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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 1  |  Page : 5-10

Deficiency of antisense lncRNA Gm48853 resulted in embryonic lethality and impaired placental development in mice


1 The Institute of Reproduction and Developmental Biology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200438, China
2 Institute of Developmental Biology and Molecular Medicine, Fudan University, Shanghai 200438, China
3 The Institute of Reproduction and Developmental Biology, Obstetrics and Gynecology Hospital; The State Key Laboratory of Genetic Engineering, MOE Key Laboratory of Contemporary Anthropology, School of Life Science, Fudan University, Shanghai 200438, China
4 The Institute of Reproduction and Developmental Biology, Obstetrics and Gynecology Hospital; Institute of Developmental Biology and Molecular Medicine; Children's Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai 200438, China

Date of Submission22-Jan-2019
Date of Web Publication11-Apr-2019

Correspondence Address:
Hong-Yan Wang
The State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai 200438
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.255984

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  Abstract 


Objective: Long noncoding RNAs (lncRNAs) play important roles in embryonic development and in various diseases. Gm48853 is a predicted natural antisense lncRNA gene of unknown function. The present study aimed to investigate its expression and function.
Methods: Quantitative reverse transcription-polymerase chain reaction and sequencing were used to detect the expression of Gm48853 antisense lncRNA. Hematoxylin and eosin staining and pathological analysis were applied to evaluate the effects of Gm48853 mutations on embryonic/placental development. 5-Ethynyl-2'-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase deoxy-UTP-nick-end labeling assays were used to analyze the status of cell proliferation and apoptosis in Gm48853- deficient embryos and placentas.
Results: PiggyBac (PB) transposon insertion into the intron of Gm48853 resulted in a significant decrease in Gm48853 expression and embryonic lethality at midgestation. Homozygous Gm48853 mutations led to severe growth retardation of embryos and impaired the morphogenesis of the placental labyrinth. In addition, cell proliferation was dramatically decreased in Gm48853PB/PB embryos and placentas, and apoptosis was radically increased in the mutant embryos but not in the mutant placentas.
Conclusion: Antisense lncRNA Gm48853 may regulate embryonic/placental development by enhancing cell proliferation and/or inhibiting apoptosis.

Keywords: Antisense lncRNA; Embryonic Development; Gm48853; Placental Development


How to cite this article:
Qin QY, Zeng WJ, Peng CY, Zheng YF, Wang HY. Deficiency of antisense lncRNA Gm48853 resulted in embryonic lethality and impaired placental development in mice. Reprod Dev Med 2019;3:5-10

How to cite this URL:
Qin QY, Zeng WJ, Peng CY, Zheng YF, Wang HY. Deficiency of antisense lncRNA Gm48853 resulted in embryonic lethality and impaired placental development in mice. Reprod Dev Med [serial online] 2019 [cited 2019 Apr 24];3:5-10. Available from: http://www.repdevmed.org/text.asp?2019/3/1/5/255984




  Introduction Top


Long noncoding RNAs (lncRNAs) (>200 nucleotides) play important roles in the regulation of gene expression during development and in the pathogenesis of a growing number of diseases, such as myopathy, some recessively inherited life-threatening pregnancy complications, congenital heart disease, cardiomyopathies, autism, and various cancers.[1],[2],[3],[4],[5] Recognizing the roles of lncRNAs in human diseases and understanding their underlying mechanisms have unveiled new diagnostic and therapeutic opportunities.[3],[5],[6]

Natural antisense RNAs are a type of lncRNA that regulate gene expression in a wide range of eukaryotic organisms. Antisense RNA may form a sense-antisense pairs with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription, mRNA stability, and protein synthesis.[7],[8],[9],[10] Mouse Gm48853 (ENSMUSG00000110740, ch9: 44332684–44334386) was predicted to encode a 217 bp antisense lncRNA by Ensembl (MGI: 6098590) (http://www.informatics.jax.org/marker/MGI: 6098590). However, its expression and physiological functions in mice remain unknown.

The placenta is a vital organ formed during mammalian embryogenesis that establishes the maternal-fetal circulatory system for nutrients and gas exchange as well as fetal metabolic waste disposal.[11],[12],[13] Developmental and/or functional defects result in fetal growth restriction or, if severe, fetal death.[14],[15],[16],[17],[18] To date, more than 100 mouse models generated by gene-targeting technology have been reported to exhibit various defects in placenta morphogenesis. A common phenotype among the different mutants is that the labyrinth is much thinner and smaller than normal.[14] However, the detailed molecular mechanisms underlying the effects of these mutations are not fully understood and whether antisense lncRNAs participate in the regulation of placenta/labyrinth development remains unknown.

