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. Author manuscript; available in PMC: 2009 Aug 26.
Published in final edited form as: Development. 2008 Jul;135(13):2321–2329. doi: 10.1242/dev.021170

β-catenin has sequential roles in the survival and specification of ventral dermis

Jennifer Ohtola 1, John Myers 1, Batool Akhtar-Zaidi 1, Diana Zuzindlak 1, Pooja Sandesara 1, Karen Yeh 1, Susan Mackem 2, Radhika Atit 1,3,4
PMCID: PMC2732188  NIHMSID: NIHMS92107  PMID: 18539925

Abstract

The dermis promotes the development and maintains the functional components of skin such as hair follicles, sweat glands, nerves, and blood vessels. The dermis is also critical for wound healing and homeostasis of the skin. The dermis originates from the somites, the lateral plate mesoderm, and the cranial neural crest. Despite the importance of the dermis in the structural and functional integrity of the skin, genetic analysis of dermal development in different parts of the embryo is incomplete. The signaling requirements for ventral dermal cell development have not been established in either the chick or mammalian embryo. We have shown previously that Wnt signaling specifies the dorsal dermis from the somites. In this study, we demonstrate that Wnt/β-catenin signaling is necessary for the survival of early ventral dermal progenitors. In addition, we show that at later stagesWnt/β-catenin signaling is sufficient for ventral dermal cell specification. Consistent with the different origins of dorsal and ventral dermal cells, our results demonstrate both conserved and divergent roles of β-catenin/Wnt signaling in dermal development.

Keywords: dermis, cell fate, cell survival, skin, sternum

INTRODUCTION

Identifying the molecular controls of dermal cell fate from diverse origins in the mammalian embryo is critical for expanding our understanding of congenital skin defects and for advancing strategies in skin tissue engineering (Paller, 2007; Supp and Boyce, 2005). Craniofacial, dorsal trunk, and ventral trunk dermis originates from different multipotential progenitor populations (Couly et al., 1992; Mauger, 1972), but is the signaling mechanism for mammalian dermal cell fate selection conserved or divergent in these different populations?

The skin consists of the epidermis, derived from the surface ectoderm, and the underlying dermis. Reciprocal interactions between the surface ectoderm and dermis induce the development of the epidermal appendages such as hair follicles and glands of the skin (Millar, 2002). Dermal fibroblasts from different parts of the embryos have distinct inductive properties (Foley et al., 2001; Hardy, 1992; Miletich and Sharpe, 2004) and maintain positional identity in adult humans (Chang et al., 2002). Diverse origins of the dermis might be the source of variation in skin patterning, pigmentation color, and types of epidermal appendages that are found on the body (Candille et al., 2004). The genetic program to establish the distinctiveness of dermal cells in different parts of the mammalian embryo is yet to be discovered.

To address the underlying basis for differences in dorsal versus ventral skin patterning, studies have been carried out in the chick embryo, and these implicate different signaling molecules that are important for ventral dermal cell development (Fliniaux et al., 2004a; Fliniaux et al., 2004b; Sengel and Kieny, 1967). Fate mapping studies in the chick embryo demonstrate that the proximal part of the somatopluere closest to the somites gives rise to the feather-forming dermis in the ventral trunk (Fliniaux et al., 2004a; Mauger, 1972). Suppression of Bone Morphogenetic Protein (BMP) signaling by endogenous Noggin expression or ectopic expression of Sonic Hedgehog can induce the differentiation of ventral trunk dermal progenitors into feather-forming dermis (Fliniaux et al., 2004a). Early during lateral plate mesoderm (LPM) cell differentiation in the chick embryo, Wnts are expressed in the ectoderm overlying the somatopleure (Fliniaux et al., 2004a; Rodriguez-Niedenfuhr et al., 2003; Schubert et al., 2002). The requirement of any signaling molecules in ventral dermal cell development has not been determined in either the chick or mammalian embryo. In this study, we examine the role of Wnt signaling in ventral dermal cell development.

