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First published online June 6, 2008
doi: 10.1242/10.1242/dev.021170

Development 135, 2321-2329 (2008)
Published by The Company of Biologists 2008
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β-Catenin has sequential roles in the survival and specification of ventral dermis

Jennifer Ohtola1, John Myers1, Batool Akhtar-Zaidi1, Diana Zuzindlak1, Pooja Sandesara1, Karen Yeh1, Susan Mackem2 and Radhika Atit1,3,4,*

1 Department of Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
2 Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA.
3 Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA.
4 Department of Dermatology, Case Western Reserve University, Cleveland, OH 44106, USA.


Figure 1
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  		Fig. 1. Wnt signaling reporter expression during ventral dermal cell development. (A-F) X-gal-stained transverse sections of TCF/Lef-lacZ embryos at the forelimb (FL) level. (A-C) lacZ expression is detectable in the somatopleure at E8.5, and in the subectodermal mesenchyme cells in the flank at E9.5 and E10.5. (D) At E11.5, expression of the TCF/Lef-lacZ transgene expands to the midline and is visible throughout the subectodermal mesenchyme (arrows and inset). (E) At E14.5, during dermal cell differentiation, lacZ is expressed extensively in dermal cells (arrows and inset), and in the hair follicle placode (hf). (F) At E11.5, in the conditional absence of β-catenin in En1-expressing cells, TCF/Lef-lacZ expression is absent in the entire subectodermal flank and ventral mesenchyme (arrows, inset), but lacZ is expressed in cells of the limb mesoderm where En1 is not expressed (small arrows). (G) When β-catenin activity is stabilized in En1-expressing cells, nuclear β-catenin is ectopically visible in the En1-lineage cells away from the ectoderm (arrows). Compare F and G with D.

 

Figure 2
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  		Fig. 2. Ventral dermal cells originate from the LPM. (A-E) X-gal-stained transverse sections. (A) At E16.5, HoxB6Cre; R26R lineage-marked cells comprise the ventral dermis. (B-E) HoxB6Cre-ERT1; R26R embryos were given Tamoxifen at E7.5 and β-gal+ cells are found in the flank mesenchyme at E11.5 (B, arrows). (C-E) Later at E17.5, β-gal+ cells are found in the dermis (small arrows) and dermal papillae (dp) of the hair follicle (hf), and in the sternum and endothelial cells (ec, arrows). Dashed line demarcates the epidermis from the dermis in the embryonic skin. (D,E) High magnification images of the boxed areas in C.

 

Figure 3
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  		Fig. 3. β-catenin activity is required early for cell survival. (A,C) Transverse sections of an E10.5 control embryo (A) and a conditional β-catenin loss-of-function mutant (C) stained with X-gal to show the distribution of lineage-labeled cells. (B,D) Alternate sections assayed for cell survival by TUNEL (brown, arrows); nuclei are counterstained with methyl green. (D) Note the significant increase in TUNEL+ cells in the HoxB6Cre; R26R; β-catlof mutants. (A-D) Images of forelimb (FL) level sections taken at the same magnification.

 

Figure 4
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  		Fig. 4. Ventral dermal progenitors in the flank mesenchyme express En1 and contribute extensively to ventral dermal cells. (A-D) In En1Cre; R26R embryos, En1 lineage-marked (blue) cells are found in the flank mesenchyme at E10.5 (A,B) and in the midline of the ventrum by E11.5 (C,D). (A,C) Whole mounts; (B,D) sections. Insets (B,D) are lower magnification views of the sections. (E) En1 lineaged-marked cells are found extensively in the epidermis and dermis of E16.5 En1Cre; R26R embryos. (F) En1Cre-ER; R26R embryos given Tamoxifen at E10.5; at E16.5 β-gal+ cells are present only in the dermis (arrows). Black dashed line demarcates the epidermis from the dermis.

 

Figure 5
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  		Fig. 5. Ventral hair placodes and dermis are lacking in the absence of β-catenin in the En1 lineage. (A) Whole-mount in situ hybridization with a Ptch1 anti-sense mRNA probe reveals ventral hair follicle placodes at E14.5 in control embryos. (B) Ventral skin hair follicle placodes are absent in En1Cre; R26R; β-catlof mutants. (C,D) Sections of E16.5 embryos stained with Hematoxylin and Eosin (H&E). (E-J) X-gal-stained sections of E16.5 embryos. (E,F) The ventral dermis is present in the control (E) but is absent from the skin of mutant fetuses (F). (G-J) Higher magnification images of boxed areas in E and F. (E,G,I) In control embryos, lineage-labeled cells are visible in the sternum, dermis and epidermis, but not in the ribs. (F,H,J) In the mutants, lineage-labeled cells are present in the sternum and epidermis, and the sternum is wider. Scale bars: in C,D, 2 mm; in E,F, 500 µm; in G-J, 200 µm.

 

Figure 6
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  		Fig. 6. Dermo1 expression in the flank and ventral subectodermal mesenchyme is induced by β-catenin. (A-L) Section in situ hybridization of Dermo1 mRNA (D-F,J-L) at E10.5 and E11.5; alternate sections are stained with X-gal to visualize En1 lineage-marked cells (A-C;G-I). (J) Dermo1 mRNA is first expressed in the subectodermal flank and ventral mesenchyme, starting at E11.5, in the control embryos. (E,K) In the β-catenin loss-of-function mutants, Dermo1 mRNA is not visible at E10.5 or E11.5. (F,L) By stabilizing β-catenin in the En1 lineage, Dermo1 mRNA is expressed earlier at E10.5, and ectopically in all the mutant cells by E11.5 (dashed line with arrows). Images are of (A-F) E10.5 and (G-L) E11.5 embryos; all insets (F,J,L) are of the same magnification. Compare E and F with D, and K and L with J.

 

Figure 7
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  		Fig. 7. Small decrease in cell proliferation in the absence of β-catenin without altering cell survival. (A-C) Cell proliferation at E11.5 assayed by BrdU incorporation (brown-stained cells). (A,B) There is a small decrease in cell proliferation in the En1Cre; R26R; β-catlof mutants but no significant difference in β-catgof mutants (see Table 1). (D-F) Apoptosis at E11.5 as assayed by TUNEL (brown-stained cells); sections are counterstained with methyl green. There is no detectable difference in cell death in the flank and ventral mesenchyme between the mutants and control embryos. (E) TUNEL+ cells are detectable in the apical ectodermal ridge of the limb (inset). (A-F) En1 lineage-marked domains are outlined in red. Composites for each panel were created from two or three images taken at the same magnification.

 

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© The Company of Biologists Ltd 2008