PgmNr Z586: Regulation of actinodin1 in embryonic fins via tissue-specific cis-acting regulatory elements: a potential mechanism for the loss of these genes during the fin-to-limb transition.

Authors:
R. Lalonde; D. Moses; J. Zhang; N. Cornell; M.-A. Akimenko


Institutes
University of Ottawa, Ottawa, CA.


Abstract:

The fin-to-limb transition is an important evolutionary step in the colonization of land and diversification of terrestrial vertebrates. Our lab has identified a gene family in zebrafish, termed actinodin (and), which codes for structural proteins in the teleost fin. These proteins are crucial for the formation of rigid fibrils known as actinotrichia. Actinotrichia support the fin fold, a structure formed from the extension of the apical ectodermal ridge (AER) during pectoral fin development. During limb development, the AER is maintained relatively longer prior to eventual regression. Interestingly, this gene family is absent from all tetrapod genomes examined to date, suggesting that it was lost during limb evolution. We propose that the disappearance of and genes resulted from modification in their regulation and we therefore characterized the regulatory elements of actinodin1 (and1), one of two longer and paralogues (actinodin1/2) in zebrafish. Within a 2kb fragment immediately upstream, we have identified tissue-specific cis-acting regulatory elements responsible for and1 expression in the developing zebrafish median and pectoral fin folds. Using transgenic reporter lines, we have identified and isolated the regulatory elements responsible for and1 activation in the ectodermal and mesenchymal cell populations, respectively. To test their functionality in tetrapods, a transgenic LacZ reporter mouse line was made. In contrast to the zebrafish transgenic line, reporter expression is restricted to the ectoderm of the fore- and hindlimb buds, localizing within the autopodal domain. One possibility suggests that while the regulatory network for and1 ectodermal activation remains intact in mice, the mechanisms for mesenchymal activation have been lost or altered during tetrapod evolution. Putative transcription factor binding sites were identified via TRANSFAC and tested for enhancer activity using site-directed mutagenesis. Removal of tcf and 5’ hoxA/D binding sites lead to the loss of ectodermal and mesenchymal enhancer activation, respectively. Dissecting the regulatory mechanisms of this gene family is crucial for uncovering the mechanisms of and loss during tetrapod evolution. Finally, and1 cis-acting regulatory elements enable us to perform tissue-specific lineage tracing and ablation of and1-expressing cells. We are currently performing lineage tracing experiments on both ectodermal and mesenchymal cell populations using the Cre-LoxP system, in addition to observing the effects of fin fold mesenchyme ablation on fin development using the Nitroreductase/Metronidazole system. This work is supported by a NSERC grant to M.-A.A.



ZFIN Genetics Index
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