Revised function of the BMP inhibtor, Gremlin, in Xenopus limb development, and its potential in hindlimb regeneration

Gremlin (Grm) is a potent antagonist of bone morphogenetic proteins (BMPs), which is highly conserved among vertebrates. A combination of functional studies and gene expression analyses in mice and chicks showed that the antagonistic relationship between Grm and the BMPs form part of epithelial-mesenchyme interactive network that plays an important part in early limb development (Zeller, et al., 2009). Together with the Sonic hedgehog-Fibroblast growth factor signalling loop, they form a self-regulatory system that controls the outgrowth, termination and patterning of the avian and murine limbs (Zeller, et al., 2009). Interestingly, a recent report by Pearl et al (2008) demonstrated Grm as one of the developmental genes that was significantly overexpressed in the regenerating blastema tissues of wildtype tadpoles compared to samples obtained from the non-regeneration competent tadpoles. The same study also revealed differential patterns of Grm expression in the developing hindlimb bud in Xenopus compared to those in the avian and murine limbs (Khokha, et al., 2003; Merino, et al., 1999; Pearl, et al., 2008). Moreover, the team also showed differential patterns of expression of the gene during normal hindlimb development and regeneration. These results raised new questions concerning the functions of Grm in amphibian limb development as well as suggesting potential involvement of the gene in regenerative success of Xenopus hindlimb. This project aims to investigate the involvement of the BMP antagonist in X. laevis hindlimb regeneration as well as the function of the gene during early amphibian limb development via a combination of expression and functional analyses. Grm was expressed in the distal region of the regenerating hindlimbs and the early limb bud stages during development. The expression data discussed here not only correlates with the results described in Pearl et al (2008), they also reveal spatial expression of the gene during development in more detail. Additionally, this study also covered the expression of Grm in stage 50 hindlimb buds, which was not tested in the previous study by (Pearl, et al., 2008). The resulting data showed that the expression pattern was vaguely similar to the patterns previously described in early limb buds of chick and mice (Khokha, et al., 2003; Merino, et al., 1999). Overexpression of Grm during early phases of hindlimb regeneration in stage 54 tadpoles using the heat shock-inducible transgenic technique developed by Beck et al (2003) reduced the ability of the animals to regenerate lost structures significantly. The induction of Grm expression also had a significant impact on autopod patterning in both hind- and forelimbs, which was manifested through a range of phenotypes. Finally, this thesis also includes a brief discussion on the project of generating a line of heat shock-inducible RNAi-mediated “knockout” frogs, which may facilitate the studies of developmental genes, such as Grm, that are essential during early development. We have successfully produced the necessary transgene construct that include the heat shock promoter, the targeted small interfering RNA-forming gene, and appropriate reporter genes. In conclusion, the results discussed here showed that stringent regulation of Grm expression in the amphibian model is important for normal patterning of the autopods as well as regenerative success of Xenopus hindlimbs. The negative effects on autopod limb patterning induced by overexpression of Grm at stage 54 were most likely caused by inhibition of BMP activities that were required for modulating the FGF-SHH signalling loop, which played important roles in normal limb outgrowth, and patterning of digits. Similarly, the decline in regenerative ability in Grm-overexpressed tadpoles might be due to disruption of BMP and, indirectly, FGF-8 activities that were required for successful regeneration in Xenopus hindlimbs. The expression data showed that the duration of Grm expression in Xenopus limbs was much shorter compared to chicks and mice, and the expression of the gene was not observed in the developing autopod structure in Xenopus, suggesting that Xenopus Grm may be involved in early stage of limb development, but not later