Abstract
Limbless morphologies in Squamata independently originated several times from ancestors that presented complete autopodium. The recurrent transitions from pentadactyl to limbless morphologies, associated to evidence for reversion of digit loss in specific lineages, characterize Squamata as a particularly interesting group for investigating associations between limb morphology and evolutionary patterns of developmental genes. Hox genes, for instance, codify transcription factors that regulate expression of several other genes and organize temporal and spatially the development of most structures in vertebrates. Some of these other genes are expressed during limb positioning and development of autopodium elements in the tetrapod embryo, and variations in their expression seem related to the evolution of new phenotypes, including the origin of snakelike organisms. Such variations may involve specific mutations in coding nucleotide sequences, which change gene roles in the signaling cascades during autopodium development, as well as mutations in regulatory regions, which likely affect cis-trans interactions that regulate gene expression. The gene Hoxd-11 in particular is expressed during tetrapod autopodium development, and is controlled by the regulatory region CsB; its expression differs between pentadactyl and non-pentadactyl species. The present proposal aims to investigate possible differences in the signaling pathways involving Hoxd-11 expression that may be associated to the evolution of limbless morphologies in Squamata. Specifically, patterns detected in Hoxd-11 regulatory and coding regions will be compared among Amphisbaenas and Snakes, both characterized by the limbless morphology. Functional essays will test for the relevance of molecular patterns detected in Hoxd-11 using bioinformatics, and results will be interpreted in the context of parallel evolution of limbless morphologies in different Squamate lineages. Coding (exon-1) and regulatory (CsB) regions of Hoxd-11 will be sequenced in representatives of both lineages, using alligators as outgroup. Based on the patterns found, one of two alternative approaches will be subsequently followed. If differences in CsB are identified, pull down essays will test for possible differences in transcription factors that bind to CsB nucleotide sequences. These tests will be implemented using CsB of one amphisbaenia, one snake and one outgroup, and will be carried out with chicken embryo developing limbs in three stages. In contrast, if we detect differences in the coding region (exon-1), their functional relevance will be investigated by transcriptome analyses of mouse cells lacking Hoxd-11 endogenous expression (negative control), compared with transcriptomes of cells tranfected with plasmids containing: I) snake Hoxd-11; II) amphisbaenia Hoxd-11; and III) mouse Hoxd-11 (positive control). Investigation of molecular evolution patterns in Hoxd-11 coding and regulatory regions, together with functional essays, will contribute for identifying the possible mechanisms of morphological diversity evolution in vertebrates that resulted in the origin of limbless morphologies in distinct clades. (AU)
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