Relating the chondrocyte gene network to growth plate morphology: from genes to phenotype

Abstract

During endochondral ossification, chondrocyte growth and differentiation is controlled by many local signalling pathways. Due to crosstalks and feedback mechanisms, these interwoven pathways display a network like structure. In this study, a large-scale literature based logical model of the growth plate network was developed. The network is able to capture the different states (resting, proliferating and hypertrophic) that chondrocytes go through as they progress within the growth plate. In a first corroboration step, the effect of mutations in various signalling pathways of the growth plate network was investigated.

Figure 1. Regulation of Col-X.

Col-X is regulated by several factors, each symbolized by a node (yellow square). Arcs represent interactions of two kinds: an activating interaction is represented by a blue arrow, whereas an inhibitory one is given by a red line with a dot.

Figure 2. Synchronous versus asynchronous updating strategy.

This example clarifies how the use of different updating strategies can change the dynamics of the system. Grey and white color indicate the node has a value of 1 and 0, respectively. Each node is regulated by its own value and that of the other. Node 1 is governed by an AND gate, i.e. it is 1 only when both inputs are 1. Node 2 is regulated by an OR gate, it is 1 when either of its inputs is 1. Under asynchronous updating, the system will reach a different stable state depending on which node is updated first. When both nodes are updated synchronously yet another stable state, unattainable by asynchronous updating, is reached.

Figure 3. The growth plate chondrocyte gene regulatory network.

Every node in the gene network is represented as a square, the interactions are represented by arrows between squares. The network as shown here was coarse grained, i.e. nodes not influenced by multiple reactions were omitted. Therefore linear signalling cascades are not represented here.

Figure 4. Representation of growth plate dynamics.

The expression values of each node as the network progresses from one stable state (framed columns) to the next by changing the inputs (indicated by a red circle). White, grey and black respectively indicate values of 0, 1 and 2. The resting (green), proliferating (dark green) and hypertrophic (red) stable states are indicated. As noted, the prehypertrophic state (yellow rectangles) is not stable but is a transient state between the proliferating and hypertrophic states. It is characterised by simultaneous expression of Runx2 and Sox9 and increased Ihh expression.

Figure 5. Predicted versus observed expression patterns in the growth plate.

The literature derived (light) and predicted (black) expression patterns in 4 growth plate zones. BMP ligands become more abundant as chondrocytes mature as is also the case in the modelled gene network. The expression pattern of Wnt4a is shown here to represent the canonical Wnts, this pattern also closely resembles that of active β-catenin which is the canonical Wnt signal transducer . TGFβ is expressed throughout the growth plate, except in the hypertrophic zone . FGFs are excreted in increasing amounts in more mature cells under influence of Runx2 , PTHrP and Ihh are expressed by resting zone and prehypertrophic chondrocytes respectively . Runx2 expression increases as chondrocytes hypertrophy , while Sox9 activity reaches its peak in proliferative chondrocytes –. Gli2 is also continuously expressed in the growth plate, but its expression tapers off in hypertrophic cartilage .