Distinct Requirements for Cranial Ectoderm and Mesenchyme-Derived Wnts in Specification and Differentiation of Osteoblast and Dermal Progenitors

Summary

The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

Introduction

The bones of the skull vault develop in close contact with the embryonic skin to enclose the brain. In the mouse embryo, both bone-forming osteoblasts and skin-forming dermal fibroblasts are derived from cranial neural crest and paraxial mesoderm [1]. At E11.5, cranial dermal fibroblast progenitors undergo specification beneath the surface ectoderm while osteoblast progenitors are specified in a deeper layer of cranial mesenchyme above the eye [2]–[4]. Subsequently, osteoblast progenitors proliferate and migrate apically beneath the dermal progenitors [1], [4]. Both cell types secrete collagen as extracellular matrix, but skull bones provide physical protection for the brain, while the overlying dermis lends integrity to the skin and houses the epidermal appendages [5].

Both paracrine and autocrine intercellular signals function in early bone and skin development. In craniofacial bone formation the mesenchyme sets the timing of ossification [6], [7], while the surface ectoderm functions in a permissive manner [8]. Likewise, during skin formation ectodermal signals are essential for formation of the trunk hair-follicle forming dermis [9], [10], but the cranial dermal mesenchyme determines epidermal appendage identity such as hair or feather [11]. Further delineation of specific ectoderm-mesenchyme signaling during early development of the bone and dermis is required to overcome challenges in the engineering of replacement connective tissues.

Mesenchymal canonical Wnt/β-catenin signal transduction is essential in the specification and morphogenesis of both craniofacial dermis and bone [2], [3], [12]–[15], and dysregulation in components of such signaling pathways is associated with diseases of bone and skin [1], [2], [16]–[18]. Wntless (Wls) functions specifically in trafficking of Wnt ligands and is required for the efficient secretion of Wnt ligands. [2]–[4], [19]–[28]. Genetic deletion of Wls in mice is likely to dramatically reduce the levels of active Wnt ligands and can recapitulate phenotypes obtained by genetic ablation of Wnt ligands in mice [1], [4], [29]. Wnt ligand binding to target cell surface receptors (Fzd and LRP5/6) results in nuclear translocation of β-catenin, which binds to TCF/LEF transcription factors and activates expression of downstream targets. Certain Wnt ligands also activate the non-canonical Wnt/Planar Cell Polarity (PCP) pathway, which influences cellular movements [5], [30], [31]. β-catenin is essential in osteoblast differentiation and inhibition of chondrogenesis [6], [7], [12]–[14]; however, deletion of individual Wnt ligands resulted only in mild effects on bone differentiation [8], [32], [33]. β-catenin is also a central regulator of early dermal specification [3], [9], [10], [34], [35], and roles for Wnt ligands so far have only been directly shown later during hair follicle initiation [9], [11], [36]. In bone and skin development, redundant functions of multiple Wnts may compensate for deletion of individual ligands. Conventional knockouts of individual ligands removed Wnt expression from all cells in the embryo, and have confounded the identification of tissue sources of Wnt ligands in bone and skin development. Thus, the relative contributions from different sources of Wnt ligands for fate selection in cranial mesenchyme remain unknown.

Previous limitations were the lack of genetic tools to spatiotemporally manipulate early surface ectoderm and mesenchyme, and an inability to circumvent the intrinsic redundancy of Wnt ligands. We took a conditional approach to ablate the efficient secretion of Wnt ligands from either surface ectoderm or cranial mesenchyme prior to fate selection of the cranial bone and dermal lineages. Our findings provide key insights into how local developmental signals are utilized during morphogenesis to generate the cranial bone and dermal lineages.

Results

We found that the genes for most Wnt ligands were expressed in the cranial mesenchyme (Figure 1A) and surface ectoderm (Figure 1B) during the specification of two separate lineages such as cranial osteoblast and dermal fibroblasts in E12.5 mouse embryos (Figure S1, S7, Table 1). To identify the cells with the potential to secrete Wnt ligands, we examined the spatiotemporal expression of Wls, the Wnt ligand trafficking regulator. We detected Wls protein expression from E11.5-E12.5 in the cranial surface ectoderm and in the underlying mesenchyme (Figure 1C, G). Both the Runx2-expressing cranial bone progenitor domain and the Dermo1/Twist2-expressing dermal progenitor domain expressed Wls [3], [37] (Figure 1C, D, E, G). Wnt signaling activation was also visualized in the cranial ectoderm, bone and dermal progenitors by expression of target gene, Lef1 and nuclear localized β-catenin (Figure 1D, F, H, I). During specification of cranial bone and dermis, ectodermal and mesenchymal tissues secreted Wnt ligands, and the dermal and bone progenitors actively transduced Wnt signaling via β-catenin (Figure 1J).

