The influence of Gremlin-induced BMP inhibition and subsequent associated genetic interactions in Xenopus laevis limb development

The tetrapod limb is one of the most extensively studied organs within the field of developmental biology, due to its highly conserved development involving key molecular pathways (Jones et al. 2013). The bone morphogenetic protein (BMP) signalling cascade is an example of such important developmental pathway, known to induce apoptotic activity and skeletal element formation within developing limbs (Chen et al. 2004). Gremlin (grem1), a BMP antagonist, is known to regulate the fibroblast growth factor-sonic hedgehog (FGF-shh) signalling loop, which controls the early outgrowth and termination of tetrapod limbs, including Xenopus laevis (Christen et al. 2012; Zeller et al. 2009). As tetrapod limb development is a tightly controlled process, modification to associated gene expression can alter downstream signalling and subsequent limb development, giving rise to limb abnormalities (Jones et al. 2013). This project aims to investigate the genetic and developmental effects that ectopic grem1 overexpression has in X. laevis limb development, focused on abnormality development, and the expression intensity and distribution of specific BMP and limb pattern marker genes. Transgenic X. laevis containing a heat shock-inducible grem1 gene, were induced to ectopically overexpress grem1 at various limb development stages. Samples were allowed to develop, skeletal elements were counted, and resultant limb developmental effects were contrasted to wild type samples using cartilage and bone staining, and in situ hybridisation methods. Grem1 overexpression during stage 49 samples, where the hindlimb bud was within the early propagation phase, was shown to have the significantly highest abnormality diversity and proportions when compared to other limb development stages. This was possibly due to decreased limb BMP activity, and subsequent increased FGF (cell proliferation) and decreased sox9 (cartilage formation) activity, resulting in limb bifurcations and truncations, respectively. Significantly fewer digits developed in stage 49 limb samples, with digit IV having significantly fewer phalanges than other digits. Earlier stage grem1 overexpression resulted in more proximally located abnormalities, such as formation of ectopic limbs, showing a time-dependent effect of grem1 within limb development. The in situ hybridisation data showed grem1 overexpression to decrease specific BMP expression intensity; increase proximodistal limb axis FGF expression distribution; increase anteroposterior limb axis shh expression distribution; and increase chondrogenesis-associated sox9 expression distribution. These results concurred with a tetrapod limb developmental model based on chick and mouse data (Verheyden & Sun 2008; Zeller et al. 2009). In conclusion, this work summarised the tetrapod limb development literature, which detailed the importance of regulated grem1 and specific BMP pathway gene expression in regards to the complex normal limb development process in the amphibian model. It also demonstrated possible means of limb abnormality development amongst the tetrapod group, such as differences between serial and mirror bifurcations, which could be useful for experimental and clinical cases. The use of a heat shock technique to overexpress a gene of interest has been proven successful. Grem1 function within X. laevis when compared to chick and mouse models was similar based on relevant studies, but not identical, suggesting limb development needs many models rather than the more extensively researched chick and mouse models. The BMPs inhibited by grem1 were not exclusive to grem1, as they are also inhibited by other BMP inhibitors, but rather the binding efficiency and timing to specific combinations of BMPs differentiates BMP inhibitors