Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse - IMCD3 screen. Mus musculus

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap – including 1700012B09Rik, 1700026L06Rik and Fam183b – we provide extended experimental evidence for a ciliary function. Overall design: Microarray experiments were conducted in dual-colour mode using amplified, fluorescently-labelled material from unstarved vs. starved cultured IMCD3 cells. Experiments were performed twice as true biological replicates (4 samples). Each pair (unstarved vs. starved) was used for dual-colour based co-hybridisation, including a technical dye-swap replicate, resulting in four dual-colour microarray hybridisations in total. In order to enable inspection of intensity values, dual-colour results were integrated into a single-color GEO matrix.