Although long non-coding RNAs (lncRNAs) have been demonstrated to regulate fundamental biologic processes, such as cell proliferation and maintenance of the pluripotent state, their patterns of expression and role during mammalian development remains poorly defined. Using RNA-Seq with a conservative pipeline, we have identified 363 lncRNAs in the lung and foregut endoderm. These lncRNAs were 3-fold more likely to be located within 20 kb of a known transcription factor compared to protein-coding genes. Three lncRNAs were selected for in-depth analysis: two lncRNAs adjacent to the critical transcription factors Foxa2 (LncFoxa2) and Nkx2.1 (Nkx2.1-Associated Non-CodIng RNA, Nanci), and a novel lncRNA (LL34) with markedly increased expression in embryonic lung. In situ hybridization revealed distinct patterns of expression in the lung for each of these transcripts, with nearly identical patterns of expression between Foxa2 and Nkx2.1 with their neighbouring lncRNAs. Lentiviral shRNA-mediated knockdown of LL34 revealed alterations in the expression of genes involved in early patterning of the foregut and lung, including genes involved in retinoic acid (RA) synthesis, and downstream targets of RA signalling including Foxa1, Gata6, Bmp2, Fgfr2, Fgfr3, and Tgfb3. Most recently, CRISPR-Cas9 technology has been used to generate stable hepatocyte knockout lines of LncFoxa2, which demonstrate that LncFoxa2 may function to regulate endoderm development by both regulation of its neighbouring transcription factor, and also through mechanisms independent of Foxa2. Taken together, these data suggest that lncRNAs play critical roles in the patterning, growth, and differentiation of the foregut and lung.
Funding for this research was provided by NIH 5 U01 HL110942 02 and NIH 5 T32GM008638 17.