- [Show abstract] [Hide abstract] ABSTRACT: Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.
- [Show abstract] [Hide abstract] ABSTRACT: We have previously shown differential regulation of components of the Retinoic acid (RA) pathway in Xenopus tadpole hindlimb regeneration. RA is thought to act as a morphogen, providing positional information during development and regeneration. We have investigated the regulation of genes involved in RA synthesis, catabolism, and binding in developing and regenerating Xenopus limbs. Our data indicate that RA is synthesised by Raldh2 in proximal cells during limb bud outgrowth. Furthermore, Cyp26b is expressed transiently in the progress zone of developing limbs and the blastema of regenerating limbs suggesting degradation of RA occurs in both processes. The RA-binding protein Crabp2 is also upregulated during regeneration. We summarise this data to predict the presence of evolving gradients of RA in the developing amphibian limb. Thus, RA from the stump cells could be responsible for the establishment of proximal-distal pattern during limb regeneration, as predicted by classical studies.
- [Show abstract] [Hide abstract] ABSTRACT: Retinoic acid (RA) is a known teratogen that is also required endogenously for normal development of the embryo. RA can act as a morphogen, through direct binding to receptors and RA response elements in the genome, and classical studies of limb development and regeneration in amphibians have shown that it is likely to provide positional information. Availability of RA depends on both metabolic synthesis and catabolic degradation, and specific binding proteins act to further modulate the binding of RA to response elements. Here, we describe the expression of seven genes involved in metabolism (Raldh1-3), catabolism (Cyp26a and b) and binding of RA (Crabp1 and 2) during organogenesis in the clawed frog Xenopus laevis. Taken together, this data indicates regions of the embryo that could be affected by RA mediated patterning, and identifies some differences with other vertebrates.