The Tissue-Specific lncRNA Fendrr Is an Essential Regulator of Heart and Body Wall Development in the Mouse The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Grote, Phillip, Lars Wittler, David Hendrix, Frederic Koch, Sandra Wahrisch, Arica Beisaw, Karol Macura, et al. “The Tissue-Specific lncRNA Fendrr Is an Essential Regulator of Heart and Body Wall Development in the Mouse.” Developmental Cell 24, no. 2 (January 2013): 206–214. © 2013 Elsevier Inc. As Published http://dx.doi.org/10.1016/j.devcel.2012.12.012 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/87007 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Developmental Cell Short Article The Tissue-Specific lncRNA Fendrr Is an Essential Regulator of Heart and Body Wall Development in the Mouse Phillip Grote,1 Lars Wittler,1,5 David Hendrix,3,4,5 Frederic Koch,1,5 Sandra Wa¨ hrisch,1 Arica Beisaw,1 Karol Macura,1 Gaby Bla¨ ss,1 Manolis Kellis,3,4 Martin Werber,1 and Bernhard G. Herrmann1,2,* 1Max Planck Institute for Molecular Genetics, Department of Developmental Genetics, Ihnestr. 63-73, 14195 Berlin, Germany 2Charite´ -University Medicine Berlin, Institute for Medical Genetics-Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany 3Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA 5These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2012.12.012 SUMMARY 2007). In particular, PRC2 catalyzes the methylation of histone H3 at lysine 27 (H3K27me3), which is repressive to gene activity, The histone-modifying complexes PRC2 and TrxG/ while the TrxG/MLL complex catalyzes the methylation of MLL play pivotal roles in determining the activation histone H3 at lysine 4 (H3K4me3), which acts as an activating state of genes controlling pluripotency, lineage mark (Margueron and Reinberg, 2011; Schuettengruber et al., commitment, and cell differentiation. Long noncod- 2011). Thus, PRC2 and TrxG/MLL have opposing activities, ing RNAs (lncRNAs) can bind to either complex, and both are essential for embryonic development (O’Carroll and some have been shown to act as modulators of et al., 2001; Yu et al., 1995). It has been shown that components of both the PRC2 and PRC2 or TrxG/MLL activity. Here we show that the Fendrr TrxG/MLL complexes are able to interact with long noncoding lateral mesoderm-specific lncRNA is essen- RNAs (lncRNAs) (Zhao et al., 2008, 2010; Wang et al., 2011). tial for proper heart and body wall development in Recent reports have suggested that lncRNAs may target PRC2 the mouse. Embryos lacking Fendrr displayed upre- or TrxG/MLL to specific genomic loci, and thus contribute to gulation of several transcription factors controlling the histone modification status and activity level of target genes lateral plate or cardiac mesoderm differentiation, (Rinn et al., 2007; Khalil et al., 2009; Bertani et al., 2011; Guttman accompanied by a drastic reduction in PRC2 occu- and Rinn, 2012). In vitro knockdown experiments have revealed pancy along with decreased H3K27 trimethylation the involvement of lncRNAs in gene regulatory networks control- and/or an increase in H3K4 trimethylation at their ling embryonic stem (ES) cell differentiation (Guttman et al., promoters. Fendrr binds to both the PRC2 and 2011), and functional studies have provided evidence for the TrxG/MLL complexes, suggesting that it acts as roles of several lncRNAs (Wang et al., 2011; Hu et al., 2011; Kretz et al., 2012; Rinn et al., 2007; Gupta et al., 2010; Ulitsky et al., modulator of chromatin signatures that define gene 2011). For instance, the knockdown of HOTTIP by an RCAS- activity. Thus, we identified an lncRNA that plays an shRNA in the developing chick limb resulted in a shortening of essential role in the regulatory networks controlling the distal bony elements of the limb, and morpholino-mediated the fate of lateral mesoderm derivatives. knockdown of megamind and cyrano in zebrafish embryos re- vealed important roles for these lncRNAs in organogenesis (Wang et al., 2011; Ulitsky et al., 2011). However, stringent INTRODUCTION genetic approaches for probing the role of lncRNAs in mouse embryogenesis using loss-of-function analyses have not yet Embryonic development commences with the formation of been reported. a group of pluripotent stem cells, which give rise to all cell types Here we have identified an lncRNA, which we have termed of the body. Development then proceeds through the coordi- Fendrr, that is specifically expressed in nascent lateral plate nated action of cellular proliferation, patterning, lineage commit- mesoderm. We inactivated Fendrr by gene targeting in ES cells ment, and differentiation, which are controlled by transcriptional and show that Fendrr is essential for proper development of regulators acting in a cell type-specific manner. The activity of tissues derived from lateral mesoderm, specifically the