- Email: [email protected]
Developmental expression patterns of fosl genes in Xenopus tropicalis
Gene Expression Patterns 34 (2019) 119056
Contents lists available at ScienceDirect
Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Developmental expression patterns of fosl genes in Xenopus tropicalis a
a
b
a
a
T
a
Xiao-Fang Guo , Zhou Zhang , Li Zheng , Yi-Min Zhou , Hai-Yan Wu , Chi-Qian Liang , Hui Zhaoc, Dong-Qing Caia, Xu-Feng Qia,∗ a b c
Key Laboratory of Regenerative Medicine, Ministry of Education, Department of Developmental & Regenerative Biology, Jinan University, Guangzhou, 510632, China School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou, 510006, China School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
A R T I C LE I N FO
A B S T R A C T
Keywords: fosl1 fosl2 Expression Development Xenopus tropicalis
Fos-like antigens (Fosl) including Fosl1 and Fosl2 exclusively heterodimerize with Jun members to form AP-1 complex, thereby participating in various cellular progresses including cell cycle regulation. However, expression patterns of these two genes during embryonic development remains largely unknown. In the present study, both temporal and spatial expression patterns of fosl1 and fosl2 were examined during embryonic development of Xenopus tropicalis. Real-time quantitative PCR results showed that the expression of the two genes was increased from stage 2 to stage 42. However, expression level of fosl1 is much higher than that of fosl2 at stage 42. Whole-mount in situ hybridization showed that fosl1 was expressed in eyes, branchial arch, notochord, otic vesicle, and liver. However, fosl2 was expressed in lung primordium from stage 34 to stage 38, in addition to the moderate expression in eyes and branchial arch at stage 42. Thus, the developmental expression patterns of these two fosl genes is different in Xenopus embryos. These results provide a basis for further functional study of these two genes.
1. Introduction The transcription factor AP-1 (activator protein 1) is essentialformany cellular processes including embryonic development, cell differentiation, proliferation, apoptosis, and inflammation (Karin et al., 1997; Subedi et al., 2019). AP-1 is a dimeric transcription factor that consists of Jun and Fos family members (Hess et al., 2004). Although Jun proteins (Jun, Junb, and Jund) can form homodimers, the Fos proteins (Fos, Fosb, Fos-like (Fosl) 1, and Fosl2) exclusively heterodimerize with Jun members to form AP-1 complexes (Schreiber et al., 2000). Previous studies have demonstrated that Jun-Fos heterodimers are more stable and have stronger DNA binding activities compared with Jun–Jun homodimers (Halazonetis et al., 1988). Therefore, Fos proteins are crucial and contribute to the robust transcription activity of AP-1. Among the four members of Fos family, individual protein shows different functions and responses to stimulation. In trophoblast cells, mRNA expression of Fos and Fosb, rather than Fosl1or Fosl2, was greatly increased by a scratch wound. In consistent, the protein levels of Fos and Fosb paralleled that of mRNA. Interestingly, Fosl1 rather than Fosl2 protein is substantially increased following introduction of the scratch wound (Renaud et al., 2014). Moreover, knockdown of Fos resulted in
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cessation of proliferation and induction of migration of trophoblast cells with robust expression of matrix metalloproteinase 1 (MMP1). Conversely, Fosl1 knockdown abrogated trophoblast migration and inhibited the production of MMP1 (Renaud et al., 2014). In the mouse, knockout of Fosl1 results in placental and extra-embryonic abnormalities leading to early embryonic death (Schreiber et al., 2000), but mice deficient in Fos are viable and fertile (Hu et al., 1994). Deficiency of Fosl2 in mice induces placental structural and signaling defects that indirectly impact differentiation of embryonic tissues (Bozec et al., 2008). Therefore, individual member of Fos family may has different biological functions during mammalian development. Fosl1, also termed Fra1 (Fos-related antigen 1), is an immediateearly gene (Cohen and Curran, 1988). Fosl1 lacks a transactivation domain and fails to activate transcription by itself (Welter et al., 1995). Functionally, Fosl1 can either increase or decrease total AP-1 activity depending on the status of the other Fos and Jun proteins in different cell types (Schreiber et al., 1997). Fosl1 is also involved in the cell cycle regulation of normal (Kovary and Bravo, 1992) and tumor (Bergers et al., 1995; Hu et al., 1994) cells. Indeed, Fosl1 and Fosl2 are significantly synthesized during the cell cycle when neither Fos nor Fosb are present (Gruda et al., 1994; Kovary and Bravo, 1992), indicating an important and unique role of Fosl1 or Fosl2 in cell growth. Although
Corresponding author. Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, China. E-mail address: [email protected] (X.-F. Qi).
https://doi.org/10.1016/j.gep.2019.119056 Received 1 May 2019; Received in revised form 19 May 2019; Accepted 20 May 2019 Available online 21 May 2019 1567-133X/ © 2019 Elsevier B.V. All rights reserved.