The canonical Wnt signaling pathway is involved in early embryonic patterning, cell fate specification, proliferation, and the maintenance of stem cell compartments (Nelson and Nusse, 2004). β-catenin is a key transducer of the Wnt signaling pathway (Nelson and Nusse, 2004). Embryos fail to gastrulate in the absence of β-catenin activity (Haegel et al., 1995), and unregulated β-catenin activity leads to cancer in adults (Giles et al., 2003). In the absence of Wnt signaling, the β-catenin protein is phosphorylated and marked for degradation (Nelson and Nusse, 2004). In the presence of Wnt signaling, the unphosphorylated form of β-catenin accumulates in the cytoplasm, translocates to the nucleus, binds to TCF/Lef family of transcription factors, and promotes the transcription of Wnt target genes (Brantjes et al., 2002). Changes in downstream target gene expression mediate the diverse roles of Wnt signaling in development and disease (Nelson and Nusse, 2004; Sancho et al., 2003).

Studies in the chick embryo demonstrate the requirement of Wnt signaling in early dorsal dermal cell development from the dermamyotome (Olivera-Martinez et al., 2002; Olivera-Martinez et al., 2004). Our previous studies in the mouse embryo on dorsal dermal fate specification from the central somite indicate that Wnts, via β-catenin provide an instructive signal for dermal fate (Atit et al., 2006). Prior to and during dorsal and ventral dermal cell specification in the mouse and chick embryo, members of the Wnt family are expressed in the entire dorsal surface ectoderm (Cauthen et al., 2001; Parr et al., 1993; Rodriguez-Niedenfuhr et al., 2003; Schubert et al., 2002). Using a transgenic Wnt signaling reporter, we now show that Wnt signaling is transduced in ventral subectodermal cells which include dermal progenitors. The role of Wnt signaling in specification and development of the ventral dermis in the mouse or chick embryo is unknown (Fuchs and Raghavan, 2002; Millar, 2002; Millar, 2005). In this study, we have used two different mouse conditional mutants to identify the role(s) of the Wnt signaling pathway in the development of the ventral trunk dermal cells. Here, we identify multiple roles for Wnt signaling/β-catenin in ventral dermal development. First, Wnt signaling/β-catenin is required for survival of the early dermal progenitors in the LPM. Later in development, Wnt signaling/β-catenin is necessary and sufficient for the specification of ventral dermal progenitors that are derived from the flank and ventral subectodermal mesenchyme. In the conditional absence of Wnt signaling/β-catenin, the ventral dermis fails to develop. Our studies on ventral dermal development reveal a new role for Wnt signaling/β-catenin in cell survival and a conserved role in dermal cell specification.

RESULTS

Wnt/β-catenin signaling is active in ventral dermal progenitors

To identify whether Wnt/β-catenin signaling is active during ventral dermal specification and differentiation, we analyzed TCF/Lef-LacZ transgenic embryos. The TCF/Lef-LacZ transgene is a reporter of endogenous Wnt signaling activity (Mohamed et al., 2004). LacZ expression was present at embryonic day 8.5 (E8.5) in the somatopleure of the LPM and in the subectodermal mesenchyme of the flank at E9.5 and E10.5 (Figure 1A–C). By E11.5, TCF/Lef-LacZ transgene expression was observed in the subectodermal mesenchyme of the ventral midline (Figure 1D). By E14.5, LacZ was expressed in a significant number of dermal cells, evenly around and between hair follicles (Figure 1E, inset). Wnt signaling reporter expression in embryonic skin suggests a role in dermal differentiation and interfollicular dermal development. In our analysis, we observed a similar expression pattern of TCF/Lef-LacZ transgene expression along the anterior-posterior axis till the level of the hind limb (data not shown). Prior to the onset of expression of the earliest dermal progenitor marker, Dermo1 at E11.5 (Figure 6J), cells process Wnt signaling in the dermal progenitors between E8.5 and E11.5 (Figure 1A–D). These results suggest that Wnt signaling may have roles in early and late ventral dermal cell development.

