J Mol Hist DOI 10.1007/s10735-009-9234-1
ORIGINAL PAPER
Conserved expression of the PRELI domain containing 2 gene (Prelid2) during mid-later-gestation mouse embryogenesis Mengya Gao • Qi Liu • Fengwei Zhang • Zhengbin Han • Tiantian Gu • Weiming Tian Yan Chen • Qiong Wu
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Received: 14 June 2009 / Accepted: 6 October 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Prelid2, which belongs to the PRELI domain containing family, is identified as a conserved evolution gene. The expression and regulation during embryonic development of the prelid2 gene is unknown. In this study, we investigated the prelid2 gene expression and regulation using mouse embryos model, by in situ hybridization analysis, RT-PCR and bisulfite sequencing. In situ hybridization analysis showed that prelid2 gene expression were found in midbrain, spinal cord, optic eminence, otic vesicle and tail at E9.5 and E10.5 embryos, in forebrain, hindbrain, heart, lung, liver and kidney at E13.5 and E15.5 embryos. Real-time quantitative RT-PCR results verified the expression pattern in the four major mouse organs, brain, heart, lung, and liver during organs differentiation and formation. Bisulfite sequencing illustrated the consistent result of expression and its unmethylation status in the genomic promoter region at E12.5, E18.5, and new born. Thus, the prelid2 gene is a widely-spread, persistently expressed and unmethylated gene in mouse embryonic development. Our results suggest that the PRELI domain containing 2 gene is involved in mouse embryonic development. Keywords prelid2 Embryonic development Unmethylated In situ hybridization Real-time quantitative RT-PCR
M. Gao Q. Liu F. Zhang Z. Han T. Gu W. Tian Y. Chen Q. Wu (&) Department of Life Science and Engineering, Harbin Institute of Technology, No.92 West Da-zhi Street Harbin, 150001 Heilongjiang, China e-mail:
[email protected]
Introduction The proteins of relevant evolutionary and lymphoid interest (PRELI) domain containing family are expected to encode a unique class of mitochondrial protein which is similar to Ups1p and MSF1/UPS2/YLR168C in Saccharomyces cerevisiae (Anantharaman and Aravind 2002; Sesaki et al. 2006; Koerner et al. 1987). This putative unknown functional protein may be involved in intra-mitochondrial sorting and required for wild-type respiratory growth (Nakai et al. 1993). This protein family in advanced vertebrates is classified into 2 classes, prelid1 (PRELI domain containing 1) and prelid2 (PRELI domain containing 1), and contains a conserved domain, PRELI superfamily, which belongs to the PRELI-like family. MSF1/YLR168C gene, which is homologues to prelid2, is defined and required for mitochondrial respiratory ability and active as a monomer in Saccharomyces cerevisiae (Koerner et al. 1987; Sanni et al. 1991). The prel was demonstrated to express ubiquitously in embryonic development in Drosophila melanogaster (Dee and Moffat 2005). The px19 and preli gene belong to the first class of the PRELI domain containing family. The px19 gene appeared to be an important developmental regulation factor and expressed highly in the blood island and liver primordial during avian embryonic development (Niu et al. 1996). The preli gene was identified to express highly only in human fetal liver, and significant for germinal center B lymphocytes development (Guzman-Rojas et al. 2000). However, there is no report about the expression and regulation of the second class in these model animals during embryonic development. In order to examine the expression of prelid2 gene during mouse embryonic development, we used the C57BL/6J strain mouse as the original materials to gain insight into the
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nature of the prelid2 gene. Additionally, we investigated the relationship between the methylation states of the prelid2 promoter region and its expression in mouse embryonic development. The identification of this novel gene provides an explanation to the function of the PRELI domain containing family in mouse embryonic development.