Gene Expression Patterns 34 (2019) 119056
X.-F. Guo, et al.
Fig. 2. Temporal expression of fosl genes in early development of X. tropicalis. The mRNA expression of fosl1 and fosl2 genes was analyzed by qPCR using total RNA isolated from X. tropicalis embryos at indicated developmental stages. Ornithine decarboxylase (odc) was used as the internal standard control. Results are presented as mean ± SEM (n = 4 experiments), ***p < 0.001. St, stage.
xFosl1) share 50% and 74% identities with human FOSL proteins, respectively (Fig. 1A and B). Among the FOSL proteins we examined, human FOSL proteins share highest identities with mouse (87% and 84%), followed by rat (86% and 93%), X. tropicalis (50% and 74%), and Danio rerio (49% and 66%). Phylogenetic tree illustrated the evolutionary distance of FOSL proteins in human, mouse, rat, X. tropicalis, and Danio rerio (Fig. 1C and D). These results reveal that Fosl proteins are conservative during the evolution of vertebrates. 2.2. Temporal expression of xenopus fosl genes The temporal expression patterns of fosl genes during early embryonic development of X. tropicalis were analyzed by quantitative RTPCR. As shown in Fig. 2, basic expression of fosl1 mRNA was detected from stage 2 to stage 27. However, the expression of fosl1 at stage 34 was significantly increased. Especially at stage 42, the expression of fosl1 was dramatically upregulated by ∼280 folds. Consistent with fosl1, a basic expression of fosl2 was detected from stage 2 to stage 27, followed by a significant increase at stage 34 and stage 42. Notably, the expression of fosl2 is significantly lower compared with fosl1 at stage 42. These results indicate the different temporal expression patterns between fosl1 and fosl2 during embryonic development of X. tropicalis, implying the potentially different functions of these two genes.
Fig. 1. Identification of two fosl genes from Xenopus tropicalis. (A and B) The identity ratio of protein sequence of human Fosl1 (A) and Fosl2 (B) compared with homologs in other vertebrates. M, mouse; r, rat; x,Xenopus tropicalis; d, Danio rerio. (C and D) Phylogenetic analysis of Fosl1(C) and Fosl2 (D) proteins from Xenopus tropicalis, human, mouse, rat,and Danio rerio.
Fosl subfamily members are widely involved in various cellular processes and in mammalian development, their expression patterns in vertebrate remains unclear. Xenopus tropicalis is an excellent model for genetic and embryonic studies depending on the true diploid genome, available genome sequence, and short generation time (Hellsten et al., 2010; Mao et al., 2018; Nakayama et al., 2013). In the present study, we studied the spatial and temporal expression patterns of fosl1 and fosl2 during the embryonic development of X. tropicalis. Our data will provide a basis for further investigations on the functions of fosl genes in Xenopus.
2.3. Spatial expression patterns of xenopus fosl genes To further determine the spatial expression patterns of fosl genes, whole-mount in situ hybridization (WISH) analysis was performed by using X. tropicalis embryos at different stages. As shown in Fig. 3, almost no discernible fosl1 expression can be detected at stage 2 (Fig. 3A). Very faint expression of fosl1 was detected from stage 8 to stage 13 (Fig. 3B and C). Fosl1 signals was detected moderately in eyes and mouth at stage 27 (Fig. 3D). At stage 34, intense expression of fosl1 was mainly detected in eyes, branchial arch and notochord in addition to moderate expression in somite (Fig. 3E). Intense expression of fosl1 in the otic vesicle was also observed at stage 38 (Fig. 3F). At stage 42, strong expression of fosl1 in liver was detected, except for in eyes, branchial arch and notochord (Fig. 3G). However, no signals were detected using a sense probe of fosl1 for each examined stage (Fig. 3A’-G′). Inconsistently, no discernible fosl2 expression can be detected from stage 2 to stage 27 (Fig. 4A–D). Intense expression of fosl2 was detected specifically in lung primordium at stage 34 (Fig. 4E). The lung specific expression of fosl2 were maintained to staged 38 (Fig. 4F). At stage 42,
2. Results 2.1. Phylogenetic analysis of fosl proteins from X. tropicalis and other species To explore the conservatism of Fosl in evolution, protein sequences were aligned among different vertebrates. By comparing the protein sequence, we found that Fosl1 and Fosl2 of X. tropicalis (xFosl1 and 2
Gene Expression Patterns 34 (2019) 119056
X.-F. Guo, et al.
Fig. 3. Spatial pattern of fosl1 analyzed by whole mount in situ hybridization. (A and A′) Stage 2, animal view. (B and B′) Stage 8, animal view. (C and C′) Stage 13, dorsal view. (D and D′) Stage 27, dorsal view. (E and E′) Stage 34, lateral view. (F and F′) Stage 38, lateral view. (G and G′) Stage 42, lateral view. Ba, branchial arches; cb, cement gland; fb, forebrain; mb, midbrain; hb, hindbrain; lv, liver; nc, notochord; ov, otic vesicle. A′-G′ indicates the sense probe control.
