Mouse knockout models reveal largely dispensable ...

Letter to the Editor

Mouse knockout models reveal largely dispensable but context-dependent functions of lncRNAs during development Xue Han1,*, Sai Luo1,2,*, Guangdun Peng3,*, J Yuyang Lu1, Guizhong Cui3, Lichao Liu1, Pixi Yan1, Yafei Yin1, Wei Liu1, Ran Wang3, Zai Chang4, Jie Na1, Naihe Jing3, and Xiaohua Shen1

1

Tsinghua-Peking Center for Life Sciences, School of Medicine, Tsinghua University, Beijing

100084, China. 2

Present address: Program in Cellular and Molecular Medicine, Boston Children's Hospital;

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 3

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science,

Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China. 4

School of Life Sciences, Tsinghua University, Beijing, China.

*

These authors contributed equally to this work.

Correspondence: Xiaohua Shen*, Naihe Jingb *

Tel: +86-10-62781029

Email: [email protected] b

E-mail: [email protected]

Running title: Spatiotemporal role of Evx1as in embryonic patterning Keywords: lncRNA, knockout mouse, Evx1as, gastrulation, development

© The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. 1

Downloaded from https://academic.oup.com/jmcb/advance-article-abstract/doi/10.1093/jmcb/mjy003/4840688 by guest on 07 February 2018

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 (Figure 1A and Supplementary Figure S1). 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 1A 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, 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 1A). 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, 2


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 KO mice homozygous for 11 out of 12 lncRNAs were born at the expected Mendelian ratio (~25%) and were viable with no obvious abnormalities (Figure 1A). 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 1A 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 (Uph) polyA knockin mice, which partially phenocopy HAND2 knockout mice (Anderson et al., 2016). Our results suggest that the Hand2as (Uph) DNA locus, rather than its transcription/transcripts, primarily controls heart development and function (data not shown). The fact that different knockout strategies produce distinct phenotypes emphasizes the necessity of utilizing complementary genetic approaches to study lncRNA function in vivo. Nevertheless, studies suggest that in general these 12 lncRNAs are dispensable for development and animal survival under laboratory growth conditions. Then, we sought to probe lncRNA function in a particular developmental setting. We reported previously that Evx1as acts in cis to promote EVX1 transcription and lineage differentiation in embryonic stem cells (ESCs) (Luo et al., 2016). Here we further demonstrated that in ESCs, the homozygous insertion of a 4× polyA stop signal downstream of the transcription start site of Evx1as abolished the expression of both Evx1as and EVX1, and also attenuated activation of mesendodermal genes upon LIF withdrawal-induced differentiation (Supplementary Figure S3A−E 3


and Table S1). Next, we sought to probe the in vivo function of Evx1as in mouse gastrulation, during which the primary germ layers are formed to establish the basic body plan of the embryo. In a gastrulating embryo, the mesoderm and endoderm cells are emerging from the primitive streak of the posterior epiblast and gradually form distinct cell types that consist of the progenitors for the respective derivatives (Fossat et al., 2007; Tam and Loebel, 2007; Arnold and Robertson, 2009; Peng and Jing, 2017). To systematically survey the expression profiles of EVX1 and Evx1as, we first performed spatial transcriptome analysis on cryosections of gastrulating embryos from E6.5 to E7.5 by low-input RNA sequencing (Geo-seq) (Figure 1B) (Peng et al., 2016; Chen et al., 2017). As illustrated in the corn plots, Evx1as and EVX1 expression is highly correlated, and both are strongly detected in the proximal region of the epiblast, with a prominent presence in the primitive streak and gradually decreased signals toward the distal (Figure 1C). This graded expression pattern of Evx1as and EVX1 is similar to that determined by RNA in situ hybridization in gastrulating embryos (E6.5−7.5) (Dush and Martin, 1992; Bell et al., 2016). Compared to the primitive streak (P) at E7.0, the nascent mesoderm (M) at E7.0 shows much weaker and graded expression of Evx1as and EVX1 (Figure 1C). The spatially and temporally restricted expression pattern of Evx1as suggests that it may have a transient role in modulating cell fate choice and proximal-to-distal patterning in the primitive streak. To probe Evx1as function in vivo, we first partially dissected the proximal (designated Pp) and distal (Pd) regions of the posterior half of E7.0 mid-gastrulation embryos containing the primitive streak (Figure 1B). Compared to heterozygous littermates obtained by heterozygous and homozygous crosses, Evx1as-/- embryos showed ~1.5-fold decreased expression of EVX1 in the Pp (Supplementary Figure S3F and Figure 1D), and exhibited altered expression of lineage genes, similar to that observed in ESCs lacking or depleted of EVX1 (Supplementary Figure S3D). Gene ontology (GO) analysis of dysregulated genes revealed significant enrichment of functional terms 4


