Progenitors oppositely polarize WNT activators and inhibitors to orchestrate tissue development
- Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, United States
- The Francis Crick Institute, Midland Road, United Kingdom
Figures
Figure 1 with 5 supplements
Two-dimensional patterning of hair placodes is severely affected upon sustained autonomous WNT activation.
(A) Sagittal views and schematic of skin section depicting a basal hair bud progenitor and underlying dermal condensate (DC, encased by blue dotted lines). Labeling is for LacZ expression knocked …
-
Figure 1—source data 1
Measurements of placodes and clusters distribution, size and shape, shown in Figure 1D.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Apc-null clusters show properties of hair follicles arrested at the placode stage.
(A) Sagittal sections of embryonic skin subjected to immunohistochemistry with antibodies against β-Catenin and APC. Note that reduced APC immunostaining coincides with broadened elevated β-Catenin, …
Figure 1—figure supplement 2
Apc-null clusters do not present signs of DNA double strand breaks.
(A–B) Immunofluorescence detection of γH2AX in developing epidermis of (A) Apc fl/+ and (B) Apc fl/fl Rosa26-lox-stop-lox-tdTOM embryos were transduced at E9.5 with lentivirus harboring a Cre …
Figure 1—figure supplement 3
Apc-null cells aggregate into clusters and are non-proliferative.
(A) Experimental design and proliferation analysis. Apcfl/fl; Rosa26-lox-stop-lox-YFP embryos were transduced at E9.5 with LVs harboring a Cre recombinase (Pgk-Cre-mRFP) and analyzed at E14.5. At 4 …
Figure 1—figure supplement 4
Apc-null cell clusters lose adherens junction transmembrane protein E-Cadherin.
Representative E14.5 whole-mount immunofluorescence images and respective orthogonal views from (A) Apc-het and (B) Apc-null transduced tdTomato+ embryonic skins showing endogenous E-Cadherin and …
Figure 1—figure supplement 5
Apc-null cell clusters lose the hemidesmossome integrin beta4 (ITGB4).
Representative E14.5 whole-mount immunofluorescence images and respective orthogonal views from (A) Apc-het and (B) Apc-null transduced tdTomato+ embryonic skins showing endogenous ITGB4 and …
Figure 2 with 2 supplements
Teasing out a WNT-sensitive molecular signature based upon transcriptome profiling of skin progenitors possessing different WNT signaling levels.
(A) LV construct, epifluorescence imaging and FACS strategy for isolating WNT signaling (GFP+) and WNTlo skin progenitors from LV-transduced E14.5 Apc fl/+ and Apcfl/fl; R26fl-stop-fl-tdTOM embryos. …
-
Figure 2—source data 1
Source data for the graphs shown in Figure 2D.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
FACS purification strategy to isolate WNThi skin progenitors from Apc embryonic skin.
(A–B) FACS purification of WNThi cells from Apcfl/+; Rosa26-lox-stop-lox-dtTomato embryos (A) and from Apcfl/fl Rosa26-lox-stop-lox-dtTomato embryos (B). Both types of embryos were transduced at …
Figure 2—figure supplement 2
Cell adhesion transcripts upregulated in Apc null WNThi cells (Geneontology – PANTHER Classification System).
-
Figure 2—figure supplement 2—source data 1
Cell adhesion transcripts upregulated in Apc null WNThi cells (Geneontology – PANTHER Classification System).
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig2-figsupp2-data1-v2.docx
Figure 3 with 4 supplements
Wild-type WNT signaling progenitor cells express high levels of WNT inhibitors.
(A) Strategy used to isolate and profile slow-cycling basal progenitors from the epidermal fraction of dispase-treated, wild-type E17.5 skin, which contains epidermis and early hair placodes/buds. …
-
Figure 3—source data 1
Source data for the graphs shown in Figure 3B.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
FACS purification strategy to isolate WNThi placode and WNTlo epidermal progenitors from wild-type embryonic skin.
(A) FACS purification plots of single cell suspensions isolated from the dispase-selected epidermal/hair bud fraction of E17.5 skin. Pups were transgenic for Lhx2-GFP, active in basal hair bud …
Figure 3—figure supplement 2
WNThi signature genes in hair follicle development.
Shown is a list of transcripts that are shared between the Apc null WNThi signature and the wild-type WNThi signature.
-
Figure 3—figure supplement 2—source data 1
WNThi signature genes in hair follicle development.
Shown is a list of transcripts that are shared between the Apc null WNThi signature and the wild-type WNThi signature.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig3-figsupp2-data1-v2.docx
Figure 3—figure supplement 3
BMP4 acts long range to perturb hair follicle patterning.
(A–B) Strong correlation between WNT signaling and Bmp4 levels in both (A) Apc-null clusters and (B) wild-type hair buds. (C) Whole-mount immunofluorescence of E14.5 mosaic Apc-null and Apc-het …
-
Figure 3—figure supplement 3—source data 1
Source data for the graphs shown in Figure 3—figure supplement 3A and B.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig3-figsupp3-data1-v2.xlsx
Figure 3—figure supplement 4
Apc-null cells express high levels of WNT inhibitors.
(A–B) Representative immunofluorescence images and respective orthogonal views from Apc-null and Apc-het transduced tdTomato+ embryonic skins showing endogenous (A) WIF1 and (B) NOTUM patterns. Note …
Figure 4 with 1 supplement
Progenitor cells apically polarize WNT inhibitors.
