Cre recombinase microinjection for single-cell tracing and localised gene targeting.

Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.

[1]  M. Torres,et al.  Live Imaging of Early Cardiac Progenitors in the Mouse Embryo. , 2022, Journal of visualized experiments : JoVE.

[2]  U. Lendahl,et al.  Highly efficient manipulation of nervous system gene expression with NEPTUNE , 2021, Cell reports methods.

[3]  Daniel E. Carlin,et al.  Unveiling Complexity and Multipotentiality of Early Heart Fields , 2021, Circulation research.

[4]  Antonio M. A. Miranda,et al.  Characterization of a common progenitor pool of the epicardium and myocardium , 2021, Science.

[5]  Samantha A. Morris,et al.  Next-Generation Lineage Tracing and Fate Mapping to Interrogate Development. , 2020, Developmental cell.

[6]  P. Vandenabeele,et al.  An Apoptotic Caspase Network Safeguards Cell Death Induction in Pyroptotic Macrophages , 2020, Cell reports.

[7]  S. Ortega,et al.  A Second Heart Field-Derived Vasculogenic Niche Contributes to Cardiac Lymphatics. , 2020, Developmental cell.

[8]  R. Benedito,et al.  iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications , 2019, Nature Communications.

[9]  M. Torres,et al.  Myc is dispensable for cardiomyocyte development but rescues Mycn-deficient hearts through functional replacement and cell competition , 2019, Development.

[10]  Matthieu Defrance,et al.  Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo , 2018, bioRxiv.

[11]  Thomas M. Norman,et al.  Molecular recording of mammalian embryogenesis , 2018, bioRxiv.

[12]  J. Junker,et al.  Simultaneous lineage tracing and cell-type identification using CRISPR/Cas9-induced genetic scars , 2018, Nature Biotechnology.

[13]  L. Carramolino,et al.  Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo. , 2014, Blood.

[14]  C. Gosse,et al.  A microdevice to locally electroporate embryos with high efficiency and reduced cell damage , 2014, Development.

[15]  Philipp J. Keller,et al.  Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells , 2013, PloS one.

[16]  F. Calegari,et al.  Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain , 2013, Development.

[17]  M. Buckingham,et al.  Asymmetric Fate of the Posterior Part of the Second Heart Field Results in Unexpected Left/Right Contributions to Both Poles of the Heart , 2012, Circulation research.

[18]  M. Buckingham,et al.  Tracing cells for tracking cell lineage and clonal behavior. , 2011, Developmental cell.

[19]  Stephen L. Johnson,et al.  Fate restriction in the growing and regenerating zebrafish fin. , 2011, Developmental cell.

[20]  Allan R. Jones,et al.  A robust and high-throughput Cre reporting and characterization system for the whole mouse brain , 2009, Nature Neuroscience.

[21]  V. Wilson,et al.  Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis. , 2009, Developmental cell.

[22]  G. Miyoshi,et al.  Cerebral Cortex doi:10.1093/cercor/bhp038 Characterization of Nkx6-2-Derived , 2009 .

[23]  Michael F. Sweeney,et al.  Microinjection of zebrafish embryos to analyze gene function. , 2009, Journal of visualized experiments : JoVE.

[24]  M. Buckingham,et al.  Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed , 2008, Development.

[25]  J. Nicolas,et al.  Methods in clonal analysis and applications. , 2005, Reproduction, nutrition, development.

[26]  G. Rousseau,et al.  Gene transfer into mouse prepancreatic endoderm by whole embryo electroporation. , 2005, JOP : Journal of the pancreas.

[27]  Manuel Serrano,et al.  Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. , 2003, Cancer cell.

[28]  R. Eisenman,et al.  N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation. , 2002, Genes & development.

[29]  S. Nagata,et al.  Efficient biallelic mutagenesis with Cre/loxP‐mediated inter‐chromosomal recombination , 2002, EMBO reports.

[30]  Jos Jonkers,et al.  A highly efficient ligand‐regulated Cre recombinase mouse line shows that LoxP recombination is position dependent , 2001, EMBO reports.

[31]  A. Nagy,et al.  The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. , 1999, Development.

[32]  F. Wardle,et al.  A gradient of BMP activity specifies dorsal-ventral fates in early Xenopus embryos. , 1999, Seminars in cell & developmental biology.

[33]  P. Tam,et al.  The allocation of epiblast cells to the embryonic heart and other mesodermal lineages: the role of ingression and tissue movement during gastrulation. , 1997, Development.

[34]  P. Tam,et al.  The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. , 1996, Developmental biology.

[35]  M. Fishman,et al.  Cardiovascular development in the zebrafish. II. Endocardial progenitors are sequestered within the heart field. , 1994, Development.

[36]  Y. Hatada,et al.  A fate map of the epiblast of the early chick embryo. , 1994, Development.

[37]  T. Davies,et al.  Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. , 1993, Development.

[38]  R. Pedersen,et al.  Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. , 1991, Development.

[39]  C. Kimmel,et al.  Origin and organization of the zebrafish fate map. , 1990, Development.

[40]  S. Fraser,et al.  A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration. , 1989, Development.

[41]  R. Pedersen,et al.  Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse. , 1987, Development.

[42]  R. Pedersen,et al.  Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer. , 1986, Developmental biology.

[43]  M. Kaufman,et al.  Early differentiation of the heart in mouse embryos. , 1981, Journal of anatomy.

[44]  D. Solter,et al.  Cell cycle analysis in the mouse EGG-cylinder. , 1971, Experimental cell research.

[45]  D. Whittingham.  Culture of mouse ova. , 1971, Journal of reproduction and fertility. Supplement.