High-throughput discovery of novel developmental phenotypes (original) (raw)

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Acknowledgements

The authors thank all IMPC members and partners for their contribution to the consortium effort, including this study, and acknowledge the contributions of J. Rossant, S. L. Adamson, and T. Bubela. This work was supported by NIH grants U42 OD011185 (S.A.M.), U54 HG006332 (R.E.B., K.S.), U54 HG006348-S1 and OD011174 (A.L.B.), HG006364-03S1 and U42 OD011175 (K.C.K.L.), U54 HG006370 (P.F., A.-M.M., H.E.P., S.D.M.B.) and additional support provided by the The Wellcome Trust, Medical Research Council Strategic Award (L.T., S.W., S.D.M.B.), Government of Canada through Genome Canada and Ontario Genomics (OGI-051)(C.M., S.D.M.B.), Wellcome Trust Strategic Award “Deciphering the Mechanisms of Developmental Disorders (DMDD)” (WT100160) (D.A., T.M.), National Centre for Scientific Research (CNRS), the French National Institute of Health and Medical Research (INSERM), the University of Strasbourg (UDS), the “Centre Européen de Recherche en Biologie et en Médecine”, the “Agence Nationale de la Recherche” under the frame programme “Investissements d’Avenir” labelled ANR-10-IDEX-0002-02, ANR-10-INBS- 07 PHENOMIN to (Y.H.), The German Federal Ministry of Education and Research by Infrafrontier grant 01KX1012 (S.M., V.G.D., H.F., M.H.d.A.)