To dissect the requirements of ectodermal and mesenchymal Wnt signals, we generated mutant mice with conditional deletion of Wls [38] in the early surface ectoderm using Crect [39] and in the whole cranial mesenchyme using Dermo1Cre [40]. Crect efficiently recombined the Rosa26 LacZ Reporter (RR) in the cranial ectoderm by E11.5 (Figure S4K), but left Wls protein expression intact in the mesenchyme (Figure 2A, E, B, F) [41]. Dermo1Cre recombination showed β-galactosidase activity and Wls deletion restricted to the cranial mesenchyme and meningeal progenitors at E12.5, and Wls protein was still expressed in the ectoderm in mutants (Figure 2C, D, G, H).

First, we compared the extent to which Wls deletion from ectoderm or mesenchyme affected formation of the craniofacial skeleton. E18.5 Crect; RR; Wls ^fl/fl mutant embryos, which experienced perinatal lethality, demonstrated a hypoplastic face with no recognizable upper or lower jaw most likely due to decrease in cell survival of branchial arch mesenchyme (Figure S5). In the remaining tissue, facial mesenchyme patterning was grossly comparable to controls for most of the markers examined (Figure S5). Notably, the mutants showed no sign of mineralization in the skull vault (Figure 2I–L). The later deletion of Wls from the ectoderm using the Keratin14Cre line resulted in comparable skull bone ossification as controls (Figure S2). Dermo1Cre; RR; Wls ^fl/fl mutant embryos exhibited lethality after E15.5, which precluded assessment of skeletogenesis by whole-mount. We generated En1Cre/+; RR; Wls ^fl/fl mutants, using a Cre that recombines in early cranial mesenchyme but lacks activity in meningeal progenitors (Figure S3 E′, F′) [3]. En1Cre/+; RR; Wls ^fl/fl mutants survived until birth, and demonstrated reduced bone differentiation and mineralization (Figure S3) as well as intact dermis in the supraorbital region with hair follicles (Figure S3). The more severe arrest in Crect; RR; Wls ^fl/fl mutants (Figure 2) suggested ectoderm Wls appears to play an earlier role than mesenchymal Wls in cranial development.

We next examined the effects of ectoderm or mesenchyme Wls deletion on cranial bone and dermal development by histology. We found Von Kossa staining for bone mineral was absent in Crect; RR; Wls ^fl/fl mutants (Figure 3A, B). The thin domain of mesenchyme above the eye in mutants appeared undifferentiated and showed no condensing dermal cells or early stage hair follicles. Additionally, the baso-apical expansion of both dermis and bone was evident by E15.5 in controls, but not in the thin cranial mesenchyme of mutants (Figure 3A–B red arrowhead). Although ossification was absent, we observed the presence of thin nodules of ectopic, alcian blue-stained cartilage (Figure 3E–F). Therefore the result of Wls deletion in the ectoderm was an absence of skull ossification and hair-inducing dermis, a failure of baso-apical expansion of mesenchyme, and the presence of ectopic chondrocyte differentiation. By comparison, Dermo1Cre; RR; Wls ^fl/fl mutants showed a reduction in mineralized bone (Figure 3C–D) without ectopic cartilage formation (Figure 3 G–H). The mutant mesenchyme nonetheless condensed and formed sufficient hair-follicle generating dermis in the supraorbital region to support the supraorbital vibrissae hair follicle and fewer primary guard hair follicles (Figure 3 C, D, C′, D′, black arrowheads). Compared to the control apical region of the head, the mutant lacked sufficient condensed dermal layer to support normal number and differentiation of hair follicles (Fig. 3 C″, D″). Reduced mineralization without ectopic chondrogenesis as well as hair-follicle formation were also present in En1Cre/+; Wls ^fl/fl mutants (Figure S3). Our data suggest that Wls deletion using the Dermo1Cre resulted in diminished bone mineralization with thinner dermis and fewer hair follicles.