Materials and methods Animals and preparation of embryos Male and female mice (C57BL/6 J) were obtained from Beijing Laboratory Animal Research Center (Beijing, China). The presence of a vaginal plug on the next morning was defined as 0.5 day post conception. The day of conception was designated as embryonic day (E). The embryos were obtained and used for real-time quantitative RT-PCR and ISH analyses. All the experiments were approved and carried out according to the ‘‘Rules for experiments’ animals’’ published by Chinese Government (Beijing, China). Phylogenetic analysis Multi-sequence alignment were performed by DNAMAN 6.0.40 Edition and confirmed by the maximum likelihood program TreePuzzle (Schmidt et al. 2002). In situ hybridization (ISH) For whole mount in situ hybridization analysis, the embryos at E9.5 and E10.5 were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and incubated at 4°C overnight. The fixed embryos (E13.5 and E15.5) were dehydrated and subsequently embedded in paraffin. Sections (10 lm) of the embryos were adhered on the slides, and ISH was performed. Digoxigenin (DIG)UTP labeled RNA probe is generated by using a DIG RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). For preparing prelid2 RNA probes, a mouse cDNA fragment (50 -TCCTCCGAAAGTATCCCAATC-30 ; 50 -GTCGTGGAGTCTACTGGTGTC-30 ; production size: 369 bp) is subcloned into pBluescriptÒ II KS (?) phagemids (Stratagene, la Jolla, California, USA) T-vector by using DNA Ligation Kit (TARAKA, Dalian, China) according to the manufacturer’s protocol. Real-time quantitative RT-PCR Total RNA were isolated from the embryos and organs which were collected from females that got pregnant on the desired day according to the standard protocols by using TRIzol Reagent (Invitrogen, San Diego, USA), (Chomczynski and
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Sacchi 1987). The cDNAs were synthesized using a SuperscriptTM III RNase H-Reverse Transcriptase kit (Invitrogene, Carlsbad, CA, USA). Subsequently, the cDNAs were used for quantitative analysis of genes expression by realtime quantitative RT-PCR system (ABI PRISM 7500 RealTime PCR System, CA, USA) with a Prefect Real Time SYBRÒ Premix Ex TaqTM Kit (TAKARA, Dalian, China). The prelid2 cDNA specific primer sequences were the same as the in situ hybridization analysis. The gapdh gene was used as a loading control. Gapdh primers sets were forward, 50 -GAGCCCTTCCACAATGCCAAA-30 , and reverse, 50 -G TCGTGGAGTCTACTGGTGTC-30 . Ct values were normalized against the Gapdh gene. All samples were analyzed at least in triplicate. Data were analyzed using SPSS 13.0 and presented as the mean ± standard deviation. DNA preparation and bisulfite sequencing DNA was isolated from the embryonic tissues by proteinase K/SDS method as described (Zuccotti and Monk 1995). The isolated DNA was treated with sodium bisulfite using CpGenome DNA Modification Kit (Intelligence, Millipore, Billerica, USA) according to the manufacturer’s protocol. The bisulfite converted DNA was amplified by nest-PCR. Prelid2 promoter specific primers were 1st, forward, 50 -GT TTGGAATTTTGGTTGTA-30 , reverse, 50 -AATACACTT CCCAAATCAA-30 ; 2nd, forward, 50 -GTAATTGGGTGG TTTAGAG-30 , reverse, 50 -TAACCCCCATATCATACA C-30 . The IG-DMR (intergenic germline-derive differentially methylated region) region V of Dlk1-Gtl2 was used as a positive control and its specific primers were the same as described in previous studies (Takada et al. 2002; Wu et al. 2006; Hiura et al. 2007).
Results and discussion Amino acid sequence analysis of the PRELI domain containing family in the phylogenetic evolution The Mus musculus prelid2 gene (GenBank databases accession number: GeneID77619) is located in mouse chromosome 18 B3, and contains 6 exons, transcripts into a length of 724 bp cDNA, and encodes a 177-amino-acid protein. The PRELI superfamily domain is located from No. 16 amino acid to No. 173 amino acid. A CpG island is predicted from -275 bp upstream to ?10 bp downstream with 14 CpG sites in the prelid2 genomic promoter region using Methprimer (http://www.urogene.org/methprimer/ index1.html, Fig. 1a). In order to examine the relationships of the members of the PRELI domain containing family, a phylogenetic analysis was performed using DNAMAN. The amino acid
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Fig. 1 Prelid2 was conserved in vertebrates. a Schematic representation of the Prelid2 gene structure (Mus musculus, Chromosome 18 B3). The length and position of the CpG island vs. the CpG sites are enlarged at the right-bottom. TSS, transcriptional start site. b Phylogeny of the PRELI/MSF1p’ protein family. The tree rooted using the yeast MSF1/YLR168C sequence as an outgroup. The branch lengths were labeled in each branch point. I, Preli domain containing 1 family, II, Preli domain containing 2 family. c A multiple sequence alignment of some members from the PRELI-like family, full-length