Author information

Author notes

  1. Mary E. Dickinson, Ann M. Flenniken, Xiao Ji, Lydia Teboul and Michael D. Wong: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, Houston, 77030, Texas, USA
    Mary E. Dickinson, Chih-Wei Hsu, Sowmya Kalaga, Lance C. Keith, Melissa L. McElwee & Leeyean Wong
  2. The Toronto Centre for Phenogenomics, Toronto, M5T 3H7, Ontario, Canada
    Ann M. Flenniken, Michael D. Wong, Hibret Adissu, Susan Newbigging, Lauryl M. J. Nutter, Ruolin Guo, Dawei Qu, Shoshana Spring, Lisa Yu, Jacob Ellegood, Lily Morikawa, Xueyuan Shang, Pat Feugas, Amie Creighton, Patricia Castellanos Penton, Ozge Danisment, R. Mark Henkelman & Colin McKerlie
  3. Mount Sinai Hospital, Toronto, M5G 1X5, Ontario, Canada
    Ann M. Flenniken
  4. Genomics and Computational Biology Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA
    Xiao Ji
  5. Medical Research Council Harwell (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, OX11 0RD, Oxfordshire, UK
    Lydia Teboul, Henrik Westerberg, James M. Brown, James Cleak, Neil R. Horner, Sara J. Johnson, Thomas N. Lawson, Zsombor Szoke-Kovacs, Michelle E. Stewart, Carol Copley, Jackie Harrison, Samantha Joynson, Sara Wells, Ann-Marie Mallon & Steve D. M. Brown
  6. Mouse Imaging Centre, The Hospital for Sick Children, Toronto, M5T 3H7, Ontario, Canada
    Michael D. Wong & R. Mark Henkelman
  7. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, Cambridge, UK
    Jacqueline K. White, Brendan Doe, Antonella Galli, Ramiro Ramirez-Solis, Edward Ryder, Nicola Griggs, Catherine L. Tudor, Angela L. Green, Cecilia Icoresi Mazzeo, Emma Siragher, Charlotte Lillistone, Elizabeth Tuck, Diane Gleeson, Debarati Sethi, Tanya Bayzetinova, Jonathan Burvill, Bishoy Habib, Lauren Weavers, Ryea Maswood, Evelina Miklejewska, Michael Woods, Evelyn Grau, Stuart Newman, Caroline Sinclair, Ellen Brown, Allan Bradley, William C. Skarnes & David J. Adams
  8. European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
    Terrence F. Meehan, Jeremy Mason, Ilinca Tudose, Jonathan Warren, Paul Flicek & Helen E. Parkinson
  9. Centre for Anatomy and Cell Biology, Medical University of Vienna, Vienna, A-1090, Austria
    Wolfgang J. Weninger
  10. The Hospital for Sick Children, Toronto, M5G 1X8, Ontario, Canada
    Hibret Adissu, Susan Newbigging, Lauryl M. J. Nutter, Monica J. Justice & Colin McKerlie
  11. The Jackson Laboratory, Bar Harbor, 04609, Maine, USA
    Candice N. Baker, L. Brianna Caddle, James M. Denegre, Mary E. Dolan, Sarah M. Edie, Kevin A. Peterson, Matthew McKay, Barbara Urban, Caroline Lund, Erin Froeter, Taylor LaCasse, Adrienne Mehalow, Emily Gordon, Leah Rae Donahue, Robert Taft, Peter Kutney, Stephanie Dion, Leslie Goodwin, Susan Kales, Rachel Urban, Kristina Palmer, Robert E. Braun, Karen L. Svenson & Stephen A. Murray
  12. Mouse Biology Program, University of California, Davis, 95618, California, USA
    Lynette Bower, Dave Clary, Louise Lanoue, Douglas J. Rowland, Amanda G. Trainor & K. C. Kent Lloyd
  13. Monterotondo Mouse Clinic, Italian National Research Council (CNR), Institute of Cell Biology and Neurobiology, Monterotondo Scalo, I-00015, Italy
    Francesco Chiani, Alessia Gambadoro & Glauco P. Tocchini-Valentini
  14. Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA
    Mark J. Daly, Monkol Lek, Kaitlin E. Samocha & Daniel G. MacArthur
  15. Program in Medical and Population Genetics, Broad Institute MIT and Harvard, Cambridge, 02142, Massachusetts, USA
    Mark J. Daly, Monkol Lek, Kaitlin E. Samocha & Daniel G. MacArthur
  16. Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics and German Mouse Clinic, Neuherberg, 85764, Germany
    Helmut Fuchs, Valerie Gailus-Durner, Susan Marschall & Martin Hrabe de Angelis
  17. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, 77030, Texas, USA
    Juan Gallegos, John R. Seavitt, Monica J. Justice & Arthur L. Beaudet
  18. SKL of Pharmaceutical Biotechnology and Model Animal Research Center, Collaborative Innovation Center for Genetics and Development, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China
    Shiying Guo & Xiang Gao
  19. Infrastructure Nationale PHENOMIN, Institut Clinique de la Souris (ICS), et Institut de Génétique Biologie Moléculaire et Cellulaire (IGBMC) CNRS, INSERM, University of Strasbourg, Illkirch-Graffenstaden, 67404, France
    Manuel Mark, Mohammed Selloum, Olivia Wendling, Fabien Pertuy, Deborah Bitz, Bruno Weber, Patrice Goetz-Reiner, Hughes Jacobs, Elise Le Marchand, Amal El Amri, Leila El Fertak, Hamid Ennah, Dalila Ali-Hadji, Abdel Ayadi, Marie Wattenhofer-Donze, Sylvie Jacquot, Philippe André, Marie-Christine Birling, Guillaume Pavlovic, Tania Sorg & Yann Herault
  20. RIKEN BioResource Center, Tsukuba, 305-0074, Ibaraki, Japan
    Masaru Tamura, Shigeharu Wakana, Atsushi Yoshiki, Shinya Ayabe, Mizuho Iwama & Ayumi Murakami
  21. Children’s Hospital Oakland Research Institute, Oakland, 94609, California, USA
    David B. West
  22. IMPC, San Anselmo, 94960, California, USA
    Mark Moore
  23. Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Freising, 81675, Germany
    Martin Hrabe de Angelis
  24. German Center for Diabetes Research (DZD), Neuherberg, 85764, Germany
    Martin Hrabe de Angelis
  25. The Francis Crick Institute Mill Hill Laboratory, The Ridgeway, Mill Hill, NW1 1AT, London, UK
    Tim Mohun
  26. Departments of Genetics and Psychiatry, Perlman School of Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA
    Maja Bućan
  27. Charles River Laboratories, Wilmington, 01887, Massachusetts, USA
    Iva Morse & Frank Benso
  28. HelmholtzZentrum Munich, Institute of Developmental Genetics, Munich-Neuherberg, 85764, Germany
    Wolfgang Wurst
  29. Technical University of Munich, Chair of Developmental Genetics, Munich-Neuherberg, 85764, Germany
    Wolfgang Wurst
  30. German Center for Neurodegenerative Diseases (DZNE) Site Munich,, Munich, 81377, Germany
    Wolfgang Wurst
  31. Munich Cluster for Systems Neurology (SyNergy), Munich, 81377, Germany
    Wolfgang Wurst

