Functional evolution of the colony-stimulating factor 1 receptor (CSF1R) and its ligands in birds.

Macrophage colony-stimulating factor (CSF1 or M-CSF) and interleukin 34 (IL34) are secreted cytokines that control macrophage survival and differentiation. Both act through the CSF1 receptor (CSF1R), a type III transmembrane receptor tyrosine kinase. The functions of CSF1R and both ligands are conserved in birds. We have analyzed protein-coding sequence divergence among avian species. The intracellular tyrosine kinase domain of CSF1R was highly conserved in bird species as in mammals but the extracellular domain of avian CSF1R was more divergent in birds with multiple positively selected amino acids. Based upon crystal structures of the mammalian CSF1/IL34 receptor-ligand interfaces and structure-based alignments, we identified amino acids involved in avian receptor-ligand interactions. The contact amino acids in both CSF1 and CSF1R diverged among avian species. Ligand-binding domain swaps between chicken and zebra finch CSF1 confirmed the function of variants that confer species specificity on the interaction of CSF1 with CSF1R. Based upon genomic sequence analysis, we identified prevalent amino acid changes in the extracellular domain of CSF1R even within the chicken species that distinguished commercial broilers and layers and tropically adapted breeds. The rapid evolution in the extracellular domain of avian CSF1R suggests that at least in birds this ligand-receptor interaction is subjected to pathogen selection. We discuss this finding in the context of expression of CSF1R in antigen-sampling and antigen-presenting cells.

Toll-Like Receptor Evolution in Birds: Gene Duplication, Pseudogenization, and Diversifying Selection.

Toll-like receptors (TLRs) are key sensor molecules in vertebrates triggering initial phases of immune responses to pathogens. The avian TLR family typically consists of ten receptors, each adapted to distinct ligands. To understand the complex evolutionary history of each avian TLR, we analyzed all members of the TLR family in the whole genome assemblies and target sequence data of 63 bird species covering all major avian clades. Our results indicate that gene duplication events most probably occurred in TLR1 before synapsids diversified from sauropsids. Unlike mammals, ssRNA-recognizing TLR7 has duplicated independently in several avian taxa, while flagellin-sensing TLR5 has pseudogenized multiple times in bird phylogeny. Our analysis revealed stronger positive, diversifying selection acting in TLR5 and the three-domain TLRs (TLR10 [TLR1A], TLR1 [TLR1B], TLR2A, TLR2B, TLR4) that face the extracellular space and bind complex ligands than in single-domain TLR15 and endosomal TLRs (TLR3, TLR7, TLR21). In total, 84 out of 306 positively selected sites were predicted to harbor substitutions dramatically changing the amino acid physicochemical properties. Furthermore, 105 positively selected sites were located in the known functionally relevant TLR regions. We found evidence for convergent evolution acting between birds and mammals at 54 of these sites. Our comparative study provides a comprehensive insight into the evolution of avian TLR genetic variability. Besides describing the history of avian TLR gene gain and gene loss, we also identified candidate positions in the receptors that have been likely shaped by direct molecular host-pathogen coevolutionary interactions and most probably play key functional roles in birds.