Deletion of Wls from the ectoderm resulted in complete absence of skull vault mineralization with failure of dermis formation, pointing to early defects in formation of the two lineages. Therefore we tested if cranial mesenchyme undergoes proper patterning, fate selection, and differentiation in the absence of Wls. Msx2 and Dlx5 that are early markers of skeletogenic patterning in cranial mesenchyme were expressed in Crect; Wls ^fl/fl mutants (Figures 4A, H, S4). The number of Msx2⁺ progenitor cells was not significantly different in controls and mutants (191±9.4 in controls and 206±24 in mutants, P-value = 0.23). However, few Runx2⁺ osteoblast progenitors formed in Crect; RR; Wls ^fl/fl mutant embryos, and expression shifted directly beneath the surface ectoderm (Figure 4B, I). During subsequent differentiation, condensing osteoblast progenitors express alkaline phosphatase (AP; Figure 4C, S4), but ectoderm Wnt-secretion deficient embryos lacked AP activity entirely (Figure 4J, S4). Markers of early osteoblast progenitors from other signaling pathways, Bmp4 and PTHrP (Figure 4D–E, K–L) were also absent in mutants, suggesting an arrest in osteoblast progenitor differentiation. The block was persistent as committed osteoblast progenitors expressing Osx were present in controls but not mutants (Figure 4F, M). Cell survival was not affected in the cranial mesenchyme prior to changes in marker expression (Figure S4A–D). We did not find significant difference in cell proliferation of the underlying mesenchyme (47%±4 in controls and 51%±2; P-value = 0.12). Whereas chondrocytes expressed Sox9 only at the skull base in controls, in mutants, ectopic Sox9-expressing chondrocyte progenitors and cartilage formed within the frontal bone domain (Figure 4G, N, Q, U). In spite of the effect of ectoderm-Wls deletion on mesenchyme, surface ectoderm expression of the differentiation marker, Keratin 14 (K14) was unaffected (Figure S4E,F). Next, we examined formation of dermal fibroblast progenitors in Crect; RR; Wls ^fl/fl mutant embryos. Cranial dermal fibroblast progenitors expressed the markers, Twist2 [3], [37] and Insulin Growth factor 2 (IGF2) by E12.5 in supraorbital mesenchyme (Figure 4O, P), but mutant embryos lacked Twist2 and IGF2 expression (Figure 4S, T). Twist2 expression became more progressively restricted to upper dermal fibroblasts during differentiation in controls, but was completely absent from cranial supraorbital mesenchyme of mutants (Figure 4 R, V). The altered cell fate marker expression at E12.5 (Figure 4, S4 I, J) immediately after deletion of ectoderm Wls (Figure S4K) was indicative of primary defects in mesenchymal cell fate selection. Together, our data suggest ectoderm Wnts form a non-cell autonomous inductive signal to the underlying mesenchyme for specification of osteoblast and dermal fibroblast progenitors, and for repression of chondrogenesis.