Prelid2 protein (177 amino acids) was aligned to multiple related sequences from Bos taurus (Bt), Drosophila melanogaster (Dm), Danio rerio (Dr), Equus caballus (Ec), Gallus gallus (Gg), Homo sapiens (Hs), Mus musculus (Mm), Pan troglodytes (Pt), Rattus norvegicus (Rn), Saccharomyces cerevisiae (Sc), and Xenopus tropicalis (Xt). Amino acids that are identical in all species are shown in white letter on a blue/red/green background. PRELI domain is indicated by black frames. The number on the right refers to amino acids numbers
sequences fell into two major monophyletic clades. The first clade which contained Bos taurus, Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Danio rerio formed the Preli domain containing 1 family with a 0.059 branch distance to MSF1/YLR168C in yeast, and the second one formed the Preli domain containing 2 family with a 0.145 branch distance (Fig. 1b). A BLASTp search of the NCBI database identified several PRELI-like family members sequences, as the px19 in Gallus gallus (Niu et al. 1996), preli in Homo sapiens and Mus musculus (Guzman-Rojas et al. 2000; Fox et al. 2004; Tahvanainen et al. 2009), prel in Drosophila melanogaster (Dee and Moffat 2005), Ups1p, YLR168C, YDR185C in Saccharomyces cerevisiae (Anantharaman and Aravind 2002; Sesaki et al. 2006; Koerner et al. 1987). Comparison of the conservation among these members and the PRELI domain containing 2 family members is generated by a multiple amino acid sequence alignment (Fig. 1c). The PRELI domain contains an approximately 158 amino acid region at the N-terminus of all the identified protein (Anantharaman and Aravind 2002; Dee and Moffat 2005). Mus musculus prelid2 showed a homology to px19
and MSF1/YLR168C and similar to preli, prel, Ups1p and YDR185C. The hostile relationship between Usp1p and Ups2p in regulating cardiolipin metabolism in mitochondria further showed that they might be involved in intramitochondrial sorting (Nakai et al. 1993; Tamura et al. 2009). Comparison of the amino acid sequences of the PRELI-like family revealed that prelid2 gene is a conservative gene in evolution. Ups1p, ortholog of human PRELI, regulates the processing of the GTPase Mgm1p in Saccharomyces cerevisiae (Sesaki et al. 2006). Since Homo sapiens PRELI could replace Usp1p in Mgm1p processing in yeast cells, the close relationship between the PRELIlike family in different species and yeast MSF1/YLR168C suggests that these proteins derive from a single ancestral gene in the last common ancestor of the respective species. Conserved expression of prelid2 gene in whole embryos To establish the expression pattern of the prelid2 gene during mouse embryonic development, whole mount in situ hybridization analysis was carried out at E9.5 and E10.5.
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Fig. 2 Prelid2 gene is expressed ubiquitously in mid-gestation mouse embryogenesis by whole-mount in situ hybridization. The mRNA expression of prelid2 gene appear in branchial arches (ba), fore limb bud (flb), gut (g), mesencephalon (ms), nasal process (np),
neural tube (nt), optic eminence (oe), otic vesicles (ov), rhombencephalon (rh), spinal cord (sc), tail bud (t), telencephalon (te) at E9.5 (a–c) and E10.5 (e–g). d (E9.5) and h (E10.5) are negative control
Compared to the negative control (Fig. 2d) at E9.5 embryo, the expression signal of prelid2 gene was detected ubiquitously in branchial arches, fore limb bud, gut, mesencephalon, nasal process, neural tube, optic eminence, otic vesicles, rhombencephalon, spinal cord, telencephalon, and tail bud (Fig. 2a–c). Consistently with the E9.5, the demarcated prelid2 gene mRNA expression was found in the same site (Fig. 2e–g), compared to the negative control (Fig. 2h) at E10.5 embryo. When organogenesis happened at E13.5 and E15.5 embryo, prelid2 expression was found universally in organs, such as forebrain, hindbrain, heart, lung, liver, and kidney etc. (Fig. 3a–d). Clearer signals could be detected in forebrain cortex, hindbrain cortex, Rathke’s pouch, tongue, spinal cord, intestines, and adrenal gland at E13.5 embryo (Fig. 3c), and more other sites like cerebellum, sublingual gland, tooth bud, sternum, skin, and thymus gland at E15.5 embryo (Fig. 3d). The prel gene in Drosophila is identified to express ubiquitously throughout embryonic development, especially in central nerve system (CNS) (Dee and Moffat 2005). Consistently to the result of prelid2 gene expression, it shows that prelid2 gene might be participate in the mouse embryonic CNS development. Several previous studies demonstrated that the avian px19 gene was highly expressed within the blood island and in the liver primordial (Niu et al. 1996; Stacey and Aalen 1998). The preli was expressed highly in the fetal liver and germinal centers (GC) in human (Guzman-Rojas et al. 2000). The preli was