In this study, we report that Gm48853 is expressed in mice and PiggyBac (PB) transposon insertion into the intron of Gm48853 resulted in a significant decrease of Gm48853 expression and embryonic lethality at midgestation. Gm48853PB/PB embryos displayed severe growth retardation, and the labyrinth of Gm48853PB/PB placenta was much thinner and contained far fewer fetal blood vessels and maternal sinusoids. We also found that cell proliferation dramatically decreased in Gm48853PB/PB embryos and placentas, and apoptosis was radically increased in the mutant embryos but not in the mutantplacentas. These results suggested that Gm48853 might affect embryonic/placental development by regulatingcell proliferation and/or apoptosis.


  Methods Top


Mice

Gm48853 PB heterozygote mutant mice (hereafter termed Gm48853+/PB mice) were generated on the FVB/NJ background[19] and maintained on 12/12-h light/dark cycles. Mapping information for the PB insertion in Gm48853PB allele can be found in the PBmice database (idm.fudan.edu.cn/PBmice). Gm48853PB/PB embryos were derived by intercrossing Gm48853+/PB mice. Experiments were conducted with consent from the Animal Care and Use Committee of the Institute of Developmental Biology and Molecular Medicine at Fudan University, Shanghai, China.

Genotyping polymerase chain reaction

The toes of neonatal mice at postnatal day (P8) or the yolk sac of embryonic day (E) 8.5 to E13.5 embryos were dissected from intercrossed Gm48853+/PB female mice and used to extract genomic DNA, followed by genotyping polymerase chain reaction (PCR). The primers used are listed in [Table 1].
Table 1: Primer sequences for genotyping and qRT-PCR used in this study

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Quantitative reverse transcription-polymerase chain reaction

Embryos were collected from intercrossed Gm48853+/PB female mice, frozen at −80°C, and subsequently ground to powder using liquid nitrogen. Total RNA was extracted using TRIzol (TIANGEN Biotech Co. Ltd., Beijing, China) and treated with RNase-free DNase to remove contaminated genomic DNA. Reverse transcription was performed with the FastQuant RT Kit (TIANGEN Biotech Co. Ltd., Beijing, China). Quantitative reverse transcription-PCR (qRT-PCR) was performed according to the instructions of the RT-PCR Kit (TIANGEN Biotech Co. Ltd., Beijing, China) on Applied Biosystems Sequence Detection System (ABI, Waltham, USA). GAPDH was used as an internal control. The primers used are listed in [Table 1].

Hematoxylin and eosin staining, terminal deoxynucleotidyl transferase deoxy-UTP-nick-end labeling, and 5-ethynyl-2'-deoxyuridine assays

For paraffin section, dissected placentas or embryos were fixed in 4% paraformaldehyde (PFA) overnight, dehydrated in alcohol and xylene solutions, and then embedded in paraffin. The sections were collected at a thickness of 5 μm.

For the 5-ethynyl-2'-deoxyuridine (EdU) assays, E10.5 embryos and placentas were dissected from mice that had been intraperitoneally injected with EdU 4 h before euthanization, fixed in 4% PFA at 4°C overnight, dehydrated using 15% sucrose and 30% sucrose in phosphate-buffered saline for 1 and 2 h, respectively, embedded in optimal cutting temperature compound (Richard-Allan Scientific, Kalamazoo, MI, USA), and quickly frozen in liquid nitrogen. The frozen sections were collected at a thickness of 12 μm. EdU-positive cells were detected using an EdU Alexa Fluor 488 Imaging Kit according to the manufacture's protocol (Invitrogen no. C10337).

For terminal deoxynucleotidyl transferase deoxy-UTP-nick-end labeling (TUNEL) assay, the frozen sections of embryos and placentas were subjected to the TUNEL assay as described by Kong et al.[20] by TUNEL assay Kit (Invitrogen no. C10617).

Statistics

Statistical analysis was performed using Chi-square test for analyzing genotype ratio of the progeny from intercrossed Gm48853+/PB female mice. An unpaired t-test was used to analyze the other data by GraphPad Prism 4 (Prism, GraphPad Software Inc., San Diego CA, USA). P < 0.05 was considered statistically significant.