Next, we determined if mesenchyme Wls deletion resulted in a later defect in differentiation of cranial bone and dermal fibroblast progenitors. In En1Cre; RR; Wls ^fl/fl mutants, Runx2 expression in osteoblast progenitors was intact without ectopic Sox9 expression, but showed diminished expression of the skeletal differentiation marker, Osx and ossification (Figure S3). Wnt responsiveness by Axin2 expression was comparable in control and mutant cranial mesenchyme at E14.5 (Figure S3). In Dermo1Cre; RR; Wls ^fl/fl mutants, Runx2 expression was also unaffected during fate selection stages (Figure 5A, G, B, H). However, during later osteoblast progenitor differentiation (E15.5), Osx was diminished in mutants at E15.5 (Figure 5C, I). In dermal progenitors undergoing specification, Twist2 expression was unaffected (Figure 5D,J), and surface ectoderm differentiation marker, K14, was appropriately expressed (Figure S6C, D). Additionally at later stages in the mutant, we observed thinner dermis, which was sufficient to support initiation of fewer guard hair follicles (data not shown) and supraorbital vibrissae hair follicle formation (Figs. 3C, D; 5E, K). Furthermore, no ectopic expression of Sox9 occurred in mesenchyme Wls-deficient mutants (Figs. 5F, L). Deletion of mesenchyme-Wls did not lead to decrease in cell survival as monitored by expression of activated-Caspase3 (Figure S6A–B). Prior to E15.5, cell proliferation of osteoblast, dermal, and surface ectoderm progenitors was not significantly different from controls (Figure S6). Based on Dermo1Cre- and En1Cre- deletion of Wls, mesenchyme-derived Wnt ligands are not required for differentiation of dermal progenitors but are indispensable for later differentiation of osteoblast progenitors. Next, we tested the spatiotemporal requirement for mesenchyme Wls in Wnt signaling transduction. Nuclear β-catenin and Axin2 expression were comparable in the mesenchyme of mutants during fate selection stages at E12.5 (Figure 5M, N, Q, R). As differentiation occurs, expression of Axin2 and Lef1 was selectively diminished in the osteoblast progenitor domain of mesenchyme-Wls mutants compared to the controls (Figure 5O, P, S, T). Thus, mesenchyme Wnt ligands appeared to be important in mesenchyme Wnt signal transduction during osteoblast differentiation and ossification as opposed to earlier lineage specification events.

Next, we examined the source of Wnts for the onset of Wnt responsiveness in the mesenchyme. During dermal and osteoblast progenitor cell fate selection, Wnt ligands, inhibitors, and target genes are expressed in spatially segregated patterns. Wnt10a and Wnt7b were expressed in surface ectoderm (Figure 6A–B), Wnt11 was expressed in sub-ectodermal mesenchyme (Figure 6C), and Wnt16 mRNA was expressed in medial mesenchyme (Figure 6D). Notably, the soluble Wnt inhibitor, Dickkopf2 (Dkk2) mRNA was localized to the deepest mesenchyme overlapping with cranial bone progenitors (Figure 6E). Wnt ligands can induce nuclear translocation of β-catenin in a dose-dependent manner leading to the expression of early target genes [42], [43]. At E11.5, expression of nuclear β-catenin was present in both dermal and osteoblast progenitors, and the highest intensity of nuclear localization was found in the surface ectoderm and dermal mesenchyme (Figure 1F). Wnt target genes Lef1, Axin2, and TCF4 were patterned in partially complementary domains. Expression of Tcf4 protein was visible in the skeletogenic mesenchyme (Figure 6F). Tcf4 expression expanded into the mesenchyme under the ectoderm in ectoderm Wls-deficient mutants (Figure 6I–J) and was diminished in mesenchyme Wls-deficient mutants compared to controls (Figure 6K–L). Lef1 and Axin2 were expressed at the highest intensity in the dermal progenitors beneath the ectoderm (Figure 6 G, H). At E12.5, Lef1 expression was completely abolished in the mesenchyme of ectoderm-Wls mutants, but was comparable to controls in the absence of mesenchyme-Wls (Figure 6M–P). The onset of Wnt signaling response in the mesenchyme as measured by Lef1, Axin2, and nuclear β-catenin expression (Figure 6O–T) required ectoderm Wls. By contrast, no single tissue source of Wnt ligands was required to maintain TCF4 expression.

Finally, we tested whether cranial surface ectoderm Wnt ligands regulate the onset of Wnt ligand mRNA expression in the underlying mesenchyme (Figure 7). The non-canonical ligands Wnt5a and Wnt11 were expressed in cranial mesenchyme, with the highest expression corresponding to dermal progenitors. Wnt4, which signals in canonical or non-canonical pathways [44], was expressed strongly in dermal progenitors, as well as in osteoblast progenitors and in the skull base (Figure 7A–C). Wnt3a and 16, which signal in the canonical pathway via β-catenin and have roles in intramembranous bone formation, were expressed medially in the cranial mesenchyme containing cranial bone progenitors (Figure 7D, E) [12]–[14], [45]. Expression of Wnt5a Wnt11, Wnt3a, Wnt16 mRNAs was absent from the mesenchyme of Crect; RR; Wls ^fl/fl mutants whereas some Wnt4 expression was maintained (Fig. 7F–J). En1Cre deletion of β-catenin in the cranial mesenchyme [12] also resulted in an absence of Wnt5a and Wnt11 expression, except in a small portion of supraorbital lineage-labeled mesenchyme, suggesting a phenocopy of Crect;Wls mutants (Figure 7K, L, M). In contrast, Wnt5a, Wnt11, and Wnt4 expression were present in the Dermo1Cre; RR; Wls^fl/fl mutants (Figure 7N–S). However, the Wnt-expressing domains were smaller and only located close to the surface ectoderm, but nonetheless were lineage-labeled (Figure 7E–G, L–N; not shown). Thus, consistent with a role as initiating factors, ectoderm Wnt ligands and mesenchyme β-catenin were required for expression of certain Wnt ligands in the cranial mesenchyme during lineage selection. Mesenchymal Wnt ligands may in turn be required later for osteoblast differentiation (Figure 7T).

