Cytoplasmic intermediate filament protein expression in tunicate development: a specific marker for the test cells.

The urochordate Ciona intestinalis is a well established system for embryological studies, and large scale EST sequences begin to emerge. We cloned five cytoplasmic intennediate filament (IF) cDNAs and made specific antibodies to the recombinant proteins. Self-assembly studies and immunofluorescence microscopy were used to study these proteins and their distribution. Confirming and extending previous studies in Styela, we found that Ciona protein IF-A is expressed in muscle and forms homopolymeric filaments while proteins IF-C and IF-D, which form only obligatory heteropolymeric filaments, resemble a keratin pair exclusively found in the entire epidermis. Protein IF-B and the new protein IF-F potentially reflect tunicate-specific IF proteins. They are found in the entire internal epithelia including the neural gland. We also extended the analysis to earlier developmental stages of Ciona. Protein IF-A is expressed in muscle from larval stages, whereas proteins IF-C and IF-D are found only in the tail epidermis. Protein IF-F is detected abundantly in the test cells of eggs, embryos and premetamorphic larvae. Our studies show that IF proteins could prove very useful markers in the study of cell fate determination in Ciona. They also support previous findings on the evolutionary relationships of different IF proteins. Non-vertebrate chordates have IF proteins which represent orthologs of vertebrate type I to III proteins, but also IF proteins that do not seem to fit into these classes. However, the intron positions of all tunicate IF genes are conserved with vertebrate type I to III genes, pointing to a common evolutionary origin.