Figure 1. Wnt signaling reporter expression during ventral dermal cell development.

Figure 1

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(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 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 panels F and G with D.

Figure 6. Dermo1 expression in the flank and ventral subectodermal mesenchyme is induced by β-catenin.

Figure 6

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(A–L) Section in situ hybridization of Dermo1 mRNA and alternate sections are stained with X-gal to visualize En1 lineage-marked cells at E10.5 and E11.5 (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 and 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 (black hatched line with arrows). Images are of (A–F) E10.5 and (G–L) E11.5 embryos; and all insets (F, J, L) are of the same magnification. Compare E and F with D, and K and L with J.

Mouse ventral dermal cells originate from the lateral plate mesoderm

We used Cre/loxP tools to conduct lineage analysis of ventral dermal cells. At the forelimb level, the HoxB6Cre transgene drives the expression of Cre recombinase early in the LPM starting at E8.0 and later (Lowe et al., 2000). HoxB6Cre; R26R lineage-marked cells contributed extensively to the ventral dermis by E16.5 (Figure 2A). When we temporally restricted the recombination of R26R by using an inducible HoxB6Cre-ERT1 driver to the LPM tissue between E7.75-E8.75, we found β-gal+ cells dispersed in the flank and ventral mesenchyme by E11.5 and then in the ventral dermis at E17.5 (Figure 2B–D). Lineage-marked cells were also present in the sternum and endothelial cells in blood vessels, within muscle, and adjacent to the sternum. (Figure 2C, E). Endothelial cells were identified by morphology and immunostaining with anti-PECAM antibody (data not shown). In the absence of Tamoxifen, we did not see β-gal expression in these lineages (data not shown). Thus, similar to the chick embryo, the ventral dermis in the mouse embryo originates from the LPM.

Figure 2. Ventral dermal cells originate from the LPM.

Figure 2

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(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). Black hatch lines demarcate epidermis from dermis in the embryonic skin. (D, E) High magnification images of the boxed areas in panel C.

The survival of early dermal progenitors requires β-catenin

To determine the function of Wnt signaling in mouse ventral dermal development, we used the HoxB6Cre driver to genetically alter Wnt signaling activity levels in the LPM and analyzed mutants with a conditional loss of β-catenin function in the flank and ventral subectodermal mesenchyme. By E9.5, HoxB6Cre mediated recombination of R26R is nearly 100% in all the flank mesenchyme cells (Lowe et al., 2000). We used a loss-of-function floxed allele of β-catenin (β-cateninlof) to eliminate Wnt signal transduction in the HoxB6Cre lineage cells of the LPM (Brault et al., 2001). HoxB6Cre; R26R; β-cateninlof mutant embryos lacked a ventral body wall and died between E12.5 and E13.5 (data not shown). We examined the role of β-catenin in cell survival of the early dermal progenitors in the flank mesenchyme. At E9.5, there was no TUNEL staining of the LPM in the control or in the HoxB6Cre; R26R; β-cateninlof mutant (data not shown). By E10.5, we found fewer HoxB6Cre, R26R lineage labeled cells and a significant increase in TUNEL staining of cells in the flank mesenchyme of the β-cateninlof mutant (Figure 3C, D). In comparison, we did not find any TUNEL staining in the flank mesenchyme of the control HoxB6Cre, R26R; β-cateninlof/+ lineage-labeled cells at E10.5 (Figure 3A, B). These data demonstrate that β-catenin is required for the cell survival of early dermal progenitors and perhaps but not necessarily progenitors of other tissues derived from the LPM.

Figure 3. β-catenin activity is required early for cell survival.

