Regulation of cartilage differentiation
Our hand skeleton develops in a series of interconnected developmental steps that lead from the initial emergence of cartilaginous condensations in the autopod to their expansion and elongation and their segmentation by joints. These events are orchestrated by a complex regulatory network that assures the correct positioning and growth of the skeletal elements. Disturbances in this network, caused for example by gene mutation, results in hand malformation syndromes.
To elucidate the genetic mechanisms of digit formation and elongation we analyze a group of human hand malformations, the brachydactylies (Greek, brachys=short, dactylos=finger) as well as other inheritable conditions. The identification of disease-causing mutations in these syndromes provided a wealth of information, which is further analyzed in different experimental setups. We are mainly interested in analyzing the pathomechanism that leads to malformation using animal models.
Genetics of Digit Elongation
In our analysis we focus on the most severe form of the brachydactylies, brachydactyly type B1. BDB1 is characterized by shortening/hypoplasia/aplasia of the distal phalanges, is caused by mutations in ROR2, encoding a receptor tyrosine kinase. BDB mutations in ROR2 truncate the protein either immediately in front of or after the tyrosine kinase domain leading to the expression of truncated proteins. In contrast, missense or nonsense mutations leading to amino acid exchange or truncation of extracellular domains of ROR2, truncation within the TK domain, or selective inactivation of TK activity lead to autosomal recessive Robinow syndrome (RRS).
Mutations causing RRS are most likely associated with a loss of function of the protein, since they show retention in the endoplasmatic reticulum, while BDB mutations that localize to the plasma membrane likely interfere with signaling at the cell surface (Schwarzer et al. 2009).
Pathomechanism of BDB: In collaboration with Aris N. Econimides (Regeneron Pharmaceuticals, Tarrytown, USA) we have analyzed a knock-in mouse mutant harboring an exact copy of a human BDB mutation (Ror2-W745X, Raz et al. 2008). The mutant exhibited a brachydactyly phenotype with missing medial phalanges. The analysis of the mutant phenotype showed a specific impairment of digit elongation during the formation of the phalangeal condensations. This was caused by a disruption of Bone morphogenetic protein (BMP) signaling detected by phosphorylation the major BMP downstream components, the transcription factors SMAD1, 5 and 8. Our results demonstrate that this signaling center is active in mammalian digit elongation and suggest that its failure is causative for human disease (Witte et al. 2010).
Is there a common pathomechanism for the brachydactylies as a molecular disease family? The brachydactylies are a syndrome family that shares overlapping features. Intriguingly, many of the mutations causing the different brachydactyly subtypes are found in members of the bone morphogenetic protein (BMP) pathway (see figure). In addition to that, BDA1 is caused by mutations in Indian hedgehog (IHH). Syndromes exhibiting overlapping features are often caused by mutations in genes whose products are involved in a common pathway or are integrated in a protein-protein interaction network (concept of molecular disease families). In collaboration with Danny Chan (University of Hong Kong) we have analyzed a knock-in mouse model for BDA1 (Ihh-E95K). We found that, as in the Ror2-W749X mutant, a defect in the elongation of the phalangeal condensations was causative for the brachydactyly phenotype (Gao et al. 2009). Moreover, a similar failure of the BMP/pSMAD1/5/8 signaling center distal to the growing digit was observed (Witte et al. 2010). This leads to the assumption that a) a signaling network converging on BMP/pSMAD1/5/9 signaling is crucial for the elongation of the phalangeal condensations and b) the brachydactyly disease family is caused by perturbation of this network. The individual yet partially overlapping phenotypes of the brachydactyly syndromes hence are explainable by the differential contribution of the affected components of this network to its final outcome, i.e. pSMAD1/5/8-driven chondrogenesis.
From digit patterning to digit shape
Our appendicular skeleton mainly consists of long bones with exception of the wrist. Directional expansion of long bones is achieved via the so-called growth plate, a structure in which chondrocytes undergo a series of stereotypical differentiation events until they are ultimately replaced by bone. Importantly, in this growth plate chondrocytes in parallel undergo a cellular movement that leads to lateral restriction and concomitant elongation of the anlage in a process similar to convergent extension movements seen in gastrulation. It was noted, that in mice with mutations in the transcription factor HOXD13, which are a model for human Synpolydactyly (SPD), metacarpals ate homeotically transformed to carpal bones (Villavicencio-Lorini et al. J. Clin. Invest. 2010). We have analyzed the molecular mechanism behind this transformation and found that HOXD13 is required for proper spatial induction of the non-canonical WNT ligand WNT5A. This signaling in turn is required for polarization of growth plate and perichondral cells and together confers longitudinal growth to skeletal elements (Kuss et al. Dev. Biol 2014).