Discussion

Here we obtained data suggesting that ectodermal and mesenchymal Wnts function distinctly in early dermal and osteoblast progenitor specification and differentiation. Wnt ligands are expressed in the cranial surface ectoderm and mesenchyme, and ectoderm Wnts are required to generate an inductive cue for the specification of multiple lineages in the cranial mesenchyme. The dermal progenitors and osteoblast progenitors closest to the ectoderm experience the highest concentrations of nuclear β-catenin, in response to Wnt ligands from overlying ectoderm. Subsequent differentiation of osteoblast and dermal fibroblast progenitors requires Wls from the mesenchyme. Thus our study demonstrates that two different sources of Wnt signals coordinate to form two separate lineages, bone and dermis.

We present evidence to demonstrate that ectoderm Wnts generate an inductive cue of Wnt signaling in the mesenchyme to specify cranial bone and dermal lineages. The mechanism remains elusive; however, there are at least three possible models. First, the spatial segregation of Wnt pathway transcription cofactors such as Lef1 and TCF4, partially by lineage, provides a mechanism to generate different lineage programs. Second, a threshold-dependent model may also exist to generate multiple lineages from the same signal. At E11.5–12.5, dermal progenitors are closest to the ectoderm Wnt source and exhibit the highest Wnt signaling reporter activity and markers induced by constitutive activation of β-catenin in mesenchyme (Figure 1) [3], [9], [46]. High levels of Wnt pathway activity preclude osteoblast marker expression in the mesenchyme [12]. Consistently, osteoblast progenitors are present farther away from the ectoderm in an overlapping domain to at least one Wnt inhibitor, Dkk2 [47] (Figure 6E). Finally, the osteoblast response to ectodermal Wnts may be indirect; osteoblast progenitors may require a separate signal relayed from dermal progenitors. Future genetic experiments with new reagents will be required to distinguish between these models and test direct or indirect requirements of Wnt sources in osteoblast and dermis formation.

During fate selection of cranial dermal and osteoblast progenitors, upstream ectodermal Wnt ligands initiate expression of a subset of mesenchymal Wnt ligands via β-catenin. Ectoderm Wnts also act upstream of mesenchyme Wnts in mouse limb development [48]. Here, ectoderm Wnts act in a temporally earlier role than mesenchyme Wnts, and other studies support a direct relationship. In at least one instance, mesenchyme Wnt ligands are direct targets of canonical Wnt signaling [49]. Alternatively, ectoderm and mesenchyme Wnts may signal in parallel pathways to the mesenchyme. The signal that acts upstream to initiate Wnt ligand expression in the cranial ectoderm remains unknown.

We report here that osteoblast differentiation requires distinct Wnt signals from surface ectoderm and mesenchyme. β-catenin deletion in the ectoderm did not inhibit skull bone mineralization [39], so autocrine effects of Wls deletion on the ectoderm were unlikely to contribute to the skull phenotype. However, removal of surface ectoderm Wls resulted in ectopic chondrogenesis (Figure 3), which phenocopied mesenchymal β-catenin deletion [12]. In contrast, mesenchymal Wls deletion did not result in ectopic cartilage formation, suggesting repression of chondrogenesis in cranial mesenchyme requires an early, ectoderm Wnt signal. Our results thus implicate β-catenin here as a Wnt pathway factor that acts in the nucleus to repress chondrogenesis and functions downstream of ectoderm ligands. Ectoderm Wnt ligands thus provide an inductive cue acting on osteoblast progenitors while the cells are closest to the ectoderm. Indeed, later deletion of Wls from the ectoderm using the K14Cre line did not give rise to a skull bone ossification phenotype (Figure S2). During osteoblast progenitor differentiation, Wls deletion with Dermo1Cre resulted in a similar but more severe differentiation arrest than the more restricted En1Cre. Consistently, using a different Wls mutant allele, deletion of mesenchymal Wnts led to absence of osteoblast differentiation expression and reduced cell proliferation [50]. We show that the mesenchyme Wnts maintain the differentiation process but require an inductive ectoderm Wnt signal.