  Results Top


PiggyBac homozygous mutation impairs Gm48853 expression

Gm48853 is predicted to encode a 217 bp long noncoding antisense RNA in mice by Ensembl (MGI: 6098590). Gm48853 contains two exons separated by a 1,486 bp intron on mouse chromosome 9. Gm48853+/PB mice carry a Gm48853 mutant allele disrupted by the insertion of a PB transposon into the intron [Figure 1]a. To ascertain the expression of Gm48853 in mice, and to study the effect of PB insertion on Gm48853 expression, we examined the expression of Gm48853 in mouse NIH3T3 cells, wild-type (WT), and Gm48853 mutantmouse embryos using qRT-PCR, followed by DNA sequencing. The results showed that Gm48853 was expressed in NIH3T3 cells and E10.5 WT mouse embryos, and the expression level of Gm48853 lncRNA was not significantly altered in Gm48853+PBembryos, but was reduced about 40% in Gm48853PB/PBembryos compared with that in the WT control [Figure 1]c.
Figure 1: PB homozygous mutant impairs Gm48853 expression. (a) Diagram of the genomic structure of the mutant Gm48853 allele containing a PB transposon insertion. Arrows above the line/box represent the primers used for genotyping PCR, and arrows beneath the box represent the primers used for qRT-PCR to amplify 217 bp Gm48853 fragment. (b) Genotyping results of embryos from intercrossed Gm48853+/PBmice. (c) Statistical analysis of qRT-PCR results for Gm48853 RNA in NIH3T3 cells, WT, heterozygous, and homozygous mutant embryos at E10.5 (three batches of NIH3T3 cells and more three mouse embryos for each genotype were used for the experiment). Values in (c) represent the mean ± standard error of the mean. *P < 0.01; ns, no significance. PB: PiggyBac; PCR: Polymerase chain reaction; qRT-PCR: Quantitative reverse transcript-PCR; WT: Wild-type.

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Growth retardation of Gm48853PB/PB embryos

To investigate the physiological function of Gm48853, we intercrossed Gm48853+/PBmice. No viable Gm48853 homozygousmice were observed at weaning (data not shown), indicating the potential embryonic lethality of Gm48853 homozygous mutants. Therefore, embryos at various stages (E9.5, E10.5, and E11.5–13.5) were collected from intercrossed Gm48853+/PBmice and genotyped by PCR [Figure 1]b. No Gm48853PB/PBembryos were recovered beyond E11.5, and homozygous embryos at E11.0 were found to have undergone resorption, indicating that these Gm48853PB/PBembryos died at the midgastrulation. Living Gm48853PB/PB embryos could be recovered at E10.5; however, they displayed severe growth retardation, including incomplete embryo turning, spinal dysraphism, and abnormal tail bending [Figure 2]A and [Figure 2]B], compared with WT and Gm48853+/PB littermates.
Figure 2: Gm48853 mutation resulted in severe embryonic growth retardation. (A) Photomicrographs of Gm48853+/+embryos (a and d), Gm48853+/PBembryos (b and e), Gm48853PB/PBembryos (c and f) at E9.5 (a-c) and E10.5 (d-f). (B) H and E-stained paraffin sections of WT and Gm48853PB/PBembryos at E10.5. (C and D) Images of embryonal cryosections with the indicated genotypes for the EdU assay (C) and TUNEL assay (D). High-magnification images of the cropped region in (B) are shown beneath each panel. Images are representative sections from at least 3 embryos per group. EdU: 5-Ethynyl-2'-deoxyuridine; TUNEL: Terminal deoxynucleotidyl transferase deoxy-UTP-nick-end labeling; H and E: Hematoxylin and eosin.

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Among the 340 E9.5–E10.5 embryos evaluated, 91 were WT, 185 were heterozygotes, and 61 were homozygotes [Table 2], suggesting that the number of homozygous mutant embryo was close to the expected Mendelian ratio (Chi-square P = 0.48).
Table 2: Summary of the embryonic genotyping results*

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Decreased proliferation and enhanced apoptosis of Gm48853PB/PB embryos

To understand the cellular mechanisms underlying the growth retardation of Gm48853PB/PB embryos, we first evaluated cell proliferation in Gm48853PB/PB embryos at E10.5 by the EdU assay. The results showed that positive EdU signals were rarely observed in Gm48853PB/PB embryos, whereas the control embryos displayed extensive EdU signals [Figure 2]C, demonstrating that the proliferation of Gm48853PB/PB embryonic cells was severely impaired. We further evaluated apoptosis of Gm48853PB/PB embryos by the TUNEL assay. We found that the number of apoptotic cells obviously increased in Gm48853PB/PBembryos at E10.5 compared with that in the control embryos [Figure 2]D. These results suggested that inhibition of cell proliferation and enhanced apoptosis might lead to the growth retardation of Gm48853PB/PB mutant embryos.

