The Role of Retinoic Acid as a Morphogenic Agent in Xenopus laevis Early Axis Formation and Limb Development

Abstract

Retinoic acid (RA) is a morphogen that functions as a patterning agent in vertebrate development. It is able to pattern tissue due to variation in its bioavailability along a gradient that is generated and regulated by controlling the synthesis and degradation of RA. Its role can also be effected due to the presence of different receptors. The use of RA as a morphogenic agent has been observed as being vital to developmental processes, such as forming the anteroposterior axis in vertebrates and limb development. Analysis of the transcriptome of the developing limb, divided into three sections along the proximal-distal axis, showed varied levels of expression of over 1000 genes. Of these 15 were investigated including two variants of the protein family CYP26, one of which had not been characterized and cyp26c1, which had not been investigated for a role in limb development. The novel sequence was compared to known sequences and was identified as being highly similar to cyp26a1, thus designated cyp26a2. Phylogenetic analysis shown it had only recently diverged, which fits with the recent whole genome duplication event in Xenopus laevis. Analysis showed that the cyp26a2 variant mapped to a unique genomic locus which no confirming it to be a novel gene. Investigating the synteny of the CYP26 loci in the areas immediately around the genes shows high levels of conservation of neighboring sequence as well showing that cyp26a1 and cyp26c1 were evolutionarily linked at the same locus in vertebrates. Both cyp26a2 and cyp26c1 were successfully cloned and their expression patterns ascertained. The novel variant cyp26a2 exhibited expression in regions its paralogue was expressed in as well as novel regions. To investigate potential RA regulated of key genes tadpoles were immersed in a solution containing RA or citral, an antagonist of RA, and their expression profiles examined. This showed alterations in expression in 13 of the 15 genes studied. Next limb expression was studied to get a more accurate image of the expression usually present and this showed that the genes were often localized to specific regions of the limb. The cyp26a2 variant was expressed at the base and had a pattern distinct from its paralogue cyp26a1 which is expressed in the distal end of the limb (McEwan, Lynch et al. 2011) indicating a divergence in function. Cyp26c1 was also confirmed as being expressed in the developing limb, first in the AER before being restricted to the tips of the developing digits and the lateral epidermis of the zeugopod. Localized response to RA was tested by inserting beads saturated in RA into the developing limb. To investigate whether this technique could enable the examination of short term genetic responses to localized RA genes the expression patterns of genes that were previously determined to be affected by RA were probed using in situ hybridization. Several of the genes investigated showed an up regulation in response to RA. This indicates the technique can probe genetic responses to RA.