also reported that upregulated in response to T cell activation, and the expression level correlates with the strength of the TCR stimulus in mouse (Fox et al. 2004; Tahvanainen et al. 2009). The hematopoiesis is regulated by hematopoietic organs, and the first definitive hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region at E10.5 and thereafter shift to the fetal liver (FL) at E12.5 during the mouse embryonic development (Ogawa 1993; Medvinsky and Dzierzak 1996; Takeuchi et al. 2002). Mouse lymphatic endothelium comes from existing venous endothelia cells, and hematopoietic stem cells (HSCs) give rise to the vascular endothelium for the maintenance and function of lymphatic endothelium (LEC) (Srinivasan et al. 2007; Peters et al. 2005; Jiang et al. 2008). Therefore, our results suggests the possibility that prelid2 may be participating in the development of mouse embryonic lymphatic system and hematopoietic system. The prelid2 gene transcripts were detected frequently throughout mouse embryonic stages. The expression levels of Prelid2 in the four major tissues at E12.5, E15.5, and E18.5 was carried out by real-time quantitative RT-PCR analysis. Prelid2 was expressed at a lower level in the brain, and at an equal level in the heart, lung, and liver (Fig. 4a). During the mouse fetal developmental stage, the general tendency expression of prelid2 was lower in these four tissues from E15.5 and E18.5. The spatio-temporal persistent expression of prelid2 gene strongly suggests that prelid2 gene is an important
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J Mol Hist Fig. 3 Prelid2 gene is expressed universally in latergestation mouse embryogenesis by section in situ hybridization at E13.5 (a) and E15.5 (b) embryos. Higher magnification views at E13.5 (c) and E15.5 (d) embryos showed the detailed expression of prelid2 gene. HE, hematoxylin-eosin staining. Negative controls on right (a, b) were treated with eosin staining. Abbreviations are: fore brain (fb), hind brain (hb), heart (h), kidney (k), liver (li), lung (lu)
molecule factor which is involved in the mouse embryonic multi-system development. DNA methylation status of the prelid2 gene in the embryo As expected to acquire a detail upstream promoter region to regulate its downstream gene expression through methylation occurrence regulation mechanism, we check out the relationship between the prelid2 gene expression and its upstream CpG island by bisulfite sequencing. We determined the methylation status of some tissues of the prelid2 gene in E12.5, E18.5, and new born, compared to its expression in these tissues. The results showed the prelid2 gene is completely unmethylated at brain (0/56 CpG sites), liver (0/42 CpG sites) and spermary (0/42 CpG sites). We
detected the IG-DMR region V is almost methylated at E12.5 brain (92/96 CpG sites) and new born spermary (93/ 96 CpG sites) genome as a positive control (Fig. 4b). The sustaining unmethylated status of prelid2 gene is consistent with its expression pattern in mid-later-gestation mouse embryogenesis. The developmental kinetics of methylation imprints showed in five mutable methylation pattern stages: primordial germ cells (PGCs) phase, 6 dpc migration phase, around 10.5 dpc phase, around birth phase and adult phase (Bourc’his and Proudhon 2008). Our results indicated that no transformation of methylation status of the prelid2 gene promoter region was found in the around 10.5 dpc phase and around birth phase. In conclusion, the present study demonstrates that the prelid2 gene is ubiquitously, continuously expressed and
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J Mol Hist Acknowledgments This work is financially supported by Ministry of Education Scholarship (Grant No. GFEQ24403001), Harbin Institute of Technology Introduction of Scientific Research Talents Activation Fee (Grant No. GFQQ18600015), and Heilongjiang Province Technological Project Program Returning Foundation (Grant No. LC08C05).
References
Fig. 4 Prelid2 gene is expressed continuously in whole mouse embryos. a Graphical representations showed prelid2 gene is expressed in four major mouse embryonic developmental organs at E12.5 (black), E15.5 (gray) and E18.5 (white) by real-time quantitative RT-PCR. Values are means ± stdev from three mouse embryos. b Prelid2 promoter region is unmethylated persistently in mouse during embryonic development. The prelid2 promoter region is completely DNA unmethylated (open circles) during mouse midlater-gestation embryonic development and the IG-DMR region V genome is almost DNA methylated (filled circles) as a positive control
unmethylated during mid-later-gestation mouse embryogenesis. Prelid2 gene may participate in the regulation of the mouse embryonic development and differentiation. In summary, the function of PRELI domain proteins may have an effect on the mouse embryonic development of CNS, lymphatic system, hematopoietic system and the organic formation. In the following study, we will investigate how prelid2 gene regulates in mouse development by using siRNA as we knew that prelid2 gene is highly expressed in mouse embryonic stem cell (data not show). It may be useful for providing an explanation to the expression and function of the PRELI domain containing family in mouse embryonic development.
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