Authors

  1. Mary E. Dickinson
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  2. Ann M. Flenniken
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  3. Xiao Ji
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  4. Lydia Teboul
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  5. Michael D. Wong
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  6. Jacqueline K. White
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  7. Terrence F. Meehan
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  8. Wolfgang J. Weninger
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  9. Henrik Westerberg
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  10. Hibret Adissu
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  11. Candice N. Baker
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  12. Lynette Bower
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  13. James M. Brown
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  14. L. Brianna Caddle
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  15. Francesco Chiani
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  16. Dave Clary
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  17. James Cleak
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  18. Mark J. Daly
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  19. James M. Denegre
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  20. Brendan Doe
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  21. Mary E. Dolan
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  22. Sarah M. Edie
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  23. Helmut Fuchs
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  24. Valerie Gailus-Durner
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  25. Antonella Galli
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  26. Alessia Gambadoro
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  27. Juan Gallegos
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  28. Shiying Guo
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  29. Neil R. Horner
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  30. Chih-Wei Hsu
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  31. Sara J. Johnson
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  32. Sowmya Kalaga
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  33. Lance C. Keith
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  34. Louise Lanoue
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  35. Thomas N. Lawson
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  36. Monkol Lek
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  37. Manuel Mark
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  38. Susan Marschall
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  39. Jeremy Mason
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  40. Melissa L. McElwee
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  41. Susan Newbigging
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  42. Lauryl M. J. Nutter
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  43. Kevin A. Peterson
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  44. Ramiro Ramirez-Solis
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  45. Douglas J. Rowland
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  46. Edward Ryder
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  47. Kaitlin E. Samocha
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  48. John R. Seavitt
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  49. Mohammed Selloum
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  50. Zsombor Szoke-Kovacs
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  51. Masaru Tamura
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  52. Amanda G. Trainor
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  53. Ilinca Tudose
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  54. Shigeharu Wakana
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  55. Jonathan Warren
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  56. Olivia Wendling
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  57. David B. West
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  58. Leeyean Wong
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  59. Atsushi Yoshiki
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  60. Wolfgang Wurst
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  61. Daniel G. MacArthur
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  62. Glauco P. Tocchini-Valentini
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  63. Xiang Gao
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  64. Paul Flicek
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  65. Allan Bradley
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  66. William C. Skarnes
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  67. Monica J. Justice
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  68. Helen E. Parkinson
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  69. Mark Moore
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  70. Sara Wells
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  71. Robert E. Braun
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  72. Karen L. Svenson
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  73. Martin Hrabe de Angelis
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  74. Yann Herault
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  75. Tim Mohun
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  76. Ann-Marie Mallon
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  77. R. Mark Henkelman
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  78. Steve D. M. Brown
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  79. David J. Adams
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  80. K. C. Kent Lloyd
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  81. Colin McKerlie
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  82. Arthur L. Beaudet
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  83. Maja Bućan
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  84. Stephen A. Murray
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The International Mouse Phenotyping Consortium