Figure 3

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

Ventral dermal cells differentiate from flank and ventral subectodermal mesenchyme

In order to study the function of β-catenin at later stages, we used the endogenous Engrailed1 (En1) promoter to drive the expression of Cre recombinase in the flank and ventral subectodermal mesenchyme at E10.5 and later (Kimmel et al., 2000). En1Cre-mediated recombination of the R26R was seen in essentially all the subectodermal cells of the flank mesenchyme starting at E10.5 (Figure 4A, B, and inset) and in the ventral mesenchyme at the midline by E11.5 (Figure 4C, D, inset). En1Cre-mediated recombination of R26R at E10.5 coincides with the spatio-temporal expression of TCF/Lef-LacZ (Figure 4B and 1C) and precedes the onset of Dermo1 expression at E11.5 (Figure 6J).

Figure 4. Ventral dermal progenitors in the flank mesenchyme express En1 and contribute extensively to ventral dermal cells.

Figure 4

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(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) Wholemount; (B, D) section. Inset (B,D) are lower magnification view of the section. (E) En1 lineaged-marked cells are found extensively in the epidermis and dermis of E16.5 En1Cre; R26R embryos. (F) En1Cre-ER; R26R embryos were given Tamoxifen at E10.5; at E16.5 β–gal+ cells are present only in the dermis (arrows). (E, F)Black dashed line demarcates the epidermis from the dermis.

Next, we examined if En1Cre; R26R cells contribute to the ventral dermis. In E16.5 En1Cre; R26R fetuses, β-galactosidase-labeled cells are present in the entire ventral dermis and epidermis (Figure 4E). To determine if the ventral epidermal and dermal cells are descendants of En1 expressing cells in the ventral trunk prior to E11.5, we temporally restricted the recombination of R26R by using a tamoxifen inducible En1Cre-ERT1 line (Sgaier et al.,2005). To lineage-mark En1 expressing cells in the ventral trunk between E10.75–11.75, we administered Tamoxifen to pregnant females carrying E10.5 En1Cre-ERT1; R26R embryos (Sgaier et al., 2005). At E16.5, β-galactosidase-labeled cells were found throughout the ventral dermis (open arrows, Figure 4F). Our inducible lineage-marking experiments demonstrated that β-galactosidase-labeled cells in the ventral trunk epidermis of En1Cre; R26R embryos must arise as a result of En1 expression in the ventral trunk ectoderm starting at E13.5 (Figure 4E, F, data not shown). Therefore, the early development of mutant dermal progenitors in the conditional En1Cre; β-catenin mutant embryos occured next to normal overlying ventral ectoderm cells up until E13.5. In addition, between E9.5–12.5, we did not see En1LacZ expression in the ventral trunk ectoderm at the forelimb level (data not shown). Taken together, these results suggest that En1Cre cells in the flank and ventral subectodermal mesenchyme at E10.5 include ventral dermal progenitors and contribute extensively to the ventral dermis.

TCF/Lef-LacZ expression confirms efficient Cre mediated removal of β-catenin in the ventral dermal precursors

In subsequent studies, we used the En1Cre line to conditionally delete or stabilize β-catenin in the ventral subectodermal mesenchyme (Brault et al., 2001; Harada et al., 1999; Kimmel et al., 2000). To determine the efficiency of En1Cre-mediated recombination of two floxed alleles of β-catenin (Brault et al., 2001; Harada et al., 1999), we examined TCF/Lef-LacZ expression at E11.5 in mutant embryos. In E11.5 En1Cre; β-catlof embryos, TCF/Lef-LacZ was completely lost in cells of the En1 lineage lacking β-catenin (Figure 1F and inset). TCF/Lef-LacZ expression was present in normal forelimb mesoderm cells that have not expressed En1Cre(Figure 1F, open arrows). Similarly, when the En1Cre line was used to stabilize β-catenin activity in the subectodermal mesenchyme, TCF/Lef-LacZ and nuclear β-catenin were ectopically expressed in the cells of the En1 lineage away from the ectoderm (Figure 1G and inset, 1D, 4D, data not shown) (Harada et al., 1999). By E11.5, En1Cre can be used to efficiently eliminate or stabilize Wnt signaling/β-catenin activity in ventral dermal precursors prior to dermal specification (Figure 6).