heart genes encoding such regulatory proteins depends largely on and the body wall. We illustrate that Fendrr acts by modifying the chromatin structure at their promoters and the associated the chromatin signatures of genes involved in the formation regulatory elements. The histone-modifying Polycomb repres- and differentiation of the lateral mesoderm lineage through sive complexes (PRC1/PRC2) and the Trithorax group/MLL binding to both the PRC2 and TrxG/MLL complexes. Further- protein complexes (TrxG/MLL) play pivotal roles in the control more, we provide evidence that an oligonucleotide correspond- of chromatin structure and, hence, gene activity of a subset of ing to part of the Fendrr transcript can bind to dsDNA in target developmentally important regulators (Schuettengruber et al., promoters. 206 Developmental Cell 24, 206–214, January 28, 2013 ª2013 Elsevier Inc. Developmental Cell lncRNA Fendrr Is Essential for Mouse Development Figure 1. Fendrr Is Transiently Expressed in Nascent LPM of Developing Mouse Embryos (A) Schematic of the genomic region of Fendrr and Foxf1. (B) Whole-mount in situ hybridization of mouse embryos at E8.25 and E9.5. (C) Transversal sections of E8.25 embryos following whole-mount in situ hybridization analysis of Foxf1 or Fendrr. (D) qPCR analysis of cDNA derived from the cytosolic or nuclear fraction of cells obtained from E9.5 embryo caudal ends; the log10 ratio of nuclear to cytosolic cDNA is presented (n = 3, SD). RESULTS not detect Fendrr expression in other tissues or in organs of later stage embryos using qPCR analysis (data not shown). In the Fendrr Expression Is Restricted to Nascent Lateral Plate caudal LPM it is coexpressed with Foxf1, while in more anterior Mesoderm LPM cells that are undergoing differentiation, Fendrr is down- We searched for differentially expressed lncRNAs in six different regulated, whereas Foxf1 expression is maintained in the tissues dissected from early somite-stage mouse embryos splanchnic mesoderm (Peterson et al., 1997; Mahlapuu et al., (TS12, E8.25, three to six somites) using RNA-seq and ChIP- 2001a). Quantitative PCR analysis of RNA extracted from the seq analyses. From this data set, we identified a gene that is nuclear or cytosolic faction of E9.5 caudal end cells showed specifically transcribed in the posterior mesoderm. We isolated that Fendrr RNA is predominantly localized in the nucleus, it by RACE-PCR from embryonic cDNA and determined its consistent with its human ortholog in cultured cells (Figure 1D) 2,397 base pair (bp) sequence and gene structure (Figure 1A). (Khalil et al., 2009). The gene consists of seven exons and is transcribed divergently from the transcription factor-coding gene Foxf1. Its transcrip- Loss of Fendrr Causes Embryonic Lethality tional start site is located 1,250 bp upstream of the 50-end of To investigate the function of Fendrr in mouse development, we Foxf1. We termed this lncRNA Fendrr (Fetal-lethal noncoding first knocked down Fendrr transcripts using a method for developmental regulatory RNA). shRNAmir-mediated RNA interference in vivo (Vidigal et al., Whole-mount in situ hybridization showed that Fendrr is 2010). A reduction in Fendrr transcripts to 40% of the wild- confined to the caudal end of the lateral plate mesoderm (LPM) type level caused no phenotype (data not shown). Therefore, of midgestation embryos, which gives rise to ventral structures we consecutively replaced the first exon of Fendrr on both chro- such as the heart and body wall (Figures 1B and 1C). We could mosomes in ES cells with a strong transcriptional stop signal Developmental Cell 24, 206–214, January 28, 2013 ª2013 Elsevier Inc. 207 Developmental Cell lncRNA Fendrr Is Essential for Mouse Development Figure 2. Heart and Body Wall Development Are Impaired in Mutants Lacking Fendrr Transcripts (A) Schematic showing the wild-type (upper) and targeted (lower) Fendrr-Foxf1 genomic regions; the first exon of Fendrr was replaced with a 3xpA stop cassette. Arrows indicate primers for qPCR. (B) E13.75 embryos derived from wild-type or homozygous Fendrr mutant ES cells (Fendrr3xpA[N)/3xpA(H]), or from mutant cells con- taining a modified rescue BAC transgene (Tg(RP23-455G4)Bgh). The tail and limbs have been removed. Scale bar, 1 mm. (C) Eosin-stained sagittal sections of E13.75 wild- type and Fendrr mutant embryos showing liver protrusion in the latter (arrowhead). (D and G) Transverse histological sections of E12.5 wild-type and mutant embryos at the midtrunk (D) or chest level (G). rv, right ventricle; is, interventricular septum; cu, atrio-ventricular endocardial cushion; la, left atrium; lv, left ventricle. Scale bar, 200 mm. (E) Tissue thickness measured from Eosin-stained transverse sections of E12.5 wild-type and homozygous mutant embryos; values from both sides were combined for paired t test analysis.