Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway

  1. Tra Thi Huong Dinh
  2. Hiroyoshi Iseki
  3. Seiya Mizuno
  4. Saori Iijima-Mizuno
  5. Yoko Tanimoto
  6. Yoko Daitoku
  7. Kanako Kato
  8. Yuko Hamada
  9. Ammar Shaker Hamed Hasan
  10. Hayate Suzuki
  11. Kazuya Murata
  12. Masafumi Muratani
  13. Masatsugu Ema
  14. Jun-Dal Kim
  15. Junji Ishida
  16. Akiyoshi Fukamizu
  17. Mitsuyasu Kato
  18. Satoru Takahashi
  19. Ken-ichi Yagami
  20. Valerie Wilson
  21. Ruth M Arkell
  22. Fumihiro Sugiyama  Is a corresponding author
  1. Laboratory Animal Resource Center, Faculty of Medicine, University of Tsukuba, Japan
  2. Ph.D. Program in Human Biology, School of Integrative and Global Majors (SIGMA), University of Tsukuba, Japan
  3. Department of Traditional Medicine, University of Medicine and Pharmacy, Viet Nam
  4. Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, Japan
  5. International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Japan
  6. Experimental Animal Division, RIKEN BioResource Research Center, Japan
  7. Doctor’s Program in Biomedical Sciences, Graduate School of Comprehensive Human Science, University of Tsukuba, Japan
  8. Department of Genome Biology, Faculty of Medicine, University of Tsukuba, Japan
  9. Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Japan
  10. Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Japan
  11. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Japan
  12. Division of Complex Bioscience Research, Department of Research and Development, Institute of National Medicine, University of Toyama, Japan
  13. Department of Experimental Pathology, Faculty of. Medicine, University of Tsukuba, Japan
  14. MRC Centre for Regenerative Medicine, School of Biological Sciences, SCRM Building, The University of Edinburgh, United Kingdom
  15. John Curtin School of Medical Research, The Australian National University, Australia
9 figures, 4 tables and 6 additional files

Figures

Cables2 expression during early mouse embryo development.

(A) Cables2 gene expression was examined by RT-PCR with ESCs, blastocyst, and E7.5 embryo samples. Gapdh was used as an internal positive control. (B–F) Wild-type embryos from E6.5 to E9.5 were …

Figure 2 with 1 supplement
Morphological and histological analyses of Cables2d embryos at early stages of development.

(A) Following VelociGene’s KOMP design, the entire protein coding sequence of the target gene was deleted by homologous recombination in C57BL/6N ESCs, therefore, the full Cables2 allele was null (Ca…

Figure 2—figure supplement 1
Genotyping of Cables2d and expression of wild-type Cables2.

(A) Genotyping by PCR analysis from whole embryo samples using three primers. Bands at 985 bp and 599 bp represent mutant and wild-type Cables2d alleles, respectively. (B) At E6.5, Cables2 was …

Expression of gastrulation markers in Cables2d embryos.

(A–T) All embryos were collected, genotyped, and used for WISH at E6.5. Several key gastrulation markers were examined using both wild-type and Cables2d embryos: T (n = 5), Wnt3 (n = 5), Fgf8 (n = 3)…

Figure 4 with 1 supplement
Enhancement of β-catenin activity by Cables2.

(A) Relative luciferase activities in 293T cells transfected with an empty control or Cables2 expression vectors together with an empty control or β-catenin expression vectors. Relative luciferase …

Figure 4—figure supplement 1
Interaction of Cables2 with endogenous β-catenin in 293T cells.

Co-IP showing the physical interaction of FLAG-Cables2 with endogenous β-catenin in 293T cells. Anti-GAPDH antibody was used as a negative control for evaluating specific interaction. Each …

Proliferating and apoptotic cells in Cables2d embryos.

(A–B) The EdU-incorporating cells represented the proliferation of cells in both wild-type and Cables2d/d embryos at E6.5 (n = 6). (C–F) Apoptotic cells were detected in both embryonic and …

Transcriptome profiling analysis of Cables2d/d embryos.

(A) Heatmap representation of 288 genes significantly different between wild-type and Cables2d/d embryo samples (fold change >= 2, FDR < 0.05). (B) List of all downregulated (blue) and upregulated …

Figure 7 with 1 supplement
Gene construction and expression in Cables2e1 mutant mouse.

(A) The Cables2e1 mice was generated using CRISPR/Cas9 system. Blue marks indicate the target sites on the left and right of exon1 of Cables2. (B) RT-qPCR using adult brain samples showed the …

Figure 7—figure supplement 1
Expression of compound embryos from Cables2d and Cables2e1 intercrossing.

Expression levels of Cables2 and Rps21 in compound mutant Cables2d/e1 embryo at E9.5. Averages of three independent experiments performed in duplicated and normalized against the expression levels …

Gene construction of Rps21d and Rps21-indel mutant mice.

Rps21d and Rps21-indel mutant mice were generated using CRISPR/Cas9 system. One-cut target site was designed to induce the mutation in Rps21-indel. Left and right target sites were introduce (red …

Defective and normal gastrulation development in Cables2d Epi KO and Cables2d Epi rescue chimeras, respectively.