β-catenin is required for ventral dermal development

To determine the requirement of Wnt/β-catenin signaling later in ventral dermal development, we used En1Cre to conditionally delete β-catenin activity in ventral dermal precursors. We bred En1Cre, R26R with the floxed β-cateninlof mice to conditionally eliminate Wnt signal transduction and genetically tag the mutant cells. En1Cre; R26R ; β-catlof mutants die at birth. At E16.5, En1Cre; R26R; β-catlof mutants are smaller in size than control embryos, and have more transparent ventral skin through which the liver can be clear view ed (data not shown). The development of epidermally-derived hair follicles is dependent on normal interaction with dermis. In the absence of normal epidermal and dermal interactions, hair follicles fail to form (Atit et al., 2006; Millar, 2002). To obtain an overview of the affected area, we probed control and mutant embryos at E14.5 with antisense mRNA for Patched1, a marker for hair follicle placodes (Oro et al.,1997). At E14.5, Patched1 is expressed in the hair follicle placodes of the control embryo (Figure 5A), but was completely absent in the ventral flank and midline of En1Cre; β-catlof mutant embryos (Figure 5B). In transverse sections of E16.5 En1Cre; R26R control fetus, the epidermis overlies the condensed dermis, ventral muscle and sternum (Figure 5C, E, G). In striking contrast, the En1Cre; β-catlof fetuses lack v entral dermis andmuscle and the epidermis was juxtaposed next to the sternum (Figure 5D, F, H). En1 lineage-marked cells contribute extensively to the sternum and were absent in the adjacent rib cartilage (Figure 5E, I). In the En1Cre; R26R ; β-catlof, we found that the sternum, with lineage-marked cells, extended across the entire ventrum (Figure 5F, H, J). The rib also lacked lineage-marked cells in the mutant embryos (Figure 5F, J). We could not study dermal development in the complementary En1Cre; β-catgof mutants due to embryonic lethality between E12.5 and E13.5. The late stage En1Cre; β-catlof phenotype illustrates that β-catenin/Wnt signaling activity is needed for ventral dermal development.

Figure 5. Ventral hair placodes and dermis are lacking in the absence of β-catenin in the En1 lineage.

Figure 5

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(A) Whole-mount in situ hybridization with a Patched1 (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-cateninlof 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 and is absent from the skin of mutant fetuses (F). (G– J) Higher magnification images of boxed areas in panels 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 bar in C, D is 2.0mm; E, F is 500µm, G is 200µm; and H–J is 200µm.

β catenin is required for ventral dermal cell specification

To identify the molecular mechanism of Wnt/β-catenin signaling in ventral dermal development, we examined dermal specification in the En1Cre conditional loss and gain of β-catenin function mutants. We bred En1Cre, R26R with the floxed β-cateninlof mice to conditionally eliminate Wnt signal transduction and genetically tag the mutant cells prior to ventral dermal specification. Between E10.5–11.5, we assayed dermal specification by the onset of Dermo1 (also known as Twist2) mRNA expression in ventral trunk dermal precursors (Atit et al., 2006; Li et al., 1995). At E10.5, Dermo1 was not expressed in the control flank subectodermal mesenchyme (Figure 6A, D). Dermo1 was normally detectable starting at E11.5 in the En1 lineage-marked flank and ventral subectodermal mesenchyme (Figure 6G, J). In the conditional absence of Wnt signaling/β-catenin activity in the En1 lineage, Dermo1 was not expressed at E10.5 or E11.5 in the flank and ventral subectodermal mesenchyme (Figure 6B, E, H, K). In contrast, conditionally stabilizing β-catenin activity in the En1 lineage leads to induction of Dermo1 mRNA expression earlier at E10.5 (Figure 6C, F) and to ectopic expression in most of the En1Cre; R26R; β-catgof mutant cells at E11.5 (Figure 6I, L). Dermo1 expression in two different β-catenin activity mutants clearly demonstrates that β-catenin activity is required for the onset of ventral dermal progenitor marker expression and dermal specification.

