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

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. 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.

脊椎动物胚胎中运动纤毛（motile cilia）的形成，对于个体正常发育与组织功能发挥至关重要。运动纤毛发生（motile ciliogenesis）的关键调控因子为转录因子FOXJ1与NOTO，二者在脊椎动物演化过程中高度保守。目前学界已在多个物种、器官及培养细胞系中鉴定出FOXJ1的下游靶基因；然而，针对小鼠胚胎及胎组织，FOXJ1与NOTO的效应因子尚未得到全面解析，我们对于驱动哺乳动物肺脏与腹侧节点运动纤毛发生的下游遗传程序的认知仍较为零散。本研究采用微阵列杂交（microarray hybridisation）技术，比较了未分化的E14.5小鼠显微解剖气道上皮与富含纤毛的E18.5小鼠显微解剖气道上皮的全基因组表达谱（genome-wide expression profiles），同时对比了Foxj1阳性与Foxj1缺陷型胎鼠（E16.5）肺脏的表达谱。在两次筛选中均出现表达失调的326个基因，可作为肺脏运动纤毛发生内源启动阶段中FOXJ1依赖型、与纤毛发生相关的候选因子，其中包含123个此前未被关联至纤毛发生过程的基因；这类新型因子中有46%缺乏哺乳动物以外的已知同源基因。对Noto阳性与Noto缺失的早期头褶胚胎（E7.75）开展微阵列筛选后，鉴定出59个肺脏候选基因为左右组织者（left-right organiser）中NOTO/FOXJ1依赖型因子，而左右组织者携带另一亚型的运动纤毛。针对该重叠基因集中的数个未表征因子——包括1700012B09Rik、1700026L06Rik及Fam183b——我们提供了其参与纤毛功能的扩展实验证据。本研究以双色模式开展微阵列实验，使用经扩增并荧光标记的未饥饿与饥饿培养IMCD3细胞产物进行杂交。实验以真实生物学重复（biological replicates）开展了两次，共计4个样本。每一组未饥饿与饥饿样本对均用于双色共杂交，并包含一次技术染料互换重复（technical dye-swap replicate），最终总计完成4次双色微阵列杂交。为便于强度值的检视与分析，双色结果被整合为单色系的基因表达综合数据库（Gene Expression Omnibus，GEO）矩阵。