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Deletion of the Sclerotome-Enriched Lncrna PEAT Augments Ribosomal Protein Expression

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Deletion of the Sclerotome-Enriched Lncrna PEAT Augments Ribosomal Protein Expression Deletion of the sclerotome-enriched lncRNA PEAT augments ribosomal protein expression David A. Stafforda, Darwin S. Dichmanna, Jessica K. Changb, and Richard M. Harlanda,1 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and bDepartment of Genetics, Stanford University, Stanford, CA 94305-5120 Contributed by Richard M. Harland, September 23, 2016 (sent for review July 22, 2016; reviewed by Margaret Buckingham and Chen-Ming Fan) To define a complete catalog of the genes that are activated during developing sclerotome by dissection from early embryos at 8- to mouse sclerotome formation, we sequenced RNA from embryonic 9.5-d postconception is technically difficult. Although a model mouse tissue directed to form sclerotome in culture. In addition to for presomitic mesoderm culture from stem cells has been de- well-known early markers of sclerotome, such as Pax1, Pax9,andthe veloped (10), this has not been exploited as an in vitro model of Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 the sclerotome. To obtain sufficient sclerotomal transcripts for enhancer antisense transcript) was induced in sclerotome-directed RNA-seq, we chemically treated explants of embryonic tissue to samples. Strikingly, PEAT is located just upstream of the Pax1 gene. bias differentiation of fluorescently marked presomitic mesoderm Using CRISPR/Cas9, we generated a mouse line bearing a complete toward the sclerotome. Following FACS, we compared control deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant and induced cultures to define the sclerotome transcriptome. In embryos showed that loss of PEAT modestly increases bone mor- addition to the previously described transcription factors, we identi- phogenetic protein target gene expression and also elevates the fied an uncharacterized noncoding RNA transcript that we named expression of a large subset of ribosomal protein mRNAs. PEAT (Pax1 enhancer antisense transcript; also known as AI646519) that is expressed in the reverse orientation to the Pax1 promoter. To sclerotome | CRISPR/Cas9 | long-noncoding RNA | ribosomal proteins | test the hypothesis that PEAT influences sclerotome specification BMP pathway through Pax1, we deleted the PEAT locus using the CRISPR/Cas9 system and performed RNA-seq on PEAT mutant embryos. he ribs, vertebrae, and centra of the axial skeleton derive Tfrom the sclerotome, a transient mesodermal population Results specified from the medial-ventral aspect of the embryonic so- The Sclerotome Transcriptome. Our objective was to comprehen- mite. Sclerotomal cells undergo an epithelial-to-mesenchymal sively catalog the genes activated during sclerotome development. transition and migration to surround the spinal cord and extend To isolate embryonic sclerotome precursors, we genetically processes and ribs. Multiple genes and pathways required for the marked the somite and manipulated this tissue toward the scle- specification of the sclerotome, as well as the subsequent dif- rotome fate with Hh agonist and BMP antagonist. The Notch li- ferentiation and elaboration of axial skeletal progenitors, have gand Dll1 is expressed in precursors of the paraxial, intermediate, been identified. Hedgehog (Hh) signals, principally Sonic (Shh), and lateral plate, as well as restricted populations in the nervous instruct sclerotome formation from the naïve somite (1). The system (11). The regulatory elements directing Dll1 (12) have been bone morphogenetic protein (BMP) antagonists Noggin and used to generate a mouse line expressing Cre (Dll1-msd) (13). We Gremlin also permit Hh-mediated activation of transcription crossed these Dll1-msd Cre animals to R26-stop-EYFP cre reporter factors required for axial skeleton development (2-4). mice to fluorescently mark all derivatives of cells bearing Dll1-msd Pax1 encodes a paired-domain transcription factor expressed in enhancer activity (Fig. 1A). We dissected competent posterior early sclerotome, and responds directly to Hh within hours of somite formation. Along with Pax9, Pax1 is required for axial Significance skeleton development (5). Other transcription factors have more restricted roles; for example, Uncx is required for formation of the The majority of transcription generates noncoding RNAs, most of transverse processes, proximal ribs, and pedicles (6). As the scle- whichareuncharacterized.UsingRNA-seqonculturedmouse rotome progenitors mature, BMP signaling loses its inhibitory sclerotome, we identified PEAT, a long-noncoding RNA (lncRNA) activity and instead assumes a positive role in axial skeleton de- adjacent to a key regulator of sclerotome, Pax1. We