1. Developmental Biology

Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates

  1. Tempei Sato
  2. Kensuke Kataoka
  3. Yoshiaki Ito
  4. Shigetoshi Yokoyama
  5. Masafumi Inui
  6. Masaki Mori
  7. Satoru Takahashi
  8. Keiichi Akita
  9. Shuji Takada
  10. Hiroe Ueno-Kudoh
  11. Hiroshi Asahara  Is a corresponding author
  1. Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan
  2. Department of Systems BioMedicine, National Research Institute for Child Health and Development, Japan
  3. Research Fellow of Japan Society for the Promotion of Science, Japan
  4. Research Core, Tokyo Medical and Dental University, Japan
  5. Laboratory of Metabolism, National Institutes of Health, United States
  6. Laboratory of Animal Regeneration Systemology, Meiji University, Japan
  7. Department of Medical Chemistry, Shiga University of Medical Science, Japan
  8. Department of Anatomy and Embryology, University of Tsukuba, Japan
  9. Department of Clinical Anatomy, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan
  10. Reproduction Center, Yokohama City University, Japan
  11. AMED-CREST, Japan Agency for Medical Research and Development (AMED), Japan
  12. Department of Molecular Medicine, The Scripps Research Institute, United States
https://doi.org/10.7554/eLife.53608
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Cite this article
  1. Tempei Sato
  2. Kensuke Kataoka
  3. Yoshiaki Ito
  4. Shigetoshi Yokoyama
  5. Masafumi Inui
  6. Masaki Mori
  7. Satoru Takahashi
  8. Keiichi Akita
  9. Shuji Takada
  10. Hiroe Ueno-Kudoh
  11. Hiroshi Asahara
(2020)
Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates
eLife 9:e53608.
https://doi.org/10.7554/eLife.53608
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5 figures, 2 tables and 2 additional files

Figures

Figure 1 with 2 supplements
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Skeletal patterning defects in Lin28a-/- mice.

(A) Whole-mount in situ hybridization of Lin28a in E9.5–11.5 embryos. (B) Lateral views of Wt (left panel) and Lin28a–/– mice (right panel) at E16.5. White arrow, the tip of the tail; white …

Figure 1—figure supplement 1
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Generation of Lin28a–/– mice.

(A) Schematic diagram of the Lin28a gene-targeting construct. The small light-blue box indicates the 5’ and 3’ untranslated regions (UTRs), and the large light-blue box indicates the coding region. …

Figure 1—figure supplement 2
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Lin28a–/– mice exhibit growth defects.

(A, B) Appearance of Wt and Lin28a–/– mice. Representative Wt (left) and Lin28a–/– (right) pups at P0 (A) and P7 (B) are shown. (C) Body weight of Wt and Lin28a–/– mice. Data are expressed as the …

Figure 2 with 2 supplements
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Hox gene dysregulation in Lin28a-/- mice.

(A) q-PCR analyses of all Hox genes. All data are expressed as the mean ± standard error of the mean (SEM) (n = 3). *p<0.05. (B) Whole-mount in situ hybridization of Hox genes in E11.5 embryos. …

Figure 2—source data 1

Source data related to panel (A).

https://cdn.elifesciences.org/articles/53608/elife-53608-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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Whole-mount in situ hybridization of Hox genes in Lin28 knockout embryos (Related to Fig.

Figure 2B). Whole-mount in situ hybridization of Wt and Lin28a–/– mice embryos at E9.5 (using Hoxa3, Hoxd3, Hoxb8, Hoxc8, Hoxa11, Hoxd12 and Hoxa13 probes), at E10.5 (using Hoxc13 probe) and at …

Figure 2—figure supplement 2
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RA sensitivity in Lin28a mutant mice.

(A) Dorsal views of thoracic vertebrae and ribs of RA-treated Lin28a–/– mice. R1–R12, 1st to 12th ribs. The asterisk indicates the ablation of the 13th rib. (B–F) Lateral view of cervical and upper …

Figure 3 with 2 supplements
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Let-7 targets the polycomb gene directly.

(A) Comparison of microRNA expression in Wt and Lin28a–/– embryos at E9.5. (B, C) q-PCR analyses of let-7-family members (B) and Hox-embedded microRNAs (C). In (B), data are expressed as the mean (n …

Figure 3—source data 1

Source data related to panel A-C, and F-H.

https://cdn.elifesciences.org/articles/53608/elife-53608-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
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Skeletal defects in Cbx2 mutant mice.