Contributions

M.E.D., A.M.F., X.J., L.T., M.D.W., J.K.W., T.F.M., W.J.W., H.W., D.J.A., M.B., and S.A.M. contributed to the data analysis and writing of the paper. A.Y., A.B., L.B., L.B.C., F.C., B.D., H.F., A. Galli, A.G., V. G.-D., S.G., S.M., S.A.M., L.M.J.N., E.R., J.R.S., M.S., W.C.S., R.R.S., L.T., S.W. and J.K.W. generated animal models and identified lethal genes. M.E.D, A.M.F., X.J., H.W., L.T., J.M.B., N.R.H., T.F.M., M.E.Dolan and S.A.M. contributed to gene list analysis. H.A., L.B, L.B.C., C.N.B., J.C., J.M.D., M.E.D, S.M.E., A.M.F. A. Galli, C.-W.H., S.J.J., S.K., L.C.K., L.L., M.M., M.L.M., T.M., S.A.M., S.N., L.M.J.N., K.A.P., D.R., E.R., Z. S.-K., M.T., L.T., A.T., O.W., W.J.W., J.K.W. and L.W. contributed to the secondary lethal screen and data analysis. J.M.B., D.C., J.G., N.R.H, T.N.L., J.M., I.T. and J.W. provided informatics support. M.D.W. and R.M.H. performed the automated 3D analysis. J.M.B, N.R.H, I.T., J.W. and H.W. developed and implemented the IMPC portal, X.J, M.J.D., S.A.M., M.L., K.E.S., D.G.M., D.J.A. and M.B. contributed to the essential gene and human disease analysis. M.E.D, A.M.F., X.J, L.T., M.D.W., J.K.W, T.F.M, W.J.W., H.W., S.W., R.R-S., J.M.D., D.G.M., D.B.W., W.W., G.P.T.-V., X.G., P.F., W.C.S., A.B, M.J.J., H.E.P., M.Moore, S.W., R.E.B., K.S., M.H.d.A, Y.H., T.M., A.-M.M., R.M.H., S.D.M.B., D.J.A., K.C.K.L., C.M., A.L.B., M.B. and S.A.M. contributed to the design, management, execution of the work and review of the manuscript.

Corresponding author

Correspondence toStephen A. Murray.

Additional information

All data are freely available from the IMPC database hosted at EMBL-EBI via a web portal (mousephenotype.org), ftp (ftp://ftp.ebi.ac.uk/pub/databases/impc) and automatic programmatic interfaces. An archived version of the database will be maintained after cessation of funding (exp. 2021) for an additional 5 years. Allele and phenotype summaries are additionally archived with Mouse Genome Informatics at the Jackson Laboratory via direct data submissions (J:136110, J:148605, J:157064, J:157065, J:188991, J:211773).

Reviewer Information Nature thanks N. Copeland, L. Niswander and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Standard IMPC allele variants included in this study.

a, Conditional-ready, knockout-first allele (tm1a) design (top) with LacZ reporter, and the Cre-converted (tm1b, bottom) version lacking the neo cassette and critical exon. The promoter driven variant is illustrated. b, Schematic of the small number of alleles included where the distal loxP had been lost during targeting (tm1e, top) and the converted (tme.1) variant with the neo cassette removed. c, Velocigene ‘definitive null’ design (top, tm1) where the LacZ cassette replaces the coding sequence of the target gene, and Cre-excised variant (bottom, tm1.1). Details of all alleles used are listed in Supplementary Table 2 and 5. Additional details and schematics of all allele variants are available at http://www.mousephenotype.org/about-ikmc/targeting-strategies.

Extended Data Figure 2 Distribution of lethal, subviable and viable lines for each IMPC Centre.