β-catenin regulates cell proliferation of ventral dermal progenitors

Next, we examined the role of β-catenin in cell proliferation and cell survival of ventral dermal progenitor cells at these later stages. Since the specification defect phenotype is evident by E11.5 in both the conditional mutants, we analyzed embryos for alterations in cell proliferation and cell survival at E10.5 and E11.5 (Figure7, and Table 1). We examined cell proliferation by BrdU incorporation. There was no statistically significant difference (P < 0.05) in the cell proliferation index between the control and the En1Cre; R26R; β-catlof and β-catgof at E10.5 (Table 1). At E11.5, we found statistically significant difference in cell proliferation between control and En1Cre; β-catlof embryos only and not with β-catgof mutants (Figure 7A, B, C, Table1). We assayed cell survival by TUNEL and could not find any significant changes in cell survival between control and En1Cre; β-catlof or β-catgof at E10.5 and E11.5 (Figure 7D–F, data not shown).

Figure 7. Small decrease in cell proliferation in the absence of beta-catenin without altering cell survival.

Figure 7

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(A–C) Cell proliferation at E11.5 is assayed by BrdU incorporation (brown stained cells). (A, B) There is a small decrease in cell proliferation in the En1Cre; R26R; β-cateninlof mutants but no significant difference in β-cateningof mutants (See Table1). (D–F) Apoptosis at E11.5 was assayed by TUNEL (brown stained cells) and 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) The En1 lineage-marked domains are outlined in red. (A–F) Composites for each panel were created from 2–3 images taken at the same magnification.

Table 1.

Proliferation of β-Catenin Mutant Cells

E10.5
β-Catenin Genotype Number of Embryos Analyzed En1 Domain Four Cells Adjacent to Ectoderm
BrdU-Positive Cells (%) SD P
Control 7 48.1 13.6 —
Loss of Function 6 52.9 14.1 0.685
Control (AR) 4 54.2 8.4 —
Gain of Function (AR) 6 55.0 8.0 0.126
E11.5
β-Catenin Genotype Number of Embryos Analyzed En1 Domain Four Cells Adjacent to Ectoderm
BrdU-Positive Cells (%) SD P
Control (AR) 5 46.1 6.8 —
Loss of Function (AR) 6 35.0 11.0 0.011
Gain of Function (AR) 5 45.6 10.5 0.887
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AR = Antigen Retrieval protocol

DISCUSSION

In this study, we investigate the origin of mammalian ventral dermis and the signaling cues necessary for the specification and survival of the ventral dermal lineage. Using four different genetic fate-mapping tools, we conclusively show that LPM cells contribute to the flank and ventral body wall mesenchyme, and that subectodermal mesenchymal cells differentiate into dermal cells. Using a Wnt signaling reporter transgenic line, we show that Wnt/β-catenin signaling is active in the somatopleure of the LPM at E8.5, and later in ventral dermal progenitors. We generated conditional β-catenin mutants early in the LPM and later in the flank and ventral mesenchyme. When β-catenin is eliminated early in the LPM between E8.0–9.0, dermal progenitors die by E10.5. However, when β-catenin is removed in the subectodermal mesenchyme between E10.0–11.5, embryos have a dramatic loss of ventral dermis and an expanded sternum by E16.5. We show that the loss of dermis phenotype occurs due to loss in cell fate specification and a small decrease in proliferation. In contrast, when β-catenin activity is stabilized in the flank and ventral mesenchyme, there is ectopic expression of dermal progenitor marker, Dermo1, in the lineage-labeled cells. Our results in the mouse embryo conclusively demonstrate that Wnt signaling/β-catenin plays multiple roles during mammalian ventral dermal development.