related to pattern specification and embryonic development (Supplementary Figure S3G and Tables S2 and S3). To reveal spatial gene expression in more detail, we performed Geo-seq analysis of cryosections of the primitive streak in E7.0 embryos (Figure 1B). In the Evx1as-/- embryo, Evx1as expression was abolished and EVX1 mRNA was dramatically reduced in sections toward the proximal end of the primitive streak (section S11) (Figure 1E). Interestingly, expression alterations of a set of developmental genes were also observed in the primitive streak of the Evx1as-/- embryo (Figure 1F and G and Supplementary Table S4). First, a subset of master regulators of mesendoderm and mesoderm development (Pfister et al., 2007; Tam and Loebel, 2007), including T, WNT3A, LEFTY2, and FST were downregulated, while others including MIXL1, EOMES, WNT3, and CITED1 were aberrantly upregulated with a graded increase toward the proximal streak (S11) in the Evx1as-/- embryo (Figure 1F). Second, in a similar way, genes that are expressed in the distal streak and involved in endoderm commitment, were down- or upregulated in the Evx1as-/- embryo (Figure 1F). For example, the expression of early maker genes for endoderm commitment, including SOX17 and NODAL, was downregulated and became less restricted to the anterior-most region (section S1) of the streak, whereas other endoderm genes, including GSC, CXCR4, and FOXA2, were hyper-activated in the distal streak (S1) of the Evx1as-/- embryo (Figure 1F). Third, genes that are expressed in the neuroectoderm or associated with pluripotency, including NANOG, POU5F1, ID1, and OLIG1, exhibited aberrant activation in the Evx1as-/- embryo (Figure 1F), indicating a mis-patterning of germ layer genes. Expression of both Evx1as and EVX1 was significantly downregulated in the nascent mesoderm (M) layer that migrated out of the primitive streak (E7.0) (Figure 1B and C). To analyze Evx1as function beyond the primitive streak, we performed transcriptome analysis on cryosections of the nascent mesoderm layer of the same embryos (E7.0) analyzed above (Figure 1B). Interestingly, despite the loss of Evx1as expression, EVX1 expression was very weakly detected and slightly upregulated in the nascent mesoderm of the Evx1as-/- embryo, suggesting delayed downregulation 5


