In this study we report on the identification of the FOXE1 gene of three bird species and the characterization of FOXE1 expression pattern during chicken embryogenesis. Both FOXE1 of chicken and zebra finch are distinctively localized on the sex-determining Z chromosome, in contrast to placental and marsupial FOXE1 genes which are localized on autosomes. In situ hybridization shows that the expression of the chicken FOXE1 gene is restricted to the developing thyroid and feathers. Its thyroid expression is initiated at the stage of placode formation when the thyroid cells evaginate from the pharyngeal floor and migrate, and is also maintained during the stage of thyroid maturation. The process of evagination is characterized by tissue remodeling, which includes modulation of cell adhesiveness and cell mobility. Based on the pattern of FOXE1 expression, we propose that the transcription factor FOXE1 may regulate evagination of thyroid primordia by regulating specific genes required for cell motility and adhesiveness. This observation is supported by previous loss- and gain-of-function studies in the mouse and cell culture, respectively. For example, in FOXE1-null mice the secondary palate remains opened , which indicates inability of the palate shelves to adhere in the mutant mice . Forced expression of mammalian FOXE1 in cell culture resulted in significantly increased expression of an actin-binding protein, tropomyosin isoform 3, and lower expression of integrin beta-1 and collagen type XI alpha-1 . Tropomyosin has been shown to be important for regulating the actin mechanics in the cell cytoskeleton, and can mediate changes in cell morphology, adhesion and migration [27, 28]. Similarly, integrin beta-1 has been shown to mediate cell migration . Moreover, it has been recently shown that human FOXE1 directly regulates the signaling molecule TGF-3β , which in turn, is involved in regulation of cellular adhesion and extracellular matrix . Thus, FOXE1 may be involved in regulation of a set of genes and signaling pathways that are required for controlling cell adhesiveness and motility during migration and morphogenesis of thyroid cells. In the future, it will be important to determine whether chicken FOXE1 directly regulates a similar set of genes during the migration and morphogenesis of the thyroid gland. We also detected expression of FOXE1 in the growing feather, which suggests the acquisition of a novel expression domain by FOXE1 in the bird ancestry; since the feather is a bird specific integumentary appendage.
Two striking features are found in the sequence of avian FOXE1 proteins, which are the sequence divergence of the C-terminus and the loss of two functional domains: a C-terminal aromatic domain and the Eh1 motif as a consequence of two microdeletions. Both domains appear to be involved in mediating transcriptional repression. The aromatic domain of the mammalian FOXE1 protein can inhibit transcription in cell culture when fused to a heterologous DNA-binding domain, thus acting as a transferable repression domain . Nothing is known about direct targets of this repression domain. Interestingly, mammalian FOXE1 represses transcriptional activation mediated by PAX8 in cell culture, which suggests that it may directly interact with transcription factor PAX8, possibly via this repression domain . We noted that the avian genomes lack transcription factor PAX8 (personal communication), which is important for thyroid formation in mammals . This is consistent with an extensive loss of genes in the chicken genome . Thus, it would be interesting to determine whether the loss of the aromatic repressive domain was associated with the loss of the PAX8 locus in birds.
The Eh1 motif is a conserved amino acid sequence , known to mediate physical interaction of other Fox proteins with Groucho/TLE co-repressors. FOXG1, SLP2 (FOXG), FOXD3 and FOXH1 have been shown to interact physically with Groucho/TLE co-repressors via the Eh1 motif ([19, 20, 34] Yaklichkin and Kessler, unpublished data). Strikingly, the Eh1 motif is conserved in all FOXE1 of fish, amphibians, and non-placental mammals, but it was lost in those of birds. Interestingly, a loss of the Eh1 motif is also observed in FOXE1 of placental mammals as an outcome of a microdeletion (Yaklichkin and Kessler, unpublished data). The loss of the Eh1 motif in the avian FOXE1 protein is likely to lead to the loss of Groucho/TLE recruiting activity mediated by the Eh1 motif, and the loss of specific repressive activity dependent on the aromatic domain. Even though the functional implication of both domain losses in avian FOXE1 proteins is not clear, it is likely to affect transcriptional function. It is intriguing that the loss of two putative repressive domains is accompanied by a gain of an N-terminal polyalanine repeat. The avian FOXE1 domain losses may be associated with either functional divergence, loss of co-factor interacting proteins, or even reduction of expression domain. In situ hybridization in the chicken embryo shows that expression of FOXE1 is restricted to the developing thyroid and feathers, and no expression was observed in other internal embryonic tissues. FOXE1 orthologs have additional domain expressions other than in developing thyroid. For instance, frog foxe1 is expressed both in the developing thyroid and pituitary . Foxe1 of zebrafish is expressed in pharyngeal skeleton, gills and thyroid . It is certainly possible that the reduction of expression of FOXE1 in birds has resulted in the loss of these functional sequences. Similarly, a loss of the Eh1 motif in FOXE1 of placental mammals can possibly be associated with a novel functional requirement.
