The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium.

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The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.

Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.