview · Dataset · Aug 2010
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    Dataset: Figure S1
    Isabelle Migeotte · Tatiana Omelchenko · Alan Hall · Kathryn V. Anderson
    [Show abstract] [Hide abstract] ABSTRACT: Gastrulation initiates in Rac1null embryos. Single confocal sections of e7.5 embryos stained with phalloidin to visualize F-actin (red). Mutant embryos generate mesoderm, which is present around the embryo between the epiblast and endoderm layers. There are numerous pyknotic nuclei in the mesoderm layer, as previously described [25]. Scale bars = 50 µm. (4.71 MB TIF)
    Preview · Dataset · Aug 2010

Publication Stats

1k Citations
126.91 Total Impact Points

Institutions

  • 2002-2012
    • Université Libre de Bruxelles
      • • Institute of Interdisciplinary Research in human and molecular Biology (IRIBHM)
      • • Department of Interdisciplinary Research
      Bruxelles, Brussels Capital, Belgium
  • 2008-2010
    • Memorial Sloan-Kettering Cancer Center
      New York, New York, United States
  • 1999
    • Vrije Universiteit Brussel
      Bruxelles, Brussels Capital, Belgium