To investigate the role of selection in the evolution of FOXE1 coding regions in the avian lineages, we used various models of codon evolution dN/dS (ω). Overall, dN/dS (ω) of FOXE1 was estimated to be less than 1, which suggested that FOXE1 were evolving under purifying selection. Significant increase of the dN/dS ratio was estimated between the branches of avian FOXE1 and those of mammals and amphibians, which is indicative of a change in the selection and of the acceleration of evolution of avian 3'FOXE1. The increase of the dN/dS ratio can be a result of either a relaxation of purifying selection or positive selection in specific sites of the C-terminal domain of FOXE1 in the avian lineage. In paralogous regulatory genes, the relaxation of purifying selection was proposed to be a result of paralogous proteins binding to a subset of interacting proteins relative to the ancestral gene copy . By this analogy, relaxation of purifying selection in avian FOXE1 could be a result of loss of ancestral protein interactions and possibly formation of interaction with novel binding proteins. Overall, the C-terminal domain is subjected to fewer functional constraints when compared to the DNA-binding forkhead domain. An increased evolutionary rate of C-terminal regions can be attributed to the capacity of trans-regulatory domains to interact with co-factors and the transcriptional machinery via short interaction motifs. In turn, interaction peptide motifs can evolve quickly due to short size and low affinity of interaction with co-factors .
Our branch-site model identified eighteen C-terminus residues under positive selection in the avian lineage, and two residues, 196Q and 203L, had the highest posterior probability, suggestive of adaptive evolution. Only a single adaptively evolving residue, 134R, was identified in the forkhead DNA-binding domain, which resulted in a non-synonymous substitution in the avian lineage, whereas all other residues lie in the C-terminus. Interestingly, the 196Q and 203L residues are located in a segment (195-207 aa) of avian FOXE1 proteins. This segment shows a strong homology to N-terminal short sequences of the homeobox HOXA13 proteins. The N-terminal portion of the HOXA13 protein contains a trans-regulatory domain, which is likely involved in regulation of transcription. Mouse HOXA13 has been shown to function as a negative regulator . Moreover, HOXA13 can inhibit Smad-mediated activation of transcription by binding directly to Smad co-factors via the N-terminus . However, refined mapping of Smad-interacting sequences have not been conducted. It is likely that the region (195-207 aa) is involved in regulation of transcription based on high homology to the N-terminal segment of homeodomain-containing HOXA13 protein, and adaptively evolving residues may have contributed to avian specific FOXE1 function.
A residue 211P under positive selection was found in the FOXE1 segment (214-225 aa) enriched with proline and alanine residues. This segment shares a high similarity with N-terminal sequences of HOXB3 and HOXA4 proteins of mammals, which are also enriched with proline and alanine residues. Interestingly, the mouse HOXB3 protein can function as a transcriptional repressor , and is expressed in the thyroid primordia and regulates its migration . Minimal repression domains of metazoan transcription factors are known to be often enriched with proline and alanine residues . It is thus predicated that this region may be involved in repression of transcription. It is possible that the avian-specific gain of proline residues has contributed to enhancement of repressive characteristics or the formation of novel avian-specific motifs. Additionally, we cannot exclude the contribution of the N-terminus to transcriptional activity of avian FOXE1, which has gained polyalanine repeats. Thus, relaxed selection in the avian lineage may be the predominant contributor to the accelerated evolution of avian FOXE1 and significant sequence divergence of the C-terminus, whereas a limited positive selection could lead to the formation of novel avian specific transcriptional motifs.
Evolution of gene expression, and thus, the evolution of transcription factors, is likely to play a major role in morphological evolution. Because of the pleiotropic effects of changes in transcription factor sequence, some have argued that changes in gene regulatory networks are predominantly mediated via changes in DNA cis-elements . However, negative pleiotropic effects can be limited by tissue-restricted expression of transcription factors and changes in the transcription factor sequences affecting their interaction with other tissue-specific co-factors . This seems to be the case for FOXE1 evolution in birds. However, directed experiments will be needed to further clarify the functional underpinnings of the evolutionary divergence of avian FOXE1.