Ken Hastings
More about TnI gene family evolution in the chordates/vertebrates
Vertebrates are a division of the Phylum Chordata. Two additional chordate groups - the cephalochordates (Amphioxus) and the urochordates (tunicates) - have features thought to resemble early vertebrate ancestors. Study of gene family structure and regulation in these primitive groups may shed light on the evolution of vertebrate gene families such as the one encoding the three TnI isoforms: TnIcardiac, TnIfast and TnIslow. The tunicates represent the earliest vertebrate ancestors and make a good starting point for this work. We have begun a molecular genetic characterization of TnI in the ascidian tunicate (sea squirt) Ciona intestinalis. Our studies indicate that Ciona contains a single TnI gene that is expressed in all the known muscles of the organism. In having a single TnI gene, Ciona may represent a stage in chordate/vertebrate history before gene duplication and divergence gave rise to the three-member TnI gene family of the higher vertebrates.
TnI gene expression has several novel features in Ciona, including tissue-specific alternative splicing that generates a longer TnI isoform in the "heart" than in the other striated muscle types. A similar sequence is present in the vertebrate TnIcardiac gene, in an exon that has no counterpart in the TnIfast and TnIslow genes. Thus it appears that ascidians and vertebrates produce homologous tissue-specific (i.e heart-specific) TnI isoforms by non-homologous mechanisms (i.e tissue-specific alternative splicing in asicidians versus tissue-specific transcriptional control in vertebrates). Moreover despite the change of regulatory mechanism during chordate/vertebrate evolution, the tissue-specificity, namely expression of the longer isoform in the heart, was maintained.
An additional novel feature of TnI gene expression in Ciona is mRNA 5’-leader trans-splicing (SL trans-splicing). SL trans-splicing is unknown in vertebrates but its discovery in the ascidians raises the possibility that SL trans-splicing may have been present in vertebrate ancestors, but lost during vertebrate evolution.
Our plans for further study of the ascidian TnI gene include an analysis of the cis-elements that drive transcription in each of the three muscle types of the organism (adult heart, adult body wall muscle, and larval tail muscle). We plan to identify the elements by electroporation of experimental gene constructs into early zygotes, followed by embryonic development, which is very rapid (<24 hr). We also plan to begin to characterize the TnI gene(s) of the other major primitive chordate group, the cephalochordates (Amphioxus) which have more advanced vertebrate characters than the tunicates. Our long-range goal is to establish the evolutionary timing of the gene duplication events that established the higher vertebrate TnI gene family, and to understand how the ancestral gene regulatory mechanism(s) gave rise to, or were subverted by, the mechanisms that direct differential expression of the TnIfast, TnIslow and TnIcardiac genes. We hope this kind of molecular genetic understanding may provide insight into the evolution of the specialized cell types themselves. We also hope to further explore SL-trans-splicing in the tunicates, and possibly in other chordate groups, to better understand the evolution of this character and its impact on genomic evolution in the chordates.
Ciona tailbud stage embryo expressing a TnI-beta-gal transgene in tail muscle cells (M), but not in notochord/nerve cord (N) or epidermal cells (E).
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