of EVX1 in absence of Evx1as or transcriptional compensation in subsequent lineage differentiation in vivo (Figure 1E and Supplementary Figure S3H). In addition, expression alterations of embryonic patterning genes in the nascent mesoderm were significantly ameliorated compared to those in the primitive steak between Evx1as-/- and Evx1as+/- embryos (Figure 1G and Supplementary Figure S3H and Table S5). Taken together, temporary transcription dysregulation observed in Evx1as-/embryos defines a short developmental window in which Evx1as is transiently required for coordinated, spatiotemporal expression in prospective mesoderm and endoderm cells in the primitive streak. Studies of the Evx1as/EVX1 locus in vitro and in vivo provide an excellent model to understand the context-dependent functions of an lncRNA. First, despite severe defects in mesendoderm differentiation observed in vitro ESC culture upon deleting or depleting Evx1as (Luo et al., 2016), Evx1as-/- mice were viable, healthy, and fertile. Second, careful analysis of mid-gastrulation Evx1as-/- embryos revealed a profound transcriptional defect in the patterning of the primitive streak, which surprisingly did not lead to a strong effect on embryonic development. There may be compensatory mechanisms in vivo that lead to transcriptional recovery from the loss of Evx1as. Third, the transcriptome of Evx1as-/- primitive streak resembled that of ESCs lacking EVX1, supporting a cis-regulatory role of Evx1as on EVX1 expression in vivo (Luo et al., 2016). Nevertheless, the transient role of Evx1as in embryonic patterning indicates that Evx1as is not a major determinant of cell fate in vivo. Out of the 12 lncRNAs, only one lncRNA Hand2as (17-kb deletion) caused a perinatal lethal phenotype (data not shown). Gastrulation is tightly regulated by a multitude of gene regulatory networks, morphogenetic interactions, and signaling pathways, which provide failsafe mechanisms to ensure correct embryonic development (Fossat et al., 2007; Tam and Loebel, 2007; Arnold and Robertson, 2009; Peng and Jing, 2017). We believe that development, especially gastrulation, is a very robust process and may be less likely to be disrupted by lncRNA mutations. Many lncRNAs may function transiently and spatiotemporally without influencing overall fitness of animals. This is 6


consistent with the proposed roles of lncRNAs in fine-tuning biological processes and regulating the spatiotemporal expression of pleiotropic developmental loci instead of being master regulators or switches of development (Morris and Mattick, 2014; Luo et al., 2016). Comprehensive and careful revelation of the in vivo functions of lncRNAs in animal models remains the main challenge for the lncRNA field.

Data availability RNA-Seq data of Evx1as mutant embryos and Evx1as polyA knockin ESCs have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE104292. Supplementary material is available at Journal of Molecular Cell Biology online.

Acknowledgements We thank L. Yu, G. Ou, Y. Chen, F. Tang, Q. Xi, and Shen Laboratory members for insightful discussion and critical reading. This study was supported by the National Natural Science Foundation of China (8141101062, 20161310854, and 31471219 to X.S., 31430058, 31571513, 31630043, 91519314, and 31661143042 to N.J.), the National Basic Research Program of China (2017YFA0504204 to X.S., 2014CB964804, 2015CB964500, and 2017YFA0102700 to N.J.), and the Center for Life Sciences (CLS) at Tsinghua University.

Figure legend Figure 1 The spatial and temporal role of Evx1as in embryonic patterning. (A) Summary of the 12 lncRNA candidates selected for targeted deletion in mouse. lncRNAs were transcribed in an antisense direction and positioned head-to-head (XH, divergent) or tail-to-tail (XT, convergent) to protein-coding genes within 5 kb, or were transcribed 5 kb away from a protein-coding gene (lincRNA). P, 5’ proximal knockout; D, 3’ distal knockout; F, full-length knockout; -/- (%), survival rate of -/- (homozygotes) out of analyzed mice. (B) Graphic representation of the transverse sections of mouse embryos at E6.5, E7.0, and E7.5 (Top). Evx1as mutant embryos at E7.0 were either partially dissected into proximal (Pp) and distal (Pd) sections of the posterior half (left), or 7


fully dissected into a serial of sections (from S1 to S11, alternative sections were used for RNA-Seq) including the posterior epiblast (P, including primitive streak) and the nascent mesoderm (M) (right). A, anterior epiblast; L, left lateral epiblast (including L1 and L2); R, right lateral epiblast (including R1 and R2). (C) Corn plots of Geo-seq analysis showing the spatial expression patterns of Evx1as and EVX1 in wild-type mouse embryos at E6.5, E7.0, and E7.5 stages. Each colored dot represents the cell sample at the respective position illustrated in B, and the color indicates the level of gene expression as determined by normalized gene expression log2 (FPKM +1). Section number is shown on the left of each corn plot. (D) qRT-PCR analysis of Evx1as mutant embryos (E7.0). The Pp and Pd regions are illustrated in B. The y-axis shows expression relative to GAPDH. Data are shown as mean ± SD (n=3 embryos per genotype). *, P＜0.05. (E) Corn plots showing the spatial expression patterns of Evx1as and EVX1 in Evx1as mutant embryos with full dissection of the primitive streak (P) and nascent mesoderm (M) illustrated in B. Scales represent levels of gene expression as FPKM. (F) Heatmap showing dysregulated genes involved in lineage development in primitive streak of Evx1as mutant embryos at E7.0. Section numbers are shown on the top. (G) Correlation co-efficiency analysis of expression of genes shown in F in the primitive streak and nascent mesoderm between Evx1as-/- and Evx1as+/- embryos.