Lineage mapping the ventral dermis in the mouse embryo

We use genetic tools to lineage map the mammaliam ventral dermis from the LPM. We have taken advantage of two different Cre and inducible Cre-ER alleles to improve the accuracy of our lineage analysis. First, we use the HoxB6Cre transgenic line that uses the LPM and limb enhancer region of the HoxB6 promoter to drive Cre or Cre-ER expression in the lateral LPM starting at E8.0 (Lowe et al., 2000). Consistent with the fate maps from the chick embryo, we find lineage-labeled cells in known derivatives of the LPM in ventral trunk tissues such as the body wall mesenchyme at E11.5 and later in the dermis, sternum, and endothelial cells by E16.5. As expected, we do not see lineage-labeled cells in the ribs derived from the somites or in the epidermis, which differentiates from the surface ectoderm. We confirmed our ventral dermis fate map by using the En1Cre and En1Cre-ERT1 lines, which drive expression later at E10.0 in the flank and ventral mesenchyme. We find β-gal+ cells extensively in the ventral dermis, dermal papillae of the hair follicles, and the sternum. Our results from multiple genetic lines and strategies demonstrate clearly that LPM cells contribute to the body wall mesenchyme by E11.5, and then later to ventral dermis in the mouse embryo. Our fate mapping results have aided our analysis of the β-catenin conditional mutant phenotypes.

Multiple roles for Wnt signaling/β-catenin in ventral dermal cell development

Wnt signaling reporter activity is evident in the somatopleure of the LPM starting at E8.5 and then in the subectodermal mesenchyme of the flank and ventrum. The Wnt signaling reporter activity is consistent with the known expression of multiple Wnt ligands in the surface ectoderm of the mouse embryo (Cauthen et al., 2001; Parr et al., 1993), and with signaling to the mesenchyme cells directly below. These subectodermal mesenchyme cells process the Wnt signal and differentiate to ventral dermis. The additional lineage-marked cells located away from the surface ectoderm must differentiate into other cell types. Our complementary results from mutants with conditional loss and gain of β-catenin function, clearly demonstrate that β-catenin has a role early in cell survival and later in the cell specification of ventral dermal progenitors. In this study, by eliminating β-catenin early in the LPM, we found a role for β-catenin in early cell survival before E10.5. When we eliminate β-catenin after E10.0, the subectodermal cells survive but fail to be specified to the ventral dermal cell fate by E11.5. In our previous studies on dorsal dermis, we eliminated β-catenin early in the multipotential progenitors of the somite and demonstrated that β-catenin has an instructive role in dermal cell fate from the somite, but we did not see a role in the cell survival of dermal progenitors (Atit et al., 2006). In this study, we found that β-catenin has a new role in ventral dermal progenitor cell survival while maintaining a functionally conserved role in dorsal and ventral dermal cell fate selection.

Our fate mapping results demonstrate that we have marked cells that originate in the LPM and that contribute to sternal cartilage, ventral dermal, and endothelial cell lineages. The En1Cre; R26R; β-cateninlof mutant shows the absence of ventral dermal specification and a significant expansion of the sternum. The sternum, containing lineage-labeled cells, traverses the width of the embryo. It is not clear whether the mutant ventral dermal progenitors are respecified to sternum fate. The small decrease in cell proliferation could account for a decrease in dermal progenitor population. Taken together with the absence of cell death, some of the dermal progenitors may also be respecified to the sternum cell fate.

It still remains to be determined how Wnt signal transduction instructs subectodermal mesenchyme in the flank and trunk to progress towards the ventral dermal cell fate. Wnt signaling/β-catenin activity is critical for Dermo1 gene expression in dorsal and ventral dermal progenitors, and Dermo1 may be the direct target gene to promote dermal cell fate (Atit et al., 2006, and Figure 6). Identifying the additional downstream target genes and defining the genetic program for dermal cell development are the next steps in understanding dermal cell differentiation.