Spine (a) and mosaic (b) plots of progress examining primary viability of IMPC lines for each IMPC Centre, segmented by ‘Lethal’, ‘Subviable’ or ‘Viable’ outcome. The mosaic plot shows the significant overrepresentation of viable lines from UCD and lethal lines from ICS, NING, and TCP. c, d, Spine and mosaic plots of primary viability outcome by chromosome, showing no significant deviation from the expected distribution. e, Comparison of the percentage of viable, subviable, and lethal lines between genes for which no targeted knockout alleles have been reported (novel) and genes for which one or more knockout alleles has been reported. BCM, Baylor College of Medicine; GMC, German Mouse Clinic; H, MRC Harwell; ICS, Institut Clinique del la Souris (PHENOMIN); J, The Jackson Laboratory; NING, Model Animal Research Center, Nanjing University; RBRC, RIKEN BioResource Center; TCP, Toronto Centre for Phenogenomics; UCD, University of California, Davis; WTSI, Wellcome Trust Sanger Institute.

Extended Data Figure 3 Multiple GOSlim categories show enrichment for lethal and subviable genes versus viable genes from the IMPC dataset.

The analysis was performed for GO Process (a), GO Function (b) and GO Component (c) categories. On the x axis is the proportion of genes in each class that are annotated for the GO Slim group for each category. d–f, Novel lethal IMPC genes, previously reported IMPC genes and all MGI genes were subject to the same analysis, showing the large effect analysis and characterization of lethal genes has on GO analysis.

Extended Data Figure 4 Schematic of the IMPC embryonic lethal phenotyping pipeline.

Lines were defined as lethal if zero homozygous animals were identified after ≥ 28 animals had been genotyped. The KOMP2/IMPC centres began with a mid-gestation (E12.5) screen, while the DMDD program initiated screening at the organogenesis phase (E14.5). If no homozygotes were identified (after ≥ 28 embryos screened), centres examined and characterized embryos at the pattern formation stage (E9.5). Homozygous embryos at this stage were scored for gross anatomical defects and imaged using OPT. If live homozygotes were identified at E12.5, centres proceeded with the screen at E15.5 or E18.5. This decision was based on the presence of any observable phenotype at E12.5 and was at the discretion of the centre. Embryos collected at E15.5 were imaged via iodine-contrast microCT. Once sufficient numbers were collected, image registration and quantitative volumetric analysis was performed. Each time point should be considered independently, as some included strains have not been completely analysed and progression through each time point is at the discretion of the centre. For each term, two mutants with the same phenotype were required to score a hit.

Extended Data Figure 5 Cardiac defects in Strn3, Atg3, and Slc39a8 mutant embryos.

a, b, Severe fetal oedema and sporadic haemorrhaging in E15.5 homozygous mutant embryos versus controls (n = 7 mutants analysed) c, d, Subtle but consistent cardiac septal defects (arrowhead) observed in transverse micro-CT volume sections in Strn3 −/− embryos (d) versus control (c) (n = 5 mutants analysed). e, f, Atg3 +/− (e) and Atg3 −/− (f) E14.5 embryos imaged by micro-CT after contrast staining showed evidence of heart morphological defects including ventricular septal defects (white arrows in f). Atg3 −/− mice also showed abnormal atrio–ventricular valves (n = 4 mutants analysed). g–j, Transverse (g, h) and coronal (i, j) sections through micro-CT volumes of mutant and control Slc39a8 E14.5 embryos revealed heart morphological defects including ventricular septal defects (white arrows in h). Slc39a8 −/− mice also showed the absence of sternum, a small chest cavity and a small liver (j) (n = 4 mutants analysed).

Extended Data Figure 6 High-resolution 3D imaging reveals phenotypes in Tmem100 and Eya4 mutant embryos.