G′).
fosl2 expression was detected in eyes and branchial arch (Fig. 4G). The transverse section confirmed that fosl2 specifically was expressed in lung primordium with moderate signals (Fig. 4H and H′). The positive expression of fosl2 was further confirmed by the sense probe (Fig. 4A’-
Fig. 4. Spatial pattern of fosl2analyzed by whole mount in situ hybridization. (A and A′) Stage 2, animal view. (B and B′) Stage 8, animal view. (C and C′) Stage 13, dorsal view. (D and D′) Stage 27, dorsal view. (E and E′) Stage 34, lateral view. (F and F′) Stage 38, lateral view. (G and G′) Stage 42, lateral view. (H) Transverse sections of embryos at the levels illustrated by broken black lines in F. (H′) Large magnification of image shown in H. Ba, branchial arch; nc, notochord. A′-G′ indicates the sense probe control. 3
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3. Discussion
database at NCBI (http://www.ncbi.nlm.nih.gov/protein/).
All AP-1 proteins are characterized by a basic leucine-zipper region for dimerisation and DNA-binding. Previous studies have demonstrated that the expression of Fos proteins is crucial for the activity of AP-1regulated genes. Target genes regulated by the AP-1 transcription factor are involved in multiple cellular progresses including proliferation, differentiation, survival, and angiogenesis. The activity of all Fos family members is also modulated by posttranslational modification. Therefore, further study of the functions of Fos proteins in vivo is more important and will probably open new perspectives for related disease therapy (Milde-Langosch, 2005). In the present study, we examined the spatial and temporal expression pattern of fosl1 and fosl2 genes during early embryonic development of X. tropicalis, and found the different expression patterns for these two genes. In vertebrates, Fosl proteins are conservative during biological evolution (Fig. 1). However, the detailed functions of these proteins are different in individual cell type or species. The modulation of fosl1 expression directly affected cell proliferation, invasiveness and motility of cells in vitro(Belguise et al., 2005). In zebrafish, the fosl2 can potentiate the rate of cardiomyocyte differentiation from SHF progenitors (Bozec et al., 2013). Moreover, it have been reported that fosl1 transgenic mice developed severe anomaly of lung structure, including fibrosis, vascular remodeling and inflammation (Belguise et al., 2005; Tsujino et al., 2017b; Tsujino and Sheppard, 2016). Artificial Fra-2 expression in αSMA-expressing lineage cells critically modulates secondary alveolar septation in developing murine lungs (Tsujino et al., 2017a). These previous studies suggest that Fosl1 and Fosl2 proteins have different detailed functions depending on cell type and species. In consistent, fosl2 rather than fosl1 were detected specifically in lung from stage 34 to stage 38 during X. tropicalis development (Fig. 4), implying the different functions of fosl genes for specific tissue development. The dramatically upregulation of fosl1in embryonic development, especially at stage 42, implies that fosl1 may play an important role in normal development of Xenopus embryos (Fig. 2). Consistently, previous study has demonstrated the pivotal role in embryonic development in mammals. Fosl1 deficiency induces defects in vascularization of the placenta, thereby resulting in severely growth retardation and lethality between E10.0 and E10.5 (Schreiber et al., 2000). Therefore, these data in previous study and our works suggest that fosl1 is crucial for embryo development in vertebrates. For fosl2 gene, previously study has reported that fosl2 are detected in several differentiating epithelial, developing cartilage and central nervous system during mouse development, with a distinct expression patterns from other fos related genes, suggesting that fosl2 has a unique role in cellular differentiation during fetal development (Carrasco and Bravo, 1995). In consistent, fosl2 indeed shown unique expression patterns during embryonic development of X. tropicalis compared with fosl1 (Figs. 2–4). In conclusion, we examined and reported different temporal and spatial expression patterns between fosl1 and fosl2 during embryonic development of X. tropicalis. This study will facilitate the future functional analysis of fosl genes during embryonic development. Moreover, our study also provides the basis for generating fos genes-related disease models using Xenopus in the future.