(A) Cbx2 targeting and the sequence of Cbx2 mutants. The start codon of Cbx2 is highlighted in red and the PAM sequence for hCas9 is highlighted in yellow. Targeting sequences are underlined. The …

Figure 3—figure supplement 2
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Expression level of Cbx2 in Lin28a mutant embryo.

Change in the expression of Cbx2 in Lin28a+/– and Lin28a–/– mice E9.5 embryos were detected by western blot analyses. β-actin is shown as a loading control. All data are expressed as the mean ± SEM …

Figure 4
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Histone modifications and polycomb occupancy at Hox loci in Lin28a-/- mice.

(A) Schematic diagram of the experimental procedure for ChIP analysis. (B) ChIP and q-PCR analyses of H2K27me3 in Hox A cluster genes in Wt embryos. (C–G) ChIP and q-PCR analyses of H3K27me3 (C), …

Figure 4—source data 1

Source data related to panel B-G.

https://cdn.elifesciences.org/articles/53608/elife-53608-fig4-data1-v1.xlsx
Figure 5
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Knockdown of let-7 can reverse Hox gene dysregulation.

(A) Morphology (top panels) and alkaline phosphatase activity (bottom panels) of Wt and Lin28a–/– ES-like cells. (B) Western blot analysis of Lin28a in ES-like cells. β-actin is shown as a loading …

Figure 5—source data 1

Source data related to panel C-E, G-H, and K.

https://cdn.elifesciences.org/articles/53608/elife-53608-fig5-data1-v1.xlsx

Tables

Table 1
Summary of skeletal abnormalities in Lin28a mutant mice.

Anterior arch of the atlas*Ribs†Sternum attachment‡Lumbar§
131276165L6/S1*
Wt(n = 16)016 (100%)016 (100%)07 (43.8%)8 (50%)1 (6.2%)
Lin28a+/-(n = 19)4 (21.1%)19 (100%)019 (100%)0018 (94.7%)1 (5.3%)
Lin28a-/-
(n = 14)		9 (64.3%)014 (100%)014 (100%)09 (64.3%)5 (35.7%)

  1. The percentages of each phenotype are shown in parenthesis.

    * The anterior arch of the atlas was formed from C2 or via fusion.

  2. † Total number of pairs of ribs.

    ‡ Total number of pairs of true ribs that were attached to the sternum.

  3. § Total number of lumbar vertebrae. L6/S1* indicates an abnormal sacral vertebra that had morphological features of a lumbar vertebra on only one side.