REFERENCES Anderson, K.M., Anderson, D.M., McAnally, J.R., et al. (2016). Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. Nature 539, 433-436. Arnold, S.J., and Robertson, E.J. (2009). Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol 10, 91-103. Batista, P.J., and Chang, H.Y. (2013). Long noncoding RNAs: cellular address codes in development and disease. Cell 152, 1298-1307. Bell, C.C., Amaral, P.P., Kalsbeek, A., et al. (2016). The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation. Sci Rep 6, 26657. Cech, T.R., and Steitz, J.A. (2014). The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94. Chen, J., Suo, S., Tam, P.P., et al. (2017). Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq. Nat Protoc 12, 566-580. Djebali, S., Davis, C.A., Merkel, A., et al. (2012). Landscape of transcription in human cells. Nature 489, 101-108. 8


Dush, M.K., and Martin, G.R. (1992). Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak. Dev Biol 151, 273-287. Engreitz, J.M., Haines, J.E., Perez, E.M., et al. (2016). Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539, 452-455. Fossat, N., Pfister, S., and Tam, P.P. (2007). A transcriptome landscape of mouse embryogenesis. Dev Cell 13, 761-762. Hanna, L.A., Foreman, R.K., Tarasenko, I.A., et al. (2002). Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev 16, 2650-2661. Hon, C.C., Ramilowski, J.A., Harshbarger, J., et al. (2017). An atlas of human long non-coding RNAs with accurate 5' ends. Nature 543, 199-204. Hu, W., Alvarez-Dominguez, J.R., and Lodish, H.F. (2012). Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO Rep 13, 971-983. Klattenhoff, C.A., Scheuermann, J.C., Surface, L.E., et al. (2013). Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell 152, 570-583. Koutsourakis, M., Langeveld, A., Patient, R., et al. (1999). The transcription factor GATA6 is essential for early extraembryonic development. Development 126, 723-732. Lee, J.T. (2012). Epigenetic regulation by long noncoding RNAs. Science 338, 1435-1439. Li, M., Gou, H., Tripathi, B.K., et al. (2015). An Apela RNA-Containing Negative Feedback Loop Regulates p53-Mediated Apoptosis in Embryonic Stem Cells. Cell Stem Cell 16, 669-683. Lufkin, T., Dierich, A., LeMeur, M., et al. (1991). Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression. Cell 66, 1105-1119. Luo, S., Lu, J.Y., Liu, L., et al. (2016). Divergent lncRNAs Regulate Gene Expression and Lineage Differentiation in Pluripotent Cells. Cell Stem Cell 18, 637-652. Morris, K.V., and Mattick, J.S. (2014). The rise of regulatory RNA. Nat Rev Genet 15, 423-437. Orom, U.A., Derrien, T., Beringer, M., et al. (2010). Long noncoding RNAs with enhancer-like function in human cells. Cell 143, 46-58. Pandolfi, P.P., Roth, M.E., Karis, A., et al. (1995). Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 11, 40-44. Pauli, A., Rinn, J.L., and Schier, A.F. (2011). Non-coding RNAs as regulators of embryogenesis. Nat Rev Genet 12, 136-149. Peng, G., and Jing, N. (2017). The genome-wide molecular regulation of mouse gastrulation embryo. Sci China Life Sci 60, 363-369. Peng, G., Suo, S., Chen, J., et al. (2016). Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo. Dev Cell 36, 681-697. Pfister, S., Steiner, K.A., and Tam, P.P. (2007). Gene expression pattern and progression of embryogenesis in the immediate post-implantation period of mouse development. Gene Expr Patterns 7, 558-573. Shawlot, W., Wakamiya, M., Kwan, K.M., et al. (1999). Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse. Development 126, 4925-4932. Srivastava, D., Thomas, T., Lin, Q., et al. (1997). Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat Genet 16, 154-160. Tam, P.P., and Loebel, D.A. (2007). Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet 8, 368-381. Ulitsky, I., and Bartel, D.P. (2013). lincRNAs: genomics, evolution, and mechanisms. Cell 154, 26-46. Yin, Y., Yan, P., Lu, J., et al. (2015). Opposing Roles for the lncRNA Haunt and Its Genomic Locus in Regulating HOXA Gene Activation during Embryonic Stem Cell Differentiation. Cell Stem Cell 16, 504-516.

