They were usually solitary microtubules that were extensively cross-linked to intermediate filaments. In addition, we display that main cilia in inner hair cells, a feature of early development, persist much later on than previously reported. The findings represent the first description of developmental cell type-specific reductions in tubulin isotypes in any system. == Intro == The ubiquitous structural protein tubulin is found in cells as microtubules consisting of and tubulin monomers. Mammalian tubulin DBPR108 is present as seven isotypes, termed I, II, III, IVa, IVb, V, and VI, each a separate gene product synthesized without option splicing (Luduea, 1998). The amino acid sequences of the seven isotypes are 7596% identical, but several of them are also among the most highly conserved in development. For example, the chicken and mouse Iisotypes differ by only two residues (Luduea, 1998). The conservation of isotype sequence in mammals and in additional vertebrates has led to the multi-tubulin hypothesis, the proposition the multiple functions of microtubules may require different forms of tubulin (Fulton & Simpson, 1976). This hypothesis predicts that isotypes will be selectively synthesized in different cell types relating to function. In post-mitotic organ of Corti development, microtubules are elaborated in a specific temporal pattern, beginning with hair cells at post-natal day time 0 (P0), then in pillar cells by P3 and Deiters cells by P6 (Hallworthet al.,2000). A recent study using four tubulin isotype-specific antibodies has shown that, in gerbil organ of Corti, the isotypes are differentially synthesized in several cell types (Hallworth & Luduea, 2000). To become specific, inner hair cells (IHCs) were found to have only Iand II, while outer hair cells (OHCs) experienced only Iand IV. Both inner and outer pillar cells (IPs and OPs) showed only IIand IV, while Deiters cells showed I, II, and IV. Selective synthesis of tubulin isotypes has also been explained in the vestibular system and in nose epithelia (Perryet al.,2003;Wooet al.,2002). We here ask, how is the adult construction of tubulin isotypes accomplished during development? Are microtubules equipped with the mature isotype composition duringde novosynthesis, or are isotypes added DBPR108 inside a cell-specific temporal sequence? Or, a further possibility, are all isotypes present in the beginning and the number of isotypes in each cell type selectively pruned in development? To answer this question, we have taken advantage of the progressive post-natal elaboration of microtubules in gerbil organ of DBPR108 Corti. We examined the distribution of tubulin isotypes in the developing organ of Corti during the first thirty days of post-natal development using isotype-specific antibodies and have compared the results to the previously explained adult pattern. == Materials and methods == The distribution of tubulin isotypes was examined in developing (P0 to P30) and adult gerbil cochlea BLR1 using indirect immunofluorescence in whole mounts and freezing sections. Gerbils were anesthetized with Nembutal and cardiac perfused with heparinized saline answer followed by 4% paraformaldehyde in phosphate buffered saline (PBS). Cochleae were dissected out, post-fixed for one hour, and decalcified if more than P6 with EDTA. The apical, middle and basal becomes were dissected out for processing as whole mounts. For sections, cochleas were equilibrated, after decalcification if necessary, in 30% sucrose in PBS like a cryoprotectant, and were then quickly freezing in O.C.T. compound (Kilometers Labs, Elkart, IN). Frozen sections, 810 m solid, were cut on a cryostat (Leica Microsystems, Bannockburn, IL). Specimens were clogged and permeabilized in PBS comprising 1% bovine albumin serum,.