Tmem100 −/− embryos had abnormal heart development compared to Tmem100+/+ controls. E9.5 Tmem100 −/− embryos had large pericardial effusion and cardiac dysmorphology and enlargement (arrow) when compared to E9.5 Tmem100+/+ (wild-type) embryos as seen by OPT imaging (a) and bright-field microscopy(c) resulting in lethality. (n = 8 Tmem100+/+ versus n = 8 Tmem100 −/−, with all 8 showing the defect). b, LacZ expression in the E12.5 Tmem100+/− embryo indicated expression in the heart (arrows), blood vessels and craniofacial regions (blue). d–i, Micro-CT imaging revealed a small cochlear volume in E15.5 Eya4 −/− embryos. E15.5 Eya4 −/− embryos were registered to an average control dataset of the same age followed by automated analysis to show that mutant embryos had a statistically smaller cochlear volume compared to Eya4+/+ (wild-type) embryos. d, Transverse, coronal, and sagittal sections through the right cochlea are marked with a horizontal and vertical dashed line in the transverse section to indicate the location of the coronal and sagittal sections, respectively. The colours correspond to areas of larger (red) and smaller (blue) volumes in the knockout embryos. The colour bar minimum corresponds to a false discovery rate (FDR) threshold of 5%. Hypoplastic bilateral cochlear structures are highlighted in blue, (n = 8 Eya4+/+ (wild-type) versus n = 8 _Eya4_−/− (knockout), with all eight showing the defect). e, LacZ imaging in the E12.5 Eya4+/− revealed Eya4 gene expression (blue) in the cochlear region (arrow). f, g, H&E stained histological sections through the right cochlea of an Eya4+/+ embryo (f) compared to an _Eya4_−/−embryo (g) confirmed the hypoplastic phenotype. h, i, Higher magnification of the region (indicated by the white boxes) showed abnormal perilymphatic (periotic) mesenchyme in mutant embryos. In the mutant embryo (i) the perilymphatic mesenchyme did not show rarefaction and had reduced vacuolation compared to control (h) (arrows), suggesting that the cochlear hypoplasia was due to delayed perilymph development.

Extended Data Figure 7 Whole brain MRI reveals many volume changes in the P7 _Tox3_−/− mice.

a, P7 _Tox3_−/− knockout mouse brains were registered to an average control dataset of P7 Tox3+/+ (wild-type) brains. The colours correspond to areas of larger (red) and smaller (blue) relative volumes in the knockout embryos. The colour bar minimum corresponds to a false discovery rate (FDR) threshold of 5%. Knockout mice exhibited altered volumes in multiple brain structures including an enlarged pons, amygdala and thalamus/hypothalamus and a decreased pontine nucleus when compared to the wild-type brains (arrows). Most striking was the decrease in the size of the cerebellum of the knockout mice (arrows). (n = 8 Tox3+/+ versus n = 10 _Tox3_−/−, with all 10 showing the defects). Histological analysis of _Tox3_−/− mice revealed abnormal development of the cerebellum. b, c, The cerebellum of P7 _Tox3_−/− mice is hypoplastic and dysplastic characterized by markedly reduced fissure formation, poor delineation of folia and disorganized cortical structure and layering (c) when compared to the P7 Tox3+/+ mice (b) (arrows). In some segments, there was complete absence of folial pattern. d, e, Higher magnification revealed that the normally transient external granular layer was absent in the _Tox3_−/− mice and the subjacent molecular layer was hypotrophic and irregular in thickness and in multiple foci very thin or absent; in these foci the Purkinje cells extended to the pial surface (arrows). The Purkinje cell layer was also jumbled with no evidence of cell polarity (e).