9


A

B E6.5 LncRNA

Class

Evx1as (Evx1os)

XH XH

Hand2as (Uph)

Nearby Convserved Total mice Gene in human analyzed yes

73

36%

HAND2

yes

43 (F)

0%

51 (P)

29%

135 (D)

22%

XH XH XH

FOXD3

yes

43

28%

GATA3

yes

61

21 %

GATA6

yes

61 (P)

16%

36 (D)

17%

LHX1

yes

12

17%

TRAF7

yes

74

20%

Bvht

XH XH XT

Carmn

no

58

26%

Haunt (Halr1)

lincRNA

HOXA1

no

72

21 %

Ape/a

lincRNA

TRIM60

yes

69

23%

Gm10451

Iinc RNA

PPP1R36

no

58

24%

1700020114Rik

lincRNA

CHP1

yes

56

23%

Foxd3as (Gm12688) Gata3as (4930412013Rik) Gata6as (1010001N08Rik)

Lhx1as (Lhx1os) Traflas (Rab26os)

@

-1- (%)

EVX1

E7.5 M

,

M 811

Partial Dissection

Distal (d )

Full Dissection

F

Evx1as·IP

3

5

7

9 11 (Section#)

Evx1as EVX1 FZ010 T HOXA1 SPRY2 LEFTY2 HOXB2 CDX1 NKX1-2 FST HOXB1 TBX6 FGF4 WNT3A HES7 SNA/1 MIXL1 EOMES WNT3 FLT1 FGFB MESP1 MSX1 DAB2 CITED1 DKK1 PKOCC

c

67..

proximal

Evx1as

11

p

A

9 7

5 4 3

2 distal 1

5 3 4 L _ __ ____J

proximal

6. • 7

EVX1

•

5

•

4

•

3 2

distal 1

11

3

9

2

7

5

0

3

• L___ __ _ j

E6.5

E7.0

FOXH1 CYP26A1 CXCR4 HHEX FZOB LHX1 FOXA2 SHB SOX17 XBP1 NNAT IFITM1 NODAL NOG

E

D EVX 1

G

GSC

E7.5

ij' c

t,

Q)

""

Q)

-o

~

0.6

"k

0.4

~ ~

0.2

0

~ ~

0

lJ.J

u 0

• Evx1as-l-

•• ••

-1-

•• •• •

0.0

•• ~ • •

4

11

3

9 7

2

5

3 0

•• ••• +/-

•

p-value=0.011

~11

'E

9

!i> 7 -o 5l 5 Q) ~ 3

0.8

11

0.6

•• •• •

Evx1as

7 6 5

4 3 2

0

•

•

9

•

•

7 0.4

5

•• ••

0.2

3

•

1.5

0.5

• 0

EVX1


0

c.. CD

3 I

co

ill CD

c..

cc CD

:::l

CD

(/)

m :::l c.. 0 c.. CD

3..., I

CD

ill CD

c..

101 103 LSM14A NGRN GAP43 NANOG POU5F1 CFOP1 JAR/02 RAB18 OLIG1 OTX2 RNF114 ZIC5 PTK2

-1-

0

..L.....---..-----r-Primitive Mesoderm streak

-1-

+/-

•

~ 0.8

ffi

+/-

1.0