1. Immunology and Inflammation

Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

  1. Sayumi Fujimori  Is a corresponding author
  2. Izumi Ohigashi
  3. Hayato Abe
  4. Yosuke Matsushita
  5. Toyomasa Katagiri
  6. Makoto M Taketo
  7. Yousuke Takahama  Is a corresponding author
  8. Shinji Takada  Is a corresponding author
  1. Division of Experimental Immunology, Institute of Advanced Medical Sciences, Tokushima University, Japan
  2. National Institute for Basic Biology, National Institutes of Natural Sciences, Japan
  3. Student Laboratory, School of Medicine, Tokushima University, Japan
  4. Division of Genome Medicine, Institute of Advanced Medical Sciences, Tokushima University, Japan
  5. Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, Japan
  6. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, United States
  7. Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Japan
  8. Department of Basic Biology in the School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Japan
https://doi.org/10.7554/eLife.69088
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Cite this article
  1. Sayumi Fujimori
  2. Izumi Ohigashi
  3. Hayato Abe
  4. Yosuke Matsushita
  5. Toyomasa Katagiri
  6. Makoto M Taketo
  7. Yousuke Takahama
  8. Shinji Takada
(2022)
Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development
eLife 11:e69088.
https://doi.org/10.7554/eLife.69088
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12 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Thymic dysplasia caused by the overexpression of β-catenin in thymic epithelial cells (TECs).

(A) Thoracic cavity of control mice and β-cat gain-of-function (GOF) mice at E15.5. Dotted lines show the outline of the thymic primordium. In many cases, blood clots were observed in the central …

Figure 1—figure supplement 1
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Immunofluorescence analysis of embryonic thymus.

Immunofluorescence staining for K5 and K8 (left) or β-catenin and β5t (right) on sagittal sections of the thymus from control mice and β-cat gain-of-function (GOF) mice at E15.5. Shown are merged …

Figure 2 with 1 supplement
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Defective thymus development in β-cat gain-of-function (GOF) embryos.

(A) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of thymic primordium in control mice and β-cat GOF mice at E11.5–E15.5. The sections were nuclear counterstained with TO-PRO3. …

Figure 2—figure supplement 1
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Immunofluorescence analysis of embryonic thymus and purity of isolated TECs.

(A) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of the thymic primordium in control mice and β-cat gain-of-function (GOF) mice at E11.5–E15.5. Shown are merged images with …

Figure 3
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Thymic dysplasia in postnatal β-cat gain-of-function (GOF) mice.

(A) Appearance of control mice and β-cat GOF mice at 8 wk. Bar: 1 cm. (B) Body weight of control mice and β-cat GOF mice at 8 wk (means and standard error of the means [SEMs], n = 4–6). N.S., not …

Figure 4
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Loss of αβT and γδT cells in secondary lymphoid organs by the overexpression of β-catenin in thymic epithelial cells (TECs).

(A) Flow cytometric analysis of splenocytes from control mice and β-cat gain-of-function (GOF) mice at 11 wk. Shown are representative dot plot profiles of CD3 and TCRβ expression (left) and CD3 and …

Figure 5
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No apparent change in thymus development in β-cat loss-of-function (LOF) embryos.

(A) Thoracic cavity of control mice and β-cat LOF mice at E15.5. Dotted lines show the outline of the thymic primordium. Representative data from three independent experiments are shown. TH: thymus, …

Figure 6
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The reduction of thymus size in postnatal β-cat loss-of-function (LOF) mice.

(A) Appearance of control mice and β-cat LOF mice at 8 wk. Bar: 1 cm. (B) Appearance of the thymus from control mice and β-cat LOF mice at postnatal stages (2–8 wk). Representative data from at …

Figure 7 with 1 supplement
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Characteristics of thymic epithelial cells (TECs) in postnatal β-cat loss-of-function (LOF) mice.

(A) Intracellular staining of β-catenin in UEA1−Ly51+ cortical thymic epithelial cells (cTECs; left) and UEA1+Ly51− medullary thymic epithelial cells (mTECs; right) from control mice and β-cat LOF …

Figure 7—figure supplement 1
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The purity of isolated cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) for quantitative RT-PCR analysis.

Flow cytometric analysis of cTECs and mTECs from indicated mice at 2 wk. Shown are representative profiles of CD45, PI, and EpCAM expression of total cells after magnetic depletion of CD45+ cells, …

Figure 8
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RNA sequencing analysis of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) isolated from β-cat loss-of-function (LOF) mice.