MATERIALS AND METHODS

Mice and Genotyping

TCF/Lef-LacZ reporter transgenic embryos were used to monitor Wnt pathway activity (Mohamed et al., 2004) (obtained from Daniel Dufort). HoxB6Cre (Lowe et al., 2000) and En1Cre mice (Kimmel et al., 2000) (obtained from Michael Keuhn and Alexandra Joyner, respectively) were crossed to the R26R mice (purchased from Jackson Laboratories) to determine the efficiency of Cre mediated recombination (Soriano, 1999). To temporally restrict the recombination of R26R, HoxB6Cre-ERT1(obtained from Susan Mackem) and En1CreERT1 (Sgaier et al., 2005) (obtained from Alexandra Joyner) mice were used. Tamoxifen was administered by intraperitoneal (IP) injection (1mg/40g mouse) of pregnant females carrying E7.5 HoxB6Cre-ERT1; R26R embryos. Females carrying En1Cre-ERT1; R26R embryos were given 3mg/40gm of Tamoxifen by gavage at E10.5. For deletion of β-catenin in the ventral dermal progenitors, HoxB6Cre; R26R or En1Cre; R26R mice were crossed with mice carrying an exon 2–6 floxed allele of β-catenin (β-catlof) (Brault et al., 2001) (purchased from Jacskon Laboratories). For activation of β-catenin in ventral dermal progenitors, En1Cre; R26R mice were crossed with mice carrying an exon 3 floxed allele of β-catenin (β -catgof) (Harada et al., 1999) (obtained from Makoto M. Taketo). To determine the efficiency of Wnt pathway activation and inactivation, TCF/Lef-LacZ; En1Cre mice were crossed with β -catlof or β -catgof mice. All the mice and embryos were genotyped as previously described. For each experiment, five to eight embryos from 2–3 litters were analyzed. All mouse experiments were done according to protocols approved by Case Western Reserve University IACUC committee.

In situ hybridization, immunohistochemistry, and histology

Tissue preparation, histology, immunohistochemistry, LacZ expression, and in situ hybridization with digoxygenin-labeled probes were performed as previously described (Atit et al., 2006). Dermo1 probe was a gift from Eric Olson (Li et al., 1995). Antibodies against β–catenin (mouse 1:1000; Sigma), and BrdU (mouse, 1:8; Roche) were used. Appropriate secondary antibodies conjugated to biotin (1:250; Vector Labs, Jackson Immunoresearch) were used. For β–catenin immunostaining, antigen retrieval was performed on paraffin sections by boiling for10 minutes in citrate buffer, followed by application of reagents from the M.O.M kit, and overnight incubation with the primary antibody (Vector Labs). M.O.M kit reagents were used as described by the manufacturer. Brightfield images were captured using the Olympus BX60 microscope and Olympus DP70 digital camera using DC Controller software. Whole mount embryo images were captured using a Leica MZ16F microscope, and a SPOT camera system, and software. Images were processed using Adobe Photoshop and Macromedia Freehand.

Cell proliferation and survival studies

Embryos were collected and processed for proliferation and survival studies as previously described (Atit et al., 2006). To assess cell proliferation in embryos, BrdU incorporation was detected by immunohistochemistry and quantified as previously described (Atit et al., 2006). In addition, antigen retrieval was performed by boiling for 10 minutes in citrate buffer prior to the application of primary antibody. Statistical significance (P > 0.05) was determined by conducting the Student-t-test. Analysis was restricted to En1 lineage-marked cells in the flank and ventral subectodermal mesenchyme. Cell survival was assayed by brightfield TUNEL staining on cryosections as previously described (Gavrieli et al., 1992), before counterstaining with 2% methyl green for two minutes at room temperature.

Acknowledgements

We thank Makoto M. Taketo, Ozimba Anyangawe, Hilary Michel, Andrew Jarrell, and Sema Sagier for their contributions. We thank present members of the lab and Ron Conlon and Kathleen Molyneaux for critically reading this manuscript. This work was supported by Startup funds from Case Western Reserve University (R.A.), the Basil O’Connor Junior Investigator Award from the March of Dimes Foundations (R.A.), and Case Western Reserve University SOURCE Program (J.O., P.S.).

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