Developmental expression of Pax1/9 genes in urochordate and hemichordate gills: insight into function and evolution of the pharyngeal epithelium

The epithelium of the pharynx contributes to the formation of gills in hemi chordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms under lying the establishment of chordate body plans. Pax1- and Pax9-related gene s encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epitheli um that forms the thymus and parathyroid glands. To explore the molecular b asis underlying the occurrence and modifications of the pharyngeal epitheli um during evolution, we isolated cDNA clones for Pax1- and Pax9-related gen es of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona i ntestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cD NA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a home odomain, as is also true of other Pax1- and Pax9-related genes. Molecular p hylogenetic analysis based on comparison of the paired domain amino-acid se quences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9 , Screening of HrPax1/9 cDNA clones yielded six different types of transcri pts which were generated by alternative splicing. Northern blot, RT-PCR/Sou thern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 a nd PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes enco de gill-specific transcription factors. The Pax1/9 genes therefore might pr ovide the first developmental genetic corroboration of hypotheses of organ- level homology that unifies hemichordates, urochordates and cephalochordate s.