Hartmann Group Research
Functions of Wnt Signaling in Formation and Patterning of the Vertebrate Skeleton
The skeleton is essential for vertebrates; it supports the body, provides the mechanical framework for physical movements, and protects internal organs. During embryonic development the sites where the future skeletal elements (bones) are formed and their size as well as the positions of articulations (joints) are determined. Most skeletal elements are formed as cartilaginous templates and need to be remodeled into bony tissue to strengthen the skeleton - this process is referred to as endochondral ossification. We use mouse as a model organism to gain insight how different aspects of skeletogenesis are regulated by Wnt-signaling pathways during embryonic and postnatal development. In addition, we use mouse embryonic stem cells to dissect the different functions of β-catenin, a core component of the canonical Wnt-pathway, in signaling and cell-adhesion.
Regulation of cell lineage differentiation
- Figure 1 (Click to view legend)
The different cell types of the appendicular skeleton, chondrocytes, osteoblasts and the cells contributing to the future synovial joints are of mesenchymal origin. Over the past years we demonstrated that the canonical Wnt/β-catenin pathway plays very important roles for the differentiation of the different skeletal lineages in the mouse (see Figure 1). In the absence of functional canonical β-catenin signaling, osteoblast precursors, the osteochondroprogenitor cells, differentiate into chondrocytes (Hill et al., 2005). Furthermore, we have shown that the canonical Wnt/β-catenin pathway is required to suppress the chondrogenic potential of cells in the joint interzone (Spaeter et al., 2006). This suggests that differentiation along the chondrocyte lineage may be the default and that increased levels of β-catenin are required to enable the differentiation along the other two lineages.
Differentiation and Maturation of Chondrocytes
- Figure 2 (Click to view legend)
The various skeletal elements of the vertebrate skeleton differ in size and shape, but little is known about the molecular mechanisms controlling these two features. Nevertheless, per turbations in the chondrocyte maturation process lead to changes in the size of skeletal elements. Studying knock-out animals for Wnt9a we uncovered a very specific requirement for this Wnt-ligand during long-bone development: Wnt9a via β-catenin controls the expression of the central regulator of chondrocyte maturation, Indian hedgehog, in prehypertrophic chondrocytes in a spatio-temporal manner (Spaeter et al.,
2006). Thus, providing a mechanism to fine-tune the size of the future skeletal element. Currently, we are interested in the regulation of hypertrophic chondrocyte maturation and of their removal and turnover into trabecular bone and study amongst others the role of β-catenin in this process.
As we have previously uncovered a potential role for Calcium/Calmodulin dependent kinase II (CaMKII) in the maturation process of chondrocytes in the chick (Taschner et al., 2008) (Figure 2), we are currently analyzing whether this kinase plays a similar role in the mouse using transgenic approaches.
Synovial joint development
Wnt9a was identified as a major player for the induction of synovial joint development in chick studies (Hartmann and Tabin, 2001). In the mouse, Wnt9a is expressed in the early joint interzone and, after the formation of the synovial joint, in the articular cartilage and joint capsule cells. Loss of Wnt9a results in ectopic cartilage nodule formation in the humeral-radial joint. In humans this phenotype is known as synovial chondroid metaplasia. Two other Wnt-genes are also expressed in the joint regions, Wnt4 and Wnt16. Wnt4;Wnt9a double-mutant mice show synovial chondroid metaplasia in two additional joints and fusions of carpal and tarsal elements (Später et al., 2006a, 2006b), but not
complete fusion of all joints. In conclusion, the embryonic phenotypes point to a role for Wnts maintaining joint integrity.
β-catenin – co-transcriptional activity versus cell adhesion function
- Figure 3 (Click to view legend)
We used mouse embryonic stem cells (mESCs) to distinguish between the requirements of β-catenin in functioning as a transcriptional co-activator and as a component of cell adherens junctions. mESCs deficient for β-catenin function show no self-renewing defects under standard conditions (LIF & Serum) and only minor cell adhesion defects (Figure 3a). However, these cells fail to differentiate into derivatives of all three germ layers and show massive cell adhesion defects during differentiation. Rescue experiments using a Tcf/Lef-signaling defective, but cell adhesion competent variant of β-catenin revealed
a requirement for the cell adhesion function of β-catenin for the derivation of neurons (an ectodermal derivative) and for the definitive endoderm, while rescuing cell-adhesion did not influence mesoderm formation (Figure 3b) (Lyashenko et al., 2011). Hence, the function of β-catenin in cell-adhesion is probably playing are marginal role compared to its function as a transcriptional co-activator in the formation of mesoderm. We are currently developing tools that may allow us to distinguish between these two roles during embryonic development.