Severely impaired labyrinth morphogenesis in Gm48853PB/PB placentas

Developmental and/or functional defects of the placenta frequently result in severe fetal growth retardation, or fetal death in severe cases, in many gene targeted mice.[12],[13],[14] To study whether Gm48853 affectsplacental development, we performed pathological analysis of Gm48853PB/PB placenta. We observed that the labyrinths of Gm48853PB/PB placentas at E9.0 and E10.5 were much thinner and contained many fewer fetal blood vessels and maternal sinusoids than those of the control [Figure 3]a and [Figure 3]b. In addition, we also evaluated cell proliferation and apoptosis in Gm48853PB/PB placentas and found that cell proliferation in the labyrinth of Gm48853PB/PB placentas was dramatically inhibited [Figure 3]c, whereas the apoptosis signal in the labyrinth was similar between Gm48853PB/PB placenta and control [Figure 3]d. These results demonstrated that disruption of Gm48853 blocks normal labyrinth morphogenesis by affecting cell proliferation, suggesting that growth retardation of Gm48853PB/PB embryos might result from defects in the labyrinth of Gm48853PB/PB placentas.
Figure 3: Gm48853 mutation impaired placental development. (a and b) Images of H and E-stained placental sections of the WT and Gm48853PB/PBembryos at E9.0 (a) and E10.5 (b). (c and d) Images of EdU assay (c) and TUNEL assay (d) of placental cryosections of the embryos at E10.5 with the indicated genotypes. High-magnification images of the cropped region in (b-d) are shown beneath the respective panel. Dashed lines indicate labyrinth layers. Arrows and arrowheads indicate fetal nucleated and maternal enucleated red blood cells, respectively. Images are representative sections from placentas of 3 embryos per group. WT: Wild-type; H and E: Hematoxylin and eosin; EdU: 5-Ethynyl-2'-deoxyuridine; TUNEL: Terminal deoxynucleotidyl transferase deoxy-UTP-nick-end labeling.

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  Discussion Top


The expression and physiological function of the predicted Gm48853 gene, encoding an antisense lncRNA, have not been previously reported. In this study, we reported it for the first time, and we verified the expression of Gm48853 gene in FVB mouse embryos at E10.5 and in NIH3T3 cells by qRT-PCR and sequencing. The mouse Gm48853 is located at about 300 bps upstream of H2afx gene on mouse chromosome 9. Although no human lncRNA Gm48853 has been annotated in human genome database, we have identified a homologous sequence of mouse lncRNA Gm48853 at the similar location (5'end of H2AFX gene) on human chromosome 11 by blasting with the exon sequence of mouse Gm48853. Our analysis showed that 67% of nucleotides of mouse Gm48853 and putative human GM48853 exons were identical whereas only 46% nucleotides of the intron form these genes were identical. These results indicate that lncRNA GM48853 may exist in human.

We observed that Gm48853PB/PBhomozygous mutant embryos died before E11.5. Gm48853PB/PB embryos at E10.5 displayed severe growth retardation, including incomplete embryo turning, spinal dysraphism, and abnormal tail bending. The morphogenesis of the placental labyrinth of Gm48853PB/PB embryos was also impaired. Severe growth retardation of Gm48853PB/PB embryos could be the direct effect of Gm48853 mutations on embryonic development, but could also be a secondary effect resulting from an insufficient supply of nutrition and oxygen caused by the severely impaired placental development of Gm48853PB/PB embryos.

Further investigation is needed to verify if the placental defect is the primary reason for the severe growth retardation of Gm48853PB/PBembryos. One of the experiments could be tetraploid aggregation assay, in which WT tetraploid cells contribute exclusively to the trophoblast cells of the placenta and extraembryonic endoderm, whereas mutant diploid cells donate to all structures of the fetus and the extraembryonic mesoderm, including the yolk sac, allantois, and the fetal blood vessels of the placenta.[11]

Our studies have demonstrated that deficiency of antisense lncRNA Gm48853 results in embryonic lethality and impaired placental development in mice; however, the underlying molecular mechanism remains elusive. Further investigation to identify the target genes of Gm48853 is required to understand the molecular mechanism by which Gm48853 regulates embryonic/placental development.

In summary, antisense Gm48853 plays an important role in the development of embryos and placentas by regulating cell proliferation and/or apoptosis.

Financial support and sponsorship

This work was supported by the grants from the National Key Research and Development Program of China (2016YFC1000500), the National Natural Science Foundation of China (81430005, 31521003), and the Commission for Science and Technology of Shanghai Municipality (13JC1407600) to H_Y Wang.

Conflicts of interest

There are no conflicts of interest.



 
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