Key resources table
Reagent type
(species) or resource		DesignationSource or
reference		IdentifiersAdditional
information
Antibodyanti-Arl4dSanta CruzSC-271274mouse monoclonal antibody, for western blot, at 1:500
Antibodyanti-b-actinSigmaA5316mouse monoclonal antibody, for western blot, at 1:2000
Antibodyanti-Cbx2Abcamab80044Rabbit polyclonal antibody, for western blot, at 1:500
Antibodyanti-CBX2Bethyl LaboratoriesA302-524ARabbit polyclonal antibody, for ChIP
Antibodyanti-Cbx5Cell Signaling Technology#2616SRabbit polyclonal antibody, for western blot, at 1:1000
Antibodyanti-DIG-AP Fab fragment antibodyRoche1-093-274sheep polyclonal antibody, for in situ hybridization
Antibodyanti-Dusp4 (MKP-2)Santa CruzSC-1200Rabbit polyclonal antibody, for western blot, at 1:250
Antibodyanti-E2f6Santa CruzSC-8366goat polyclonal antibody, for western blot, at 1:500
Antibodyanti-Lin28aCell Signaling Technology#3978SRabbit polyclonal antibody, for western blot, at 1:1000
Antibodyanti-mouse IgG HRP-conjugatedSigmaA2304goat affinity isolated antibody, for western blot, at 1:2000
Antibodyanti-rabbit IgG HRP-conjugatedSigmaA6154goat affinity isolated antibody, for western blot, at 1:2000
Antibodyanti-trimethyl-histone H3 (Lys27)Millipore#07–449Rabbit Polyclonal Antibody, for ChIP
Antibodyanti-trimethyl-histone H3 (Lys4)Millipore#07–473Rabbit Polyclonal Antibody,for ChIP
Antibodynormal rabbit IgGSanta CruzSC-2027Rabbit Polyclonal Antibody, for ChIP
AntibodyRING1B (D22F2) XP rabbit monoclonal antibody (mAb)Cell Signaling Technology#5694Srabbit monoclonal antibody, for ChiP
Cell LinesHEK293T cellsATCCRRID:CVCL_0063
Cell LinesWt or Lin28a-/-ES like cellsMaterials and methods sectionN/A
Chemical compound, drug2-mercaptoethanolGibco#21985023
Chemical compound, drugacetic anhydrideWako#011–00276
Chemical compound, drugAlcian BlueSigmaA5268-10G
Chemical compound, drugAlizarin Red SSigmaA5533-25G
Chemical compound, drugChapsDojindo Molecular Technologies349–04722
Chemical compound, drugCHIR 99021Wako034–23103
Chemical compound, drugFast Green FCFSigmaF7258-25G
Chemical compound, drugFast Red Violet LB SaltSigmaF3381-5G
Chemical compound, drugformamideSigmaSIGF5786
Chemical compound, drugG-418 SulfateWako074–05963
Chemical compound, drugglycineWako#077–00735
Chemical compound, drugheparinNacalai Tesque17513–96
Chemical compound, drugNBT/BCIPRoche#1697471
Chemical compound, drugPD0325901Wako162–25291
Chemical compound, drugPFAWako#162–16065
Chemical compound, drugRetinoic acid (all-trans)Wako182–01111
Chemical compound, drugsodium pyruvateGibco#11360070
Chemical compound, drugtriethanolamineWako142–05625
Commercial assay, kitChemi-Lumi OneNacalai Tesque#07880
Commercial assay, kitDirectPCR Lysis reagentViagen Biotech#102 T
Commercial assay, kitExoSAP-IT Express PCR Cleanup ReagentsThermoFisher scientific#75001
Commercial assay, kitFugeneHDPromegaE2312
Commercial assay, kitGoTaq Flexi DNA PolymerasePromegaM8298
Commercial assay, kitLipofectamine 2000Invitrogen#11668019
Commercial assay, kitMegaClear Transcription Clean-Up KitInvitrogenAM1908
Commercial assay, kitmMESSAGE mMACHINE T7 KitInvitrogenAM1344
Commercial assay, kitSuperSignal West Femto Maximum Sensitivity SubstrateThermo Fisher Scientific#34095
Commercial assay, kitSYBR Green PCR Master MixApplied Biosystems#4309155
Commercial assay, kitTaqMan MicroRNA AssaysApplied Biosystemslet-7a (#000377), let-7b (#002619), let-7c (#000379), let-7d (#002283), let-7e (#002406), let-7f (#000382), let-7g (#002282), let-7i (#002221), mir-98 (#000577), mir-10a (#000387), mir-10b (#002218), mir-196a (#241070), mir-196b (#002215),
RNU6B (#001093)
Commercial assay, kitTaqMan Rodent MicroRNA Array A and BApplied Biosystems#4398979
Commercial assay, kitTaqMan Rodent MicroRNA Array BApplied Biosystems#4398980
Commercial assay, kitTaqMan Universal Master Mix II, no UNGApplied Biosystems#4440040
Commercial assay, kitthe TaqMan MicroRNA Reverse Transcription kitApplied Biosystems#4366597
Peptide, recombinant proteinESGRO Recombinant Mouse LIF ProteinMerck MilliporeESG1107
Peptide, recombinant proteinProteinase K recombinant PCR GradeRoche03-115-887-001
StrainsCbx2 deficient miceMaterials and methods sectionN/A
StrainsLin28a deficient miceMaterials and methods sectionN/A
StrainsMeox2 CreThe Jackson LaboratoryN/A
OtherDulbecco’s Modified Eagle’s medium (DMEM)SigmaD5796
OtherGlutamaxGibco#35050061
OtherImmobilonMilliporeWBKLS0100
Othernonessential amino acids (NEAAs)Gibco#11140050
Othersheep serumThermo Fisher Scientific535–81301
Otherskim milkWako#190–12865
OthertRNARoche109–495

Additional files

Supplementary file 1

Survival rate of Lin28a mutant mice at various stages.

https://cdn.elifesciences.org/articles/53608/elife-53608-supp1-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/53608/elife-53608-transrepform-v1.docx

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