4.2. Quantitative reverse transcription-PCR (qPCR) analysis Total RNA was extracted from Xenopus embryos at indicated developmental stages using the TRI reagent (Molecular Research Center Inc., USA). cDNA was synthesized from total RNA (2 μg) and oligo (dT) 18 primers (0.5 μg) using the ReverTra Ace® qPCR RT Kit (Toyobo, Japan). Real-time PCR was performed using a Light Cycler 480 SYBR Green I Master (Roche, USA) and the MiniOpticon Real-Time PCR System (Bio-Rad, CA, USA). The sequences of the qPCR primers were listed as following, fosl1 Fw, 5′-ATG GGA CAC AGC GTC AGA CT-3’; fosl1 Re, 5′-CCG AGG GCT TTG GTA TGG TG-3’; fosl2 Fw, 5′-GCA GGA GAA GTC CGG GTT AC-3’; fosl2 Re, 5′-AAA CCT CCG GCG ATG TTG AT3’; odc Fw, 5′-TGT TCT GCG CAT AGC AAC TG-3’; odc Re, 5′-ACA TCG TGC ATC TGA GAC AGC-3’. Ornithine decarboxylase (odc) was used as the internal standard control. After denaturation for 10 min at 95 °C, the reactions were subjected to 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s odc was used as the internal standard control to normalize gene expression using the △△Ct method. 4.3. Probe preparation and whole-mount in situ hybridization The whole ORFs of X. tropicalisfosl1 (accession number: XM_002939331.4) and fosl2 (accession number: XM_002938419.4) were amplified using cDNA templates from st42 tadpoles. Primers used for RT-PCR are as follows: fosl1 Fw (BamH1): 5′-CGC GGA TCC ATG TAC AGA GAC TTC ACT GGA GCC TT-3’; fosl1 Re (Xhol): 5′-CCG CTC GAG CTA AAG AGT CAG TAG GCT GTT AGA ACT T-3’; fosl2 Fw: 5′-ATG TAC CAG GAT TAT CCA GGG AAT TTC GAC AG-3’; fosl2 Re: 5′-TTA CAA AGC CAG GAG GGT GGG CGA GTT C-3’. PCR fragments were then subcloned into pBlueScript II SK (+) plasmid and verified by sequencing. Plasmids were linearized and then used as templates for synthesis of digoxigenin-labeled antisense probes with T7 RNA polymerase (Roche, Indianapolis, IN). Whole-mount in situ hybridization was performed using digoxigenin-labeled antisense RNA probe and anti-digoxigenin monoclonal antibody labeled with alkaline phosphatase. Probe signals were developed using NBT/BCIP (Roche, USA) as previously described (MonsoroBurq, 2007). Vibratome sectioning was performed as previously described (Wang et al., 2011). 5. Conflicts of interest There are no conflicts of interest. Acknowledgments This work was supported by grants from the National Key R&D Program of China (2016YFE0204700), the National Natural Science Foundation of China (31802025, 81570222, 81770240, and 81270183), the Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306011), the Guangdong Science and Technology Planning Project (2014A050503043), the New Star of Pearl River on Science and Technology of Guangzhou (2014J2200002), the Top Young Talents of Guangdong Province Special Support Program (87315007), and the Fundamental Research Funds for the Central Universities (21617436), China.
4. Experimental procedures 4.1. Blast searches and phylogenetic analysis
References Percentages of identical amino acids of human Fosl proteins sequence with different species were calculated by BLAST (BLASTP) searches in the RefSeq protein database at NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi). Protein sequence alignments and phylogenetic tree were performed using the neighbor-joining method in MEGA 6.0.6 software. Protein conserved domains were retrieved by protein
Belguise, K., Kersual, N., Galtier, F., Chalbos, D., 2005. FRA-1 expression level regulates proliferation and invasiveness of breast cancer cells. Oncogene 24, 1434–1444. Bergers, G., Graninger, P., Braselmann, S., Wrighton, C., Busslinger, M., 1995. Transcriptional activation of the fra-1 gene by AP-1 is mediated by regulatory sequences in the first intron. Mol. Cell. Biol. 15, 3748–3758. Bozec, A., Bakiri, L., Hoebertz, A., Eferl, R., Schilling, A.F., Komnenovic, V., Scheuch, H.,