Extended Data Figure 8 Whole brain MRI reveals enlarged ventricles in the P7 _Rsph9_−/− mice.

a, P7 _Rsph9_−/− mouse brains showed enlarged left and right lateral ventricles (arrows) when virtually sectioned from rostral to caudal and compared to an average of P7 Rsph9+/+ mouse brains. (n = 8 Rsph9+/+ versus n = 10 _Rsph9_−/−, with all 10 showing the defects). Histological analysis of _Rsph9_−/− (knockout) mice confirmed abnormal brain development. b, c, Arrows indicate severe hydrocephalus of the left and right lateral ventricles of the _Rsph9_−/− P7 mice (c) compared to the Rsph9+/+ mice (b). The third ventricle was also enlarged but not seen in this section. d, e, Higher magnification of the cerebrum showed marked rarefaction, cavitation, and loss of periventricular cortical tissue (arrow) in the knockout mice (e) compared to wild type (d). f, g, Coronal section through the nasal region revealed that the sinuses of the knockout mice were filled with pus (asterisks) (g).

Extended Data Figure 9 Phenotype hit rates from the adult phenotyping pipeline for lethal, subviable and viable lines.

a, Comparison of hit rates between lethal and subviable line heterozygotes versus viable line homozygotes. b, Homozygous subviable cohorts showed a much higher hit rate than lethal line heterozygotes or viable line homozygotes.

Extended Data Figure 10 Multiple phenotypes in Gyg and Kdm8 null embryos.

LacZ expression in Gyg heterozygous and homozygous embryos at E12.5 showed specific, strong expression in the heart and surrounding major vessels (that is, the dorsal aorta, the carotid artery and umbilical artery) (a, b), consistent with smooth muscle cells at this stage. Homozygous embryos were collected at expected proportions at E12.5, E15.5 and E18.5 and could not be distinguished from wild-type and heterozygous embryos by outward appearance. However, inspection of cross-sections through the whole embryo micro-CT images of E18.5 and E15.5 embryos showed abnormalities in several areas of the developing embryo. Thickened myocardium was evident in the hearts of 2 out of 3 homozygotes examined at E15.5 as shown in Fig. 5. Coronal cross-sections also confirmed thickened myocardium in E18.5 mutant hearts (arrows; n = 5 mutants), compare wild type (c) to Gyg tm1b/tm1b(d). From the E18.5 sections, it was also obvious that the thymus was enlarged in mutants (n = 5 mutants) compared with controls (*), but the thymus appeared normal in E15.5 mutant embryos (data not shown). E18.5 mutant embryos also exhibited abnormal gaps in the brain and spinal cord that we interpret as neural degeneration; compare wild-type littermates (e) to Gyg tm1b/tm1b mutants (f) (n = 5 mutants). Abnormalities in the nervous system, similar to abnormalities in the heart, were obvious at E15.5. Representative images are shown from sagittal cross-sections through a wild-type (g) and a homozygous Gyg mutant E15.5 embryo (h) (n = 3). E15.5 Gyg tm1b/tm1b mutant embryos have a flattened forebrain with reduced lateral ventricles, as well as excess space within the cephalic and cervical flexures. i–t, Tm1a and tm1b alleles can lead to phenotypes of differing strength in Kdm8 mutants. Abnormal phenotype of Kmd8 tm1a/tm1a mice at E18.5: i, k, m, o, q, wild-type fetuses; j, l, n, p, r, mutant fetuses. i, j, gross morphological appearance of E18.5 fetuses. k–n, Photomicrographs of the palate and heart taken during necropsy. g–j, Histological sections at similar levels of the trachea and the nasal cavities, (n = 4 mutants analysed at E18.5). Morphology of wild-type (s) and mutants (t) Kmd8 embryos at E9.5 captured by OPT showing developmental delay at that stage, including small size and lack of turning. Arrows, unfused palatal shelves; arrowheads, arch of the aorta. n = 7 mutants analysed at E9.5, scale bar = 1 mm.

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Dickinson, M., Flenniken, A., Ji, X. et al. High-throughput discovery of novel developmental phenotypes.Nature 537, 508–514 (2016). https://doi.org/10.1038/nature19356

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