We demonstrate that dermal progenitors require ectodermal Wls for specification and mesenchymal Wls for normal differentiation (Figs. 4–5). Cranial dermal progenitors located beneath the ectoderm require β-catenin for specification [3], but the tissue contribution of Wnt sources remained previously undetermined. Here, a mesenchymal Wls source is indispensable in the dermal lineage for normal differentiation, thickness, and hair follicle patterning. Previous reports in murine trunk skin development suggested that ectoderm Wnts alone are essential in hair follicle induction [9], [10]. Differential requirements may exist for mesoderm-derived trunk dermal progenitors and cranial neural crest-derived dermal progenitors. Future studies will be needed to uncover the requirements for a mesenchymal Wnt signal in dermal fibroblast differentiation in different parts of the embryo.

Conditional Wls deletion resulted in a failure of cranial dermal and osteoblast progenitors to undergo baso-apical extension (Figure 3), a process that occurs independently of β-catenin [12]. Since Wls deletion blocked secretion of canonical and non-canonical Wnt ligands, extension defects in the mesenchyme are consistent with known roles for non-canonical Wnt ligands in orienting cell movements [51]. Homozygous null mutants of core planar cell polarity (PCP) components lacked proper skull tissue development and neural tube closure [52]. However, mutants for individual non-canonical Wnt ligands lack a cranial PCP phenotype. In the cranial mesenchyme, non-canonical Wnt5a or Wnt11 ligands were expressed in overlapping expression domains, suggesting the ligands function redundantly [53] (Figure 7). Therefore, the role of PCP signaling remains to be rigorously tested in conditional mutant mice. The non-canonical and canonical Wnt signaling pathways interact extensively. In our study, canonical β-catenin transduction, in response to ectodermal Wnts, initiates non-canonical Wnt ligand expression (Figure 7), consistent with reports from other systems [30], [49], [51]. Our results reinforce the role of non-canonical Wnt ligands in the pathogenesis of craniofacial anomalies [54], [55]. The ability of exogenous non-canonical Wnts to compensate for Wls deletion in the baso-apical extension of dermal and osteoblast progenitors remains to be tested.

Our results from tissue-specific deletion of Wls have implications in diseases with dysregulation of dermal fibroblasts or osteoblasts, and in understanding the pathogenesis of craniofacial birth defects. Removal of Wls from the ectoderm by E12.5 of mouse development reveals a default state for formation of cartilage in the cranial skeleton and dermis if all Wnt secretion were absent from the ectoderm. This forms an important baseline state that can be used to interpret less severe genetic conditions resulting from loss or mutation of individual Wnt ligands. In this respect, we hypothesize that mutations in the Wnt secretory pathway may underlie diseases of osteoblasts, and dermal fibroblasts, warranting continued investigation into the role of Wnt production in bone and skin formation and homeostasis [15], [17], [18], [45], [56]–[58]. Understanding the signals surrounding osteoblast and dermal fibroblast formation is crucial to meet the demands of engineering appropriate connective tissues.

Materials and Methods

Mice and genotyping

Conditional functional studies were conducted using Crect, Keratin 14Cre; Dermo1Cre, En1Cre, β-catenin deleted, conditional β-catenin floxed mice [39], [40], [59]–[62]. Mice and embryos were genotyped as described previously. The conditional loss-of-function floxed allele for Wls (Wls^fl/fl) was described previously [38]. RR/RR mice harboring a LacZ transgene downstream of a floxed stop transcription signal in the ubiquitous Rosa26 locus were obtained for lineage tracing [41]. For timed matings the vaginal plug day was assigned as E0.5. At desired time points, embryos were harvested and processed for frozen sections as previously described [34]. For each experiment, at least three to five different mutants with littermate controls from 2–3 litters were analyzed. At least three to five litters were used for all analyses. Case Western Reserve Institutional Animal Care and Use Committee approved all animal procedures.