(A) Volcano plot analysis of mRNA expression for cTECs (left) and mTECs (right) isolated from control (Ctrl) mice and β-cat LOF mice at 2 wk. Detected genes are plotted as log2 fold change …

Figure 9
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Reduced total thymocyte production in β-cat loss-of-function (LOF) mice during the postnatal development.

(A) Plots show the number (means and standard error of the means [SEMs], n = 4–6) of total thymocytes in the thymus of indicated mice at 2 wk (left), 4 wk (middle), and 8 wk (right). (B) Flow …

Figure 10
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The thymus of β-cat loss-of-function (LOF) mice at 6 mo.

(A) Bars show body weight (left) and thymus weight (right) at 6 mo in control female mice and β-cat LOF female mice (means and standard error of the means [SEMs], n = 4). (B) Appearance of thymus …

Figure 11
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Thymus responses to poly(I:C) administration in β-cat loss-of-function (LOF) mice.

(A) Schematic diagram of poly(I:C) administration. Control mice or β-cat LOF mice at 4 wk were intraperitoneally injected with 250 μg of poly(I:C) on days 0 and 3, and the thymus was analyzed on the …

Figure 12
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Phenotypic differences between conditional β-cat loss-of-function (LOF) and gain-of-function (GOF) mice during the postnatal period.