(A) Schematic of whole-mount analysis from embryonic skin with examples of optical sections showing interfollicular epidermis (IFE), bud and germ; position of pixel intensity measurement. Z, plane …
-
Figure 4—source data 1
Source data for the graphs shown in Figure 4.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Apical WIF1 localization is dependent on N-Glycosylation.
(A) Strategy to test the importance of N-Glycosylation for WIF1 apical localization. E15.5 wild-type embryonic skin explants were treated with different Tunicamycin concentrations for 10 hr and …
-
Figure 4—figure supplement 1—source data 1
Source data for the graphs shown in Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 3 supplements
Evidence of oppositely polarized and short-range action of WNTs and WNT inhibitors in hair bud progenitors that are actively signaling through WNTs.
(A) LV-constructs and strategy to monitor WNT inhibitors and WNTs. M, myc-tag. TRE, tetracycline regulatory element. Krt14rtTA is a transgenic mouse line expressing a doxycycline (DOX)-inducible …
-
Figure 5—source data 1
Source data for the graphs shown in Figure 5D and E.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
WIF1 expression during early hair follicle development.
(A) Strategy to monitor WIF1 expression by MYC-tagging (M). Depicted LV was introduced into the amniotic cavities of E9.5 Krt14rtTA embryos, and transduced genes were activated by doxycycline (DOX). …
Figure 5—figure supplement 2
NOTUM expression during early hair follicle development.
(A) Strategy to monitor NOTUM by MYC-epitope tagging (M). Depicted LV was introduced into the amniotic cavities of E9.5 Krt14rtTA embryos and the transduced Notum gene was activated by doxycycline …
Figure 5—figure supplement 3
Differential polarization of WNT inhibitors and activators.
Strategy to monitor WNT inhibitors (DKK4 and APCDD1) and ligands (WNT3) by MYC-epitope tagging (M). LV-constructs were introduced into the amniotic cavities of E9.5 Krt14rtTA embryos, and transduced …
-
Figure 5—figure supplement 3—source data 1
Source data for the graphs shown in Figure 5—figure supplement 3A.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig5-figsupp3-data1-v2.xlsx
Figure 6 with 5 supplements
Hair bud progenitors apically polarize WNT inhibitors to protect their own identity and differentially confer WNT signaling to their neighbors.
(A) Whole-mount immunofluorescence and quantifications reveal that elevating NOTUM across the epidermal plane results in significantly fewer hair follicles (Mean ± SD; n > 10 mm2 skin analyzed from ≥…
-
Figure 6—source data 1
Source data for the graphs shown in Figure 6A, B, C, and D.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
WNT inhibitor overexpression perturbs hair follicle formation.
(A) Whole mount immunofluorescence and quantifications reveals that elevating WIF1 across the epidermal plane results in significantly fewer developing hair follicles beginning at the placode stage. …
-
Figure 6—figure supplement 1—source data 1
Source data for the graphs shown in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Hair bud progenitors cannot maintain their fate upon WNT inhibition.
(A) Experimental design and results of pharmacological WNT inhibition in embryonic skin explants exposed to the porcupine inhibitor LGK974. Note that placode/bud numbers were significantly reduced …
-
Figure 6—figure supplement 2—source data 1
Source data for the graphs shown in Figure 6—figure supplement 2A, C, and E.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig6-figsupp2-data1-v2.xlsx
Figure 6—figure supplement 3
Hair bud progenitors revert their fate after WNT inhibitor washout.
(A) Experimental design and results of pharmacological WNT inhibition and washout in embryonic skin explants exposed to 1 mM porcupine inhibitor LGK974. Immunofluorescence and quantifications of …
-
Figure 6—figure supplement 3—source data 1
Source data for the graphs shown in Figure 6—figure supplement 3A.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig6-figsupp3-data1-v2.xlsx
Figure 6—figure supplement 4
Hair follicle progenitor cells co-express WNT reporter, LEF1, TCF1/7 and WNT target gene products FZD10 and WIF1.
E9.5 skin was transduced with lentivirus harboring the WNT-reporter-GFP (12xTCF-TK-EGFP) and harvested at E15.5. Representative whole-mount immunofluorescence images show WNT reporter GFP positive …
Figure 6—figure supplement 5
NOTUM and WIF1 mis-localization leads to impaired development of hair follicles.
(A) Experimental setup to induce mis-localization of WNT inhibitors by targeting them to the basal membrane. Transgenes were designed to express NOTUM and WIF1 as proteins tagged with an AQP4 …
-
Figure 6—figure supplement 5—source data 1
Source data for the graphs shown in Figure 6—figure supplement 5B, C, and E.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig6-figsupp5-data1-v2.xlsx
Figure 7
Notum regulates the formation of sharp boundaries between neighboring cell fates.
(A) In utero lentiviral delivery strategy to conditionally ablate Notum in R26dtTomato embryos. (B–C) Representative whole-mount immunofluorescence showing LEF1 intensity profile and population size …
-
Figure 7—source data 1
Source data for the graphs shown in Figure 7C.
- https://cdn.elifesciences.org/articles/54304/elife-54304-fig7-data1-v2.xlsx
Figure 8
Summarizing model.
WNT signaling basal progenitors form opposing intracellular morphogen gradients of WNT inhibitors and WNT ligands/activators. In so doing, they preserve their own WNT signaling and identity and …
Additional files
-
Supplementary file 1
Key Resources Table.
- https://cdn.elifesciences.org/articles/54304/elife-54304-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/54304/elife-54304-transrepform-v2.pdf