In situ hybridization, immunohistochemistry, and histology

Embryos were fixed in 4%PFA, cryopreserved, and sectioned at 8–12 µm. In situ hybridization, β-galactosidase with eosin counter-staining, and immunohistochemistry were performed essentially as described [34], [35]. Alcian blue staining of sections was performed as described. For Von Kossa staining of frozen sections, slides were fixed with 4% PFA, incubated in the dark with 2% silver nitrate, rinsed, exposed to light, and counterstained with eosin. In situ probes for Twist2 (Eric Olson, Dallas, TX), Pthrp, Wnt4 (V. Lefebvre, Cleveland, OH), Wnt5a (Andrew McMahon, Boston, MA), Wnt11 (Steve Potter, Cincinnati, OH), Axin2 (Brian Bai), BMP4, Wnt7b, Dlx5 (Gail Martin, San Francisco, CA), Wnt16 (Yingzi Yang, Bethesda, MD) and Osx (Matthew Warmann, Boston, MA) were gifts. For Wnt10a, cDNA was amplified from E12.5 RNA using primer F: GCTATTTAGGTGACACTATAGGCGCTCTGGGTAAACTGAAG, primer R: TTGTAATACGACTCACTATAGGGAGAGCCAACCACCTCTCTCA, and in vitro transcription of antisense mRNA with T7 polymerase. For Dkk2, PCR primers DKK2-F(5′-GACATGAAGGAGACCCATGCCTACG-3′ and DKK2-T7R 5′-TGTAATACGACTCACTATAGGGCATAGATGAGGCACATAACGGAAG-3′ were used.

Primary antibodies for Runx2, Sox9, Twist2, Lef1, Osx, Msx2, Ki67, IGF2, Wls, and β-catenin (goat anti-Runx2; 1∶20, R&D Biosystems; rabbit anti-Sox9; 1∶100; Millipore; mouse anti-Twist2, 1∶500, Santa Cruz; rabbit anti-Lef1, 1∶100, Abcam; rabbit anti-Osx, 1∶400, Abcam; mouse Msx1/2, 1∶50, DSHB; activated Caspase3, 1∶250, Abcam; rabbit Ki67; 1∶500 Abcam; rabbit IGF2 1∶400, Cell Signaling); rabbit anti-Wls, 1∶2000, gift from Richard Lang; mouse β-catenin 1∶100 BD Biosciences) were used for indirect immunofluorescence and immunohistochemistry. All control/mutant pairs were photographed at the same magnification. Number of Msx2⁺ cells was counted from a fixed field in 10 different sections from 4 embryos.

Proliferation index was assessed by percent of cells with Ki67 expression in the Runx2 expression domain, in the dermal mesenchyme in the Twist2 domain, and surface ectoderm in the Keratin14 expressing cells. Similar numbers of cells in each domain were analyzed between four controls and mutants. Statistical significance for all quantifications was calculated using two-tailed Student t-test.

Alcian blue and Alizarin red and AP staining

Embryos were sacrificed, skinned and eviscerated, fixed in 95% ethanol, then stained for 24 hours each in 0.03% Alcian blue and 0.005% Alizarin red. Stained embryos were subsequently cleared in graded series of potassium hydroxide and glycerol until photography, after which they were stored in 0.02% Sodium Azide in glycerol. Whole mount Alkaline phosphatase staining was performed as previously described [63] with the addition of a 70% ethanol overnight incubation step after fixation in 4% PFA.

RT-PCR

Cranial mesenchyme and surface ectoderm were micro-dissected from E12.5 embryos and flash frozen in liquid nitrogen. Total RNA was isolated using the Qiagen RNEasy micro kit, and cDNA was reverse transcribed using the ABI kit. RT-PCR for most of the Wnt ligands was amplified for 35 cycles of 94°C for 15 seconds, 66°C for 30 seconds, and 72°C for 60 seconds and the products were resolved on a 3% agarose gel. For Wnt1, 5b, 8a, 8b, 10b the annealing temperature was 55°C for 30 seconds. Primer sequences for RT-PCR are listed in Table 1.

Supporting Information

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