deleted the velopment, where Nkx3-2 and Sox9 drive cartilage gene expression PEAT entire -transcribed unit using CRISPR/Cas9 and analyzed RNA- BIOLOGY in an autoregulatory loop maintained by BMP (7). seq from mutant embryos. While some lncRNAs regulate the DEVELOPMENTAL The axial skeleton provides support and locomotive potential, expression of their proximal genes, our analysis showed Pax1 as well as protection for the internal organs. Variation of axial expression to be unchanged. However, we identified 60 ribo- skeletal developmental programs has enabled the successful ra- somal proteins with elevated expression, and found evidence that diation of vertebrates into diverse environments. In humans, the bone morphogenetic protein signaling is slightly elevated in PEAT axial skeleton supports the entire body anterior to the pelvis, a mutants. This study reveals a role for the lncRNA PEAT in scle- task for which it functions imperfectly. Globally, back pain is the rotome development and shows next-generation sequencing to be single most prevalent disability (8), with an economic burden of a powerful tool to reveal surprising functions for lncRNAs. billions of dollars annually (9). In addition, an aging population will increase the incidence of skeletal diseases along with costs to Author contributions: R.M.H. and D.A.S. designed research; D.A.S. performed research; R.M.H., the healthcare system. To date, there is no effective therapy to D.A.S., D.S.D., and J.K.C. analyzed data; and R.M.H., D.A.S., D.S.D., and J.K.C. wrote the paper. replace lost bone. Despite the evolutionary and biomedical sig- Reviewers: M.B., Pasteur Institute; and C.-M.F., Carnegie Institution of Washington. nificance of this organ system, our knowledge of sclerotome The authors declare no conflict of interest. formation and axial skeleton development lacks a comprehensive Data deposition: The data reported in this paper have been deposited in the Gene Ex- catalog of transcripts, so our knowledge of the transcriptional pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE86112). network underlying sclerotome differentiation is incomplete. 1To whom correspondence should be addressed. Email: [email protected]. Next-generation sequencing is the ideal tool to quantitatively This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. define transcripts in the sclerotome lineage. However, obtaining 1073/pnas.1612069113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1612069113 PNAS | January 3, 2017 | vol. 114 | no. 1 | 101–106 Downloaded by guest on September 29, 2021 paraxial mesoderm from Dll1-msd Cre;R26-stop-EYFP embryos at A Scatter plot of sclerotome and control transcript abundances 9-d postconception and cultured explants for 12 h with a “midline cocktail” containing sonic agonist (SAG), to mimic Shh from the notochord and floorplate (1), and the BMPR1 inhibitor LDN slope = 1.095 193189 (LDN), to mimic notochord-derived Noggin and derma- – tome derived Gremlin (2 4). Because we isolated explants from 15 the posterior mesoderm before somite patterning, we hypothe- sized that any effects would include changes in gene expression Nkx3−1 coincident with or upstream of sclerotome induction. By quanti- A tative PCR (qPCR) we observed a 14-fold relative induction of 10 Heyl Pax1 ed counts) Pax1 in LDN and SAG-treated samples and a concomitant re- z Bmp3 Id1 Id2 duction in markers of other cell types derived from the Dll1 Pax9 Id3 lineage (Fig. 1B), confirming that sclerotome was induced. We mali + Sclerotome AI646519 collected EYFP cells from dissociated SAG/LDN-treated and 5 Pax8 vehicle control cultures and analyzed gene expression by RNA-seq from four biological replicates (Fig. 1C). log2(Nor Using DESeq2, we identified 264 genes with statistically sig- Myf5 < nificant (adjusted P 0.05), greater than twofold differential 0 expression between control and sclerotome-directed cultures, with 98 transcripts elevated in response to SAG and LDN and 166 transcripts reduced by the treatment (Table S1). Known early sclerotome-specific genes featured prominently in the 0 51015 sclerotome-directed dataset. Pax1 and Pax9 expression was ele- Control log2(Normalized Counts) vated in directed samples 6.2- and 2.4-fold, respectively. Ex- pression of the paralog of Bapx/Nkx3-2, Nkx3-1, was 7.2 times B WT E9.5 whole mount ISH for sclerotome-induced transcripts that of control samples. Similar to Pax1, Nkx3-1 expression was 1 2 3 4 detected in somites 2 h after formation of the somite (Fig. 2B). Although Nkx3-1 is dispensable for skeletal formation, the se- verity of the Bapx/Nkx3-2 skeletal phenotype is enhanced in the absence of Nkx3-1 (14). Heyl, a member of the hairy/enhancer- of-split related family of transcription factors, was expressed at 6.0 times the level of controls. Heyl is expressed in the early Nkx3.1 Nkx3.2
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