Christopher R. Lage

The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions

Mammalian immune responses to LPS exposure are typified by the robust induction of NF-κB and IFN-β responses largely mediated by TLR4 signal transduction pathways. In contrast to mammals, Tlr4 signal transduction pathways in nontetrapods are not well understood. Comprehensive syntenic and phylogenetic analyses support our hypothesis that zebrafish tlr4a and tlr4b genes are paralogous rather than orthologous to human TLR4. Furthermore, we provide evidence to support our assertion that the in vivo responsiveness of zebrafish to LPS exposure is not mediated by Tlr4a and Tlr4b paralogs because they fail to respond to LPS stimulation in vitro. Zebrafish Tlr4a and Tlr4b paralogs were also unresponsive to heat-killed Escherichia coli and Legionella pneumophila. Using chimeric molecules in which portions of the zebrafish Tlr4 proteins were fused to portions of the mouse TLR4 protein, we show that the lack of responsiveness to LPS was most likely due to the inability of the extracellular portions of zebrafish Tlr4a and Tlr4b to recognize the molecule, rather than to changes in their capacities to transduce signals through their Toll/IL-1 receptor (TIR) domains. Taken together, these findings strongly support the notion that zebrafish tlr4a and tlr4b paralogs have evolved to provide alternative ligand specificities to the Tlr immune defense system in this species. These data demonstrate that intensive examination of gene histories when describing the Tlr proteins of basally diverging vertebrates is required to obtain fuller appreciation of the evolution of their function. These studies provide the first evidence for the functional evolution of a novel Tlr.

Evidence for Evolving Toll-IL-1 Receptor-Containing Adaptor Molecule Function in Vertebrates

In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-κB and IFN-β responses. TICAM1 activates NF-κB through two different pathways involving its interactions with TNFR-associated factor 6 and receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1, leading to the robust up-regulation of IFN-β. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating NF-κB and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being α- or β-like. Through comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog. Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated factor 6, thus NF-κB activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway.

Arsenic ecotoxicology and innate immunity.

Understanding the ecotoxicological effects of arsenic in the environment is paramount to mitigating its deleterious effects on ecological and human health, particularly on the immune response. Toxicological and long-term health effects of arsenic exposure have been well studied. Its specific effects on immune function, however, are less well understood. Eukaryotic immune function often includes both general (innate) as well as specific (adaptive) responses to pathogens. Innate immunity is thought to be the primary defense during early embryonic development, subsequently potentiating adaptive immunity in jawed vertebrates, whereas all other eukaryotes must rely solely on the innate immune response throughout their life cycle. Here, we review the known ecotoxicological effects of arsenic on general health, including immune function, and propose the adoption of zebrafish as a vertebrate model for studying such effects on innate immunity.

Reduced Genetic Diversity and Effective Population Size in an Endangered Atlantic Salmon (Salmo Salar) Population from Maine, USA

Atlantic salmon (Salmo salar) populations in Maine, USA, are listed as a Distinct Population Segment under the U.S. Endangered Species Act due to reduced spawning runs and juvenile densities. Whenever possible, optimal conservation strategies for endangered populations should incorporate both present and historical knowledge of genetic variation. We assayed genetic diversity at seven microsatellite loci and at the mitochondrial ND1 gene in an endangered wild population of Atlantic salmon captured from the Dennys River from 1963 to 2001 using DNA’s extracted from archival scale and tissue samples. We examined temporal trends of genetic diversity, population structure, and effective population size (Ne). Overall temporal trends of diversity and Ne show significant reductions from 1963 to 2001 raising the possibility that current restoration efforts may be impacted by historical loss of diversity potentially critical to adaptation. Although our results suggest genetic stability in this population from 1963 to 1981, significant differentiation was observed for both the 1995 and 2001 samples compared with all other temporal samples. The presence of an ND1 mtDNA haplotype in this population, historically observed only in European and Newfoundland stocks, may represent previously unrecognized local wild diversity or, alternatively, may represent introgression from non-native fish.

Evolutionary divergence of the vertebrate TNFAIP8 gene family: Applying the spotted gar orthology bridge to understand ohnolog loss in teleosts

Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.