GOF of β-catenin using Foxn1-Cre and β5t-iCre causes severe defect in thymic epithelial cell (TEC development and arrest in T-cell development at DN1), whereas Foxn1-Cre-mediated GOF of β-catenin …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6NSLC JapanRRID:MGI:5295404
Strain, strain background (Mus musculus)β5t-iCreOhigashi et al., 2013N/A
Strain, strain background (Mus musculus)Ctnnb1 floxed(β-catfl)Brault et al., 2001RRID:IMSR_JAX:004152
Strain, strain background (Mus musculus)Ctnnb1 exon 3 floxed(β-catex3fl)Harada et al., 1999N/A
Strain, strain background (Mus musculus)Rosa26-CAG-loxP-stop-loxP-tdTomato(R26R-tdTomato)Madisen et al., 2010RRID:IMSR_JAX:007914
AntibodyAnti-Foxn1 (Rabbit polyclonal)Itoi et al., 2006N/AIHC (1:100)
AntibodyAnti-β5t(Rabbit polyclonal)Murata et al., 2007N/AIHC (1:200)
AntibodyAnti-CCL21/6Ckine(Rat monoclonal, Clone 59106)R&D systemsCat# MAB457, RRID:AB_2259799IHC (1:10)
AntibodyAnti-Aire-eFluor 660(Rat monoclonal,Clone 5H12)e-BioscienceCat# 50-5934-82, RRID:AB_2574257IHC (1:20)
AntibodyAnti-β-catenin(Mouse monoclonal, Clone 14)BD Transduction LaboratoriesCat# 610154, RRID:AB_397555IHC (1:200)FC (1:100)
AntibodyAnti-Keratin 5(Chicken polyclonal,Clone Poly9059)BioLegendCat# 905901, RRID:AB_2565054IHC (1:500)
AntibodyAnti-Keratin 8(Mouse monoclonal, Clone IE8)BioLegendCat# 904804, RRID:AB_2616821IHC (1:500)
AntibodyAnti-CD45-Biotin(Rat monoclonal, Clone 30-F11)BioLegendCat# 103104, RRID:AB_312969IHC (1:100)
AntibodyAnti-mouse CD45-PE/Cy5(Rat monoclonal, Clone 30-F11)BioLegendCat# 103110, RRID:AB_312975IHC (1:100)
AntibodyAnti-CD45-eFluor 450(Rat monoclonal, Clone 30-F11)eBioscienceCat# 48-0451-80, RRID:AB_1518807FC (1:200)
AntibodyCD45 MicroBeads(Rat monoclonal)Miltenyi BiotecCat# 130-052-301, RRID:AB_2877061
AntibodyAnti-EpCAM-PE/Cy7(Rat monoclonal, Clone G8.8)BioLegendCat# 118216, RRID:AB_1236471FC (1:100)
AntibodyAnti-Ly51-Alexa Fluor 647(Rat monoclonal, clone 6C3)BioLegendCat# 108312, RRID:AB_2099613FC (1:250)
AntibodyAnti-Ly51-PE(Rat monoclonal, clone 6C3)BioLegendCat# 108308, RRID:AB_313365FC (1:100)
AntibodyUlex Europaeus Agglutinin I (UEA1)-BiotinVector LaboratoriesCat# B-1065, RRID:AB_2336766FC (1:1000)
AntibodyUlex Europaeus Agglutinin I (UEA1)-DyLight 649Vector LaboratoriesCat# DL-1068FC (1:500)
AntibodyAnti-CD4-APC (Rat monoclonal, Clone RM4-5)eBioscienceCat# 17-0042-81, RRID:AB_469322FC (1:100)
AntibodyAnti-CD8α-eFluor 450 (Rat monoclonal, Clone clone 53-6.7)InvitrogenCat# 48-0081-80, RRID:AB_1272235FC (1:200)
AntibodyAnti-CD25-FITC(Rat monoclonal, Clone PC61)BioLegendCat# 102006, RRID:AB_312855FC (1:250)
AntibodyAnti-CD44-PE/Cy7 (Rat monoclonal, Clone IM7)eBioscienceCat# 25-0441-81, RRID:AB_469622FC (1:100)
AntibodyAnti-TCRβ-PE (Armenian hamster monoclonal, Clone H57-597)BioLegendCat# 109207, RRID:AB_313430FC (1:100)
AntibodyAnti-TCRδ-Biotin (Armenian hamster monoclonal, Clone GL3)BioLegendCat# 118103, RRID:AB_313827FC (1:250)
AntibodyAnti-Vγ5-FITC (Hamster monoclonal, Clone 536)BD PharmingenCat# 553229, RRID:AB_394721FC (1:100)
AntibodyAnti-Vγ4-FITC (Armenian hamster monoclonal, Clone UC3-10A6)BD PharmingenCat# 553226, RRID:AB_394720FC (1:100)
AntibodyAnti-Vγ1-FITC(Armenian hamster monoclonal, Clone 2.11)BioLegendCat# 141103, RRID:AB_10694242FC (1:100)
AntibodyAnti-CD3ε-APC(Armenian hamster monoclonal, Clone 145-2C11)BioLegendCat# 100312, RRID:AB_312677FC (1:20)
AntibodyMouse IgG1 (Mouse monoclonal, Clone MOPC-21)BD PharmingnenCat# 554121, RRID:AB_395252FC (1:200)
AntibodyAnti-chicken IgG-Alexa Fluor 488 (Goat polyclonal)Molecular ProbesCat# A-11039, RRID:AB_142924IHC (1:500)
AntibodyAnti-mouse IgG-Alexa Fluor 488 (Goat polyclonal)InvitrogenCat# A-11001, RRID:AB_2534069IHC (1:500)
AntibodyAnti-mouse IgG-Alexa Fluor Plus 488 (Goat polyclonal)InvitrogenCat# A32723, RRID:AB_2633275FC (1:800)
AntibodyAnti-mouse IgG2a-Alexa Fluor 555 (Goat polyclonal)Molecular ProbesCat# A-21137, RRID:AB_2535776IHC (1:500)
AntibodyAnti-rabbit IgG-Alexa Fluor 488 (Goat polyclonal)InvitrogenCat# A-11034, RRID:AB_2576217IHC (1:500)
AntibodyAnti-rabbit IgG-Alexa Fluor 555 (Goat polyclonal)InvitrogenCat# A-21429, RRID:AB_2535850IHC (1:500)
AntibodyAnti-rat IgG-Alexa Fluor 555 (Goat polyclonal)InvitrogenCat# A-21434, RRID:AB_2535855IHC (1:500)
Peptide, recombinant proteinStreptavidin-Alexa Fluor 488InvitrogenCat# S32354, RRID:AB_2315383IHC (1:500)
Peptide, recombinant proteinStreptavidin-APC-eFluor 780InvitrogenCat# 47-4317-82, RRID:AB_10366688FC (1:80)
Peptide, recombinant proteinStreptavidin-PEInvitrogenCat# S866FC (1:400)
Peptide, recombinant proteinDNase IRocheCat# 04716728001(0.01%)
Commercial assay or kitRNeasy Plus Micro KitQiagenCat# 74,034
Commercial assay or kitSMART-seq v4 Ultra Low Input RNA Kit for SequencingTakara BioCat# 634,888
Commercial assay or kitNextera XT DNA Library Preparation KitIlluminaCat# FC-131-1024
Commercial assay or kitNextSeq 500/550 High Output Kit v2.5 (75 cycles)IlluminaCat# 20024906
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798v7.0e
Software, algorithmCLC Genomics WorkbenchQiagenRRID:SCR_011853v12.0
Otherpoly(I:C) HMWInvivoGenCat# tlrl-pic
OtherTO-PRO3Thermo Fisher ScientificCat# T3605IHC (1:1000)
OtherLiberase TMRocheCat# 5401127001(0.5 or 1 unit/ml)

Additional files

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https://cdn.elifesciences.org/articles/69088/elife-69088-transrepform1-v1.docx

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