Analysis of initial step of multiciliogenesis during the differentiation of adult airway progenitors. Mus musculus

Multiciliated cells are crucial for fluid and ion transport in epithelia of a variety of organs and their impaired development and function are seen in human diseases affecting the brain, respiratory, and reproductive tracts. Multiciliogenesis requires activation of a specialized transcription program coupled to complex cytoplasmic events that lead to large-scale centriole amplification to generate multicilia. Yet, it remains unclear how these events are coordinated to initiate multiciliogenesis in epithelial progenitors. Here we identify an unsuspected mechanism orchestrated by the transcription factor E2f4 essential to integrate these processes. We show that after inducing a transcriptional program of centriole biogenesis, E2f4 translocates to the cytoplasm to become a core component of structures classically identified as fibrous granules (FG), acting as organizing centers for deuterosome assembly and centriole amplification. Remarkably, loss of cytoplasmic E2f4 prevents FG aggregation, deuterosome assembly and multicilia formation even when E2f4’s transcriptional function is preserved. Moreover, in E2f4-deficient cells multiciliogenesis is rescued only if both nuclear and cytoplasmic E2f4 activities are restored. Thus, E2f4 integrates previously unrelated nuclear and cytoplasmic events of the multiciliated cell program. Overall design: E2f4f/f; R26 CreERT2 derived mTECs were cultured on transwells and treated with vehicle or 4-hydroxytamoxifen (Tm) from day -5 to day 0. Triplicate samples (each 2 wells/sample) of each treatment were incubated at 4 degree with RNA-later (Sigma) overnight at each time point (day 0, day 2, day 4). We used Affymetrix mouse GeneChip® Gene 1.0 ST arrays.