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Mao, C.Z., Zheng, L., Zhou, Y.M., Wu, H.Y., Xia, J.B., Liang, C.Q., Guo, X.F., Peng, W.T., Zhao, H., Cai, W.B., Kim, S.K., Park, K.S., Cai, D.Q., Qi, X.F., 2018. CRISPR/Cas9mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis. FASEB J. 32, 6495–6509. Milde-Langosch, K., 2005. The Fos family of transcription factors and their role in tumourigenesis. Eur. J. Cancer 41, 2449–2461. Monsoro-Burq, A.H., 2007. A rapid protocol for whole-mount in situ hybridization on Xenopus embryos. CSH protocols. https://doi.org/10.1101/pdb.prot4809. Nakayama, T., Fish, M.B., Fisher, M., Oomen-Hajagos, J., Thomsen, G.H., Grainger, R.M., 2013. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51, 835–843. Renaud, S.J., Kubota, K., Rumi, M.A.K., Soares, M.J., 2014. The FOS transcription factor family differentially controls trophoblast migration and invasion*. J. Biol. Chem. 289, 5025–5039. Schreiber, M., Poirier, C., Franchi, A., Kurzbauer, R., Guenet, J.L., Carle, G.F., Wagner, E.F., 1997. Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis). Oncogene 15, 1171–1178. Schreiber, M., Wang, Z.Q., Jochum, W., Fetka, I., Elliott, C., Wagner, E.F., 2000. Placental vascularisation requires the AP-1 component fra1. Development 127, 4937–4948. Subedi, L., Lee, J.H., Yumnam, S., Ji, E., Kim, S.Y., 2019. Anti-inflammatory effect of sulforaphane on LPS-activated microglia potentially through JNK/AP-1/NF-kappaB inhibition and Nrf2/HO-1 activation. Cells 8. Tsujino, K., Li, J.T., Tsukui, T., Ren, X., Bakiri, L., Wagner, E., Sheppard, D., 2017a. Fra-2 negatively regulates postnatal alveolar septation by modulating myofibroblast function. Am. J. Physiol. Lung Cell Mol. Physiol. 313, L878–L888. Tsujino, K., Reed, N.I., Atakilit, A., Ren, X., Sheppard, D., 2017b. Transforming growth factor-beta plays divergent roles in modulating vascular remodeling, inflammation, and pulmonary fibrosis in a murine model of scleroderma. Am. J. Physiol. Lung Cell Mol. Physiol. 312, L22–L31. Tsujino, K., Sheppard, D., 2016. Critical appraisal of the utility and limitations of animal models of scleroderma. Curr. Rheumatol. Rep. 18, 4. Wang, C., Liu, Y., Chan, W.Y., Chan, S.O., Grunz, H., Zhao, H., 2011. Characterization of three synuclein genes in Xenopus laevis. Dev. Dynam. 240, 2028–2033. Welter, J.F., Crish, J.F., Agarwal, C., Eckert, R.L., 1995. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity. J. Biol. Chem. 270, 12614–12622.
Priemel, M., Stewart, C.L., Amling, M., Wagner, E.F., 2008. Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia. Nature 454, 221–U261. Bozec, A., Bakiri, L., Jimenez, M., Rosen, E.D., Catala-Lehnen, P., Schinke, T., Schett, G., Amling, M., Wagner, E.F., 2013. Osteoblast-specific expression of Fra-2/AP-1 controls adiponectin and osteocalcin expression and affects metabolism. J. Cell Sci. 126, 5432–5440. Carrasco, D., Bravo, R., 1995. Tissue-specific expression of the fos-related transcription factor fra-2 during mouse development. Oncogene 10, 1069–1079. Cohen, D.R., Curran, T., 1988. fra-1: a serum-inducible, cellular immediate-early gene that encodes a fos-related antigen. Mol. Cell. Biol. 8, 2063–2069. Gruda, M.C., Kovary, K., Metz, R., Bravo, R., 1994. Regulation of Fra-1 and Fra-2 phosphorylation differs during the cell cycle of fibroblasts and phosphorylation in vitro by MAP kinase affects DNA binding activity. Oncogene 9, 2537–2547. Halazonetis, T.D., Georgopoulos, K., Greenberg, M.E., Leder, P., 1988. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell 55, 917–924. Hellsten, U., Harland, R.M., Gilchrist, M.J., Hendrix, D., Jurka, J., Kapitonov, V., Ovcharenko, I., Putnam, N.H., Shu, S., Taher, L., Blitz, I.L., Blumberg, B., Dichmann, D.S., Dubchak, I., Amaya, E., Detter, J.C., Fletcher, R., Gerhard, D.S., Goodstein, D., Graves, T., Grigoriev, I.V., Grimwood, J., Kawashima, T., Lindquist, E., Lucas, S.M., Mead, P.E., Mitros, T., Ogino, H., Ohta, Y., Poliakov, A.V., Pollet, N., Robert, J., Salamov, A., Sater, A.K., Schmutz, J., Terry, A., Vize, P.D., Warren, W.C., Wells, D., Wills, A., Wilson, R.K., Zimmerman, L.B., Zorn, A.M., Grainger, R., Grammer, T., Khokha, M.K., Richardson, P.M., Rokhsar, D.S., 2010. The genome of the Western clawed frog Xenopus tropicalis. Science 328, 633–636. Hess, J., Angel, P., Schorpp-Kistner, M., 2004. AP-1 subunits: quarrel and harmony among siblings. J. Cell Sci. 117, 5965–5973. Hu, E., Mueller, E., Oliviero, S., Papaioannou, V.E., Johnson, R., Spiegelman, B.M., 1994. Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes. EMBO J. 13, 3094–3103. Karin, M., Liu, Z., Zandi, E., 1997. AP-1 function and regulation. Curr. Opin. Cell Biol. 9, 240–246. Kovary, K., Bravo, R., 1992. Existence of different Fos/Jun complexes during the G0-toG1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins. Mol. Cell. Biol. 12, 5015–5023.
