Dear Editor, In mammalian genomes, pervasive transcription produces thousands of long noncoding RNA (lncRNA) transcripts (Djebali et al., 2012; Hon et al., 2017). Compared to protein-coding mRNAs, lncRNAs are less conserved, and often exhibit low-level, developmental stage-and tissue-specific expression (Pauli et al., 2011; Hu et al., 2012; Lee, 2012; Ulitsky and Bartel, 2013; Cech and Steitz, 2014; Hon et al., 2017). Many lncRNAs are strongly correlated with their neighboring mRNA genes in terms of expression and function, and tend to regulate nearby transcription (Orom et al., 2010; Engreitz et al., 2016; Luo et al., 2016). It has been implicated that lncRNAs play versatile roles in regulating diverse aspects of cell biology through mechanisms at multiple levels (Pauli et al., 2011; Lee, 2012; Batista and Chang, 2013). However, as most lncRNA studies are performed in cell lines, direct genetic evidence of their functional significance in vivo remains limited. To explore the general relevancy of lncRNAs in development, we chose to delete 12 representative lncRNAs from the mouse genome. We selected the lncRNAs based on their genomic positions, conservation, expression, and functions in cell culture as well as their nearby genes of developmental importance (Figure 1 A and Supplementary Figure S1). Eight of the lncRNAs are located within 5 kb of the nearest gene. They include seven divergent lncRNAs (Evx1as, Hand2as, Foxd3as, Gata3as, Gata6as, Lhx1as, and Traf7as) and one convergent lncRNA (Bvht). The other four lncRNAs are intergenic and are located> 5 kb away from the nearest protein-coding gene (Haunt, Apela, Gm10451, and 1700020I14Rik). These lncRNAs are expressed at various developmental stages and in different tissue types (Supplementary Figure S1).

Importantly, 9 out of 12 lncRNAs are conserved syntenically in the human genome (Figure 1 A). The four lncRNAs Evx1as, Haunt, Bvht, and Apela have been reported to play critical roles in regulating lineage differentiation and gene expression in cultured cells (Klattenhoff et al., 2013; Li et al., 2015; Yin et al., 2015; Luo et al., 2016). Knocking out the protein-coding genes positioned next to six lncRNAs in this list, including HAND2, FOXD3, GATA3, GATA6, LHX1, and HOXA1, results in embryonic lethality (Lufkin et al., 1991; Pandolfi et al., 1995; Srivastava et al., 1997; Koutsourakis et al., 1999; Shawlot et al., 1999; Hanna et al., 2002). To effectively abolish lncRNA expression and function, we deleted large genomic DNA fragments (0.5–17 kb), which included the regulatory promoter sequences responsible for lncRNA expression and/or the majority of the lncRNA sequences (Supplementary Figures S1 and S2). For example, we removed the bulk of the RNA sequence of Evx1as. Despite the overall conservation and importance of the nearby mRNA genes in development, we found to our surprise that knockout mice homozygous for 11 out of 12 lncRNAs were born at the expected Mendelian ratio (∼ 25%) and were viable with no obvious abnormalities (Figure 1 A). The full-length (F) deletion of Hand2as (17-kb deletion), but not deletion of the promoter and 5’sequences (P) or a distal element (D), caused a perinatal lethal phenotype with dysregulated cardiac gene expression and heart hypoplasia (Figure 1 A and Supplementary Figure S1; data not shown). This phenotype is in contrast to the reported failure of right ventricle formation and lethality at E10. 5 of Hand2as