(A) Schematic diagram of tetraploid complementation experiment for Cables2d Epi KO chimera. (B–E) Bright field (B, D) and YFP fluorescent (C, E) images of wild-type and Cables2d Epi KO chimeric …

Tables

Table 1
Survival rate and Mendelian ratio of Cables2-mutant embryos.
Embryonic days (E)Total number of embryosGenotypes
+/++/--/-
E6.5437132 (30.2)*221 (50.6)80 (18.3)
E7.57018 (25.7)32 (45.7)20† (28.6)
E8.5219 (42.9)9 (42.9)3† (14.3)
E9.5187 (38.9)7 (38.9)4† (22.2)
E12.562 (33.3)4 (66.7)0 (0)
Adult9024 (26.7)66 (73.3)0 (0)
  1. * Number of embryos (percentage), † Abnormal phenotype.

Table 2
Generation of Rps21 mutant mice.
StrainElectroporated embryosTransfer embryosTotal number of mice
NewbornFounder
MaleFemale
Rps21d2071993874
Rps21-indel2742000*--
  1. * At E16.5, there were eight implanted sites.

Table 3
Phenotypes in Cables2d Epi KO and Cables2d Epi rescue chimeras.
Tetraploid embryo + Cables2d/d; ROSA26YFP/+ ESC
(Epi KO chimera)
Wild-type chimera
Embryonic days (E)Total number of embryosPhenotypeTotal number of embryosPhenotype
NormalAbnormalNormalAbnormal
E6.5211431
E7.51541114113
E8.513013*761
Tetraploid embryo + Cables2d/d; ROSA26YFP/+; CAG-tdTomato-2A-Cables2-3xFLAG ESC
(Epi rescue chimera)
Wild-type chimera
Embryonic days (E)Total number of embryosPhenotypeTotal number of embryosPhenotype
NormalAbnormalNormalAbnormal
E7.5541211
E8.5431541
E9.5220321
  1. * All embryos had A-P axis specification.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(M. musculus)
Cables2PMID:11955625MGI:2182335
Gene
(M. musculus)
Rps21PMID:10022917MGI:1913731
Strain, strain background (M. musculus)B6.Cg-Tg(TOPGAL)Riken BRCRBRC05918
Cell line
(M. musculus)
Cables2tm1(KOMP)VlcgKOMPID: VG16085, clone: 16085A-D3
RRID:MMRRC_052978-UCD
ESC line
Cell line (Homo-sapiens)293TATCCCRL-3216
RRID:CVCL_0063
embryonic kidney
AntibodyAnti-FLAG (M2) (mouse monoclonal)Sigma-AldrichCat# F1804, RRID:AB_262044WB(1:1000), IP (1:650)
AntibodyAnti-β-catenin (D10A8) (rabbit monoclonal)Cell Signaling TechnologyCat# 8480, RRID:AB_11127855WB(1:1000)
AntibodyAnti-GAPDH (rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-25778, RRID:AB_10167668WB(1:1000)
AntibodyNormal mouse IgG (mouse isotype control)Santa Cruz BiotechnologyCat# sc-2025, RRID:AB_737182IP(1:250)
AntibodyAnti-Mouse IgG-HRP (secondary antibody)GE healthcareCat# NA931, RRID:AB_772210WB(5000)
AntibodyAnti-Rabbit IgG-HRP (secondary antibody)GE healthcareCat# NA934, RRID:AB_772206WB(5000)
Peptide, recombinant proteinTrueCut Cas9 ProteinThermo Fisher ScientificCat# A36498
Peptide, recombinant proteinDynabeads Protein GThermo Fisher ScientificCat# 10003D
Commercial assay or kitGeneArt Precision gRNA Systhesis KitThermo Fisher ScientificCat# A29377
Commercial assay or kitLipofectamine 3000 ReagentThermo Fisher ScientificCat# L3000015
Commercial assay or kitAmpliTag Gold 360 Master MixThermo Fisher ScientificCat# 4398886
Commercial assay or kitRNeasy Mini KitQiagenCat# 74104
Commercial assay or kitTB Green Premix Ex Taq IITakaraCat# RR820B
Commercial assay or kitDual-Glo Luciferase assay systemPromegaCat# E2920
Commercial assay or kitClick-iT Plus EdU Imaging KitThermo Fisher ScientificCat# C10638
Commercial assay or kitClick-iT Plus TUNEL Assay for In situ Apoptosis Detection kitThermo Fisher ScientificCat# C10619
SoftwareCLC Genomics WorkbenchQiagenRRID:SCR_011853

Additional files

Supplementary file 1

Differential gene expression in wild-type and Cables2d embryos.

Statistical significance was determined using ANOVA test (FDR < 0.05). This gene list is related to heatmap in Figure 6A.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp1-v1.xlsx
Supplementary file 2

Differential gene expression in wild-type and Cables2d embryos at E6.5 and KEGG pathway, GO term enrichments in upregulated genes.

Statistical significance was determined using EDGE test (FDR < 0.05). This gene list is related to Figure 6B–D.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp2-v1.xlsx
Supplementary file 3

Differential gene expression in wild-type and Cables2d embryos at E7.5 and KEGG pathway, GO term enrichments.

Statistical significance was determined using EDGE test (FDR < 0.05). This gene list is related to Figure 6E–G.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp3-v1.xlsx
Supplementary file 4

CRISPR target sites for gene-modified mice generation.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp4-v1.xlsx
Supplementary file 5

Primers for genotyping, RT-PCR and RT-qPCR.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp5-v1.xlsx
Supplementary file 6

Mendelian ratio of Cables2d and Cables2e1 intercross at E9.5.

https://cdn.elifesciences.org/articles/50346/elife-50346-supp6-v1.xlsx

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