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Contents lists available at ScienceDirect
Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Developmental expression patterns of fosl genes in Xenopus tropicalis a
a
b
a
a
T
a
Xiao-Fang Guo , Zhou Zhang , Li Zheng , Yi-Min Zhou , Hai-Yan Wu , Chi-Qian Liang , Hui Zhaoc, Dong-Qing Caia, Xu-Feng Qia,∗ a b c
Key Laboratory of Regenerative Medicine, Ministry of Education, Department of Developmental & Regenerative Biology, Jinan University, Guangzhou, 510632, China School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou, 510006, China School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
A R T I C LE I N FO
A B S T R A C T
Keywords: fosl1 fosl2 Expression Development Xenopus tropicalis
Fos-like antigens (Fosl) including Fosl1 and Fosl2 exclusively heterodimerize with Jun members to form AP-1 complex, thereby participating in various cellular progresses including cell cycle regulation. However, expression patterns of these two genes during embryonic development remains largely unknown. In the present study, both temporal and spatial expression patterns of fosl1 and fosl2 were examined during embryonic development of Xenopus tropicalis. Real-time quantitative PCR results showed that the expression of the two genes was increased from stage 2 to stage 42. However, expression level of fosl1 is much higher than that of fosl2 at stage 42. Whole-mount in situ hybridization showed that fosl1 was expressed in eyes, branchial arch, notochord, otic vesicle, and liver. However, fosl2 was expressed in lung primordium from stage 34 to stage 38, in addition to the moderate expression in eyes and branchial arch at stage 42. Thus, the developmental expression patterns of these two fosl genes is different in Xenopus embryos. These results provide a basis for further functional study of these two genes.
1. Introduction The transcription factor AP-1 (activator protein 1) is essentialformany cellular processes including embryonic development, cell differentiation, proliferation, apoptosis, and inflammation (Karin et al., 1997; Subedi et al., 2019). AP-1 is a dimeric transcription factor that consists of Jun and Fos family members (Hess et al., 2004). Although Jun proteins (Jun, Junb, and Jund) can form homodimers, the Fos proteins (Fos, Fosb, Fos-like (Fosl) 1, and Fosl2) exclusively heterodimerize with Jun members to form AP-1 complexes (Schreiber et al., 2000). Previous studies have demonstrated that Jun-Fos heterodimers are more stable and have stronger DNA binding activities compared with Jun–Jun homodimers (Halazonetis et al., 1988). Therefore, Fos proteins are crucial and contribute to the robust transcription activity of AP-1. Among the four members of Fos family, individual protein shows different functions and responses to stimulation. In trophoblast cells, mRNA expression of Fos and Fosb, rather than Fosl1or Fosl2, was greatly increased by a scratch wound. In consistent, the protein levels of Fos and Fosb paralleled that of mRNA. Interestingly, Fosl1 rather than Fosl2 protein is substantially increased following introduction of the scratch wound (Renaud et al., 2014). Moreover, knockdown of Fos resulted in
∗
cessation of proliferation and induction of migration of trophoblast cells with robust expression of matrix metalloproteinase 1 (MMP1). Conversely, Fosl1 knockdown abrogated trophoblast migration and inhibited the production of MMP1 (Renaud et al., 2014). In the mouse, knockout of Fosl1 results in placental and extra-embryonic abnormalities leading to early embryonic death (Schreiber et al., 2000), but mice deficient in Fos are viable and fertile (Hu et al., 1994). Deficiency of Fosl2 in mice induces placental structural and signaling defects that indirectly impact differentiation of embryonic tissues (Bozec et al., 2008). Therefore, individual member of Fos family may has different biological functions during mammalian development. Fosl1, also termed Fra1 (Fos-related antigen 1), is an immediateearly gene (Cohen and Curran, 1988). Fosl1 lacks a transactivation domain and fails to activate transcription by itself (Welter et al., 1995). Functionally, Fosl1 can either increase or decrease total AP-1 activity depending on the status of the other Fos and Jun proteins in different cell types (Schreiber et al., 1997). Fosl1 is also involved in the cell cycle regulation of normal (Kovary and Bravo, 1992) and tumor (Bergers et al., 1995; Hu et al., 1994) cells. Indeed, Fosl1 and Fosl2 are significantly synthesized during the cell cycle when neither Fos nor Fosb are present (Gruda et al., 1994; Kovary and Bravo, 1992), indicating an important and unique role of Fosl1 or Fosl2 in cell growth. Although
Corresponding author. Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, 510632, China. E-mail address: [email protected] (X.-F. Qi).
https://doi.org/10.1016/j.gep.2019.119056 Received 1 May 2019; Received in revised form 19 May 2019; Accepted 20 May 2019 Available online 21 May 2019 1567-133X/ © 2019 Elsevier B.V. All rights reserved.
Gene Expression Patterns 34 (2019) 119056
X.-F. Guo, et al.
Fig. 2. Temporal expression of fosl genes in early development of X. tropicalis. The mRNA expression of fosl1 and fosl2 genes was analyzed by qPCR using total RNA isolated from X. tropicalis embryos at indicated developmental stages. Ornithine decarboxylase (odc) was used as the internal standard control. Results are presented as mean ± SEM (n = 4 experiments), ***p < 0.001. St, stage.
xFosl1) share 50% and 74% identities with human FOSL proteins, respectively (Fig. 1A and B). Among the FOSL proteins we examined, human FOSL proteins share highest identities with mouse (87% and 84%), followed by rat (86% and 93%), X. tropicalis (50% and 74%), and Danio rerio (49% and 66%). Phylogenetic tree illustrated the evolutionary distance of FOSL proteins in human, mouse, rat, X. tropicalis, and Danio rerio (Fig. 1C and D). These results reveal that Fosl proteins are conservative during the evolution of vertebrates. 2.2. Temporal expression of xenopus fosl genes The temporal expression patterns of fosl genes during early embryonic development of X. tropicalis were analyzed by quantitative RTPCR. As shown in Fig. 2, basic expression of fosl1 mRNA was detected from stage 2 to stage 27. However, the expression of fosl1 at stage 34 was significantly increased. Especially at stage 42, the expression of fosl1 was dramatically upregulated by ∼280 folds. Consistent with fosl1, a basic expression of fosl2 was detected from stage 2 to stage 27, followed by a significant increase at stage 34 and stage 42. Notably, the expression of fosl2 is significantly lower compared with fosl1 at stage 42. These results indicate the different temporal expression patterns between fosl1 and fosl2 during embryonic development of X. tropicalis, implying the potentially different functions of these two genes.
Fig. 1. Identification of two fosl genes from Xenopus tropicalis. (A and B) The identity ratio of protein sequence of human Fosl1 (A) and Fosl2 (B) compared with homologs in other vertebrates. M, mouse; r, rat; x,Xenopus tropicalis; d, Danio rerio. (C and D) Phylogenetic analysis of Fosl1(C) and Fosl2 (D) proteins from Xenopus tropicalis, human, mouse, rat,and Danio rerio.
Fosl subfamily members are widely involved in various cellular processes and in mammalian development, their expression patterns in vertebrate remains unclear. Xenopus tropicalis is an excellent model for genetic and embryonic studies depending on the true diploid genome, available genome sequence, and short generation time (Hellsten et al., 2010; Mao et al., 2018; Nakayama et al., 2013). In the present study, we studied the spatial and temporal expression patterns of fosl1 and fosl2 during the embryonic development of X. tropicalis. Our data will provide a basis for further investigations on the functions of fosl genes in Xenopus.
2.3. Spatial expression patterns of xenopus fosl genes To further determine the spatial expression patterns of fosl genes, whole-mount in situ hybridization (WISH) analysis was performed by using X. tropicalis embryos at different stages. As shown in Fig. 3, almost no discernible fosl1 expression can be detected at stage 2 (Fig. 3A). Very faint expression of fosl1 was detected from stage 8 to stage 13 (Fig. 3B and C). Fosl1 signals was detected moderately in eyes and mouth at stage 27 (Fig. 3D). At stage 34, intense expression of fosl1 was mainly detected in eyes, branchial arch and notochord in addition to moderate expression in somite (Fig. 3E). Intense expression of fosl1 in the otic vesicle was also observed at stage 38 (Fig. 3F). At stage 42, strong expression of fosl1 in liver was detected, except for in eyes, branchial arch and notochord (Fig. 3G). However, no signals were detected using a sense probe of fosl1 for each examined stage (Fig. 3A’-G′). Inconsistently, no discernible fosl2 expression can be detected from stage 2 to stage 27 (Fig. 4A–D). Intense expression of fosl2 was detected specifically in lung primordium at stage 34 (Fig. 4E). The lung specific expression of fosl2 were maintained to staged 38 (Fig. 4F). At stage 42,
2. Results 2.1. Phylogenetic analysis of fosl proteins from X. tropicalis and other species To explore the conservatism of Fosl in evolution, protein sequences were aligned among different vertebrates. By comparing the protein sequence, we found that Fosl1 and Fosl2 of X. tropicalis (xFosl1 and 2
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Fig. 3. Spatial pattern of fosl1 analyzed by whole mount in situ hybridization. (A and A′) Stage 2, animal view. (B and B′) Stage 8, animal view. (C and C′) Stage 13, dorsal view. (D and D′) Stage 27, dorsal view. (E and E′) Stage 34, lateral view. (F and F′) Stage 38, lateral view. (G and G′) Stage 42, lateral view. Ba, branchial arches; cb, cement gland; fb, forebrain; mb, midbrain; hb, hindbrain; lv, liver; nc, notochord; ov, otic vesicle. A′-G′ indicates the sense probe control.
G′).
fosl2 expression was detected in eyes and branchial arch (Fig. 4G). The transverse section confirmed that fosl2 specifically was expressed in lung primordium with moderate signals (Fig. 4H and H′). The positive expression of fosl2 was further confirmed by the sense probe (Fig. 4A’-
Fig. 4. Spatial pattern of fosl2analyzed by whole mount in situ hybridization. (A and A′) Stage 2, animal view. (B and B′) Stage 8, animal view. (C and C′) Stage 13, dorsal view. (D and D′) Stage 27, dorsal view. (E and E′) Stage 34, lateral view. (F and F′) Stage 38, lateral view. (G and G′) Stage 42, lateral view. (H) Transverse sections of embryos at the levels illustrated by broken black lines in F. (H′) Large magnification of image shown in H. Ba, branchial arch; nc, notochord. A′-G′ indicates the sense probe control. 3
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3. Discussion
database at NCBI (http://www.ncbi.nlm.nih.gov/protein/).
All AP-1 proteins are characterized by a basic leucine-zipper region for dimerisation and DNA-binding. Previous studies have demonstrated that the expression of Fos proteins is crucial for the activity of AP-1regulated genes. Target genes regulated by the AP-1 transcription factor are involved in multiple cellular progresses including proliferation, differentiation, survival, and angiogenesis. The activity of all Fos family members is also modulated by posttranslational modification. Therefore, further study of the functions of Fos proteins in vivo is more important and will probably open new perspectives for related disease therapy (Milde-Langosch, 2005). In the present study, we examined the spatial and temporal expression pattern of fosl1 and fosl2 genes during early embryonic development of X. tropicalis, and found the different expression patterns for these two genes. In vertebrates, Fosl proteins are conservative during biological evolution (Fig. 1). However, the detailed functions of these proteins are different in individual cell type or species. The modulation of fosl1 expression directly affected cell proliferation, invasiveness and motility of cells in vitro(Belguise et al., 2005). In zebrafish, the fosl2 can potentiate the rate of cardiomyocyte differentiation from SHF progenitors (Bozec et al., 2013). Moreover, it have been reported that fosl1 transgenic mice developed severe anomaly of lung structure, including fibrosis, vascular remodeling and inflammation (Belguise et al., 2005; Tsujino et al., 2017b; Tsujino and Sheppard, 2016). Artificial Fra-2 expression in αSMA-expressing lineage cells critically modulates secondary alveolar septation in developing murine lungs (Tsujino et al., 2017a). These previous studies suggest that Fosl1 and Fosl2 proteins have different detailed functions depending on cell type and species. In consistent, fosl2 rather than fosl1 were detected specifically in lung from stage 34 to stage 38 during X. tropicalis development (Fig. 4), implying the different functions of fosl genes for specific tissue development. The dramatically upregulation of fosl1in embryonic development, especially at stage 42, implies that fosl1 may play an important role in normal development of Xenopus embryos (Fig. 2). Consistently, previous study has demonstrated the pivotal role in embryonic development in mammals. Fosl1 deficiency induces defects in vascularization of the placenta, thereby resulting in severely growth retardation and lethality between E10.0 and E10.5 (Schreiber et al., 2000). Therefore, these data in previous study and our works suggest that fosl1 is crucial for embryo development in vertebrates. For fosl2 gene, previously study has reported that fosl2 are detected in several differentiating epithelial, developing cartilage and central nervous system during mouse development, with a distinct expression patterns from other fos related genes, suggesting that fosl2 has a unique role in cellular differentiation during fetal development (Carrasco and Bravo, 1995). In consistent, fosl2 indeed shown unique expression patterns during embryonic development of X. tropicalis compared with fosl1 (Figs. 2–4). In conclusion, we examined and reported different temporal and spatial expression patterns between fosl1 and fosl2 during embryonic development of X. tropicalis. This study will facilitate the future functional analysis of fosl genes during embryonic development. Moreover, our study also provides the basis for generating fos genes-related disease models using Xenopus in the future.
4.2. Quantitative reverse transcription-PCR (qPCR) analysis Total RNA was extracted from Xenopus embryos at indicated developmental stages using the TRI reagent (Molecular Research Center Inc., USA). cDNA was synthesized from total RNA (2 μg) and oligo (dT) 18 primers (0.5 μg) using the ReverTra Ace® qPCR RT Kit (Toyobo, Japan). Real-time PCR was performed using a Light Cycler 480 SYBR Green I Master (Roche, USA) and the MiniOpticon Real-Time PCR System (Bio-Rad, CA, USA). The sequences of the qPCR primers were listed as following, fosl1 Fw, 5′-ATG GGA CAC AGC GTC AGA CT-3’; fosl1 Re, 5′-CCG AGG GCT TTG GTA TGG TG-3’; fosl2 Fw, 5′-GCA GGA GAA GTC CGG GTT AC-3’; fosl2 Re, 5′-AAA CCT CCG GCG ATG TTG AT3’; odc Fw, 5′-TGT TCT GCG CAT AGC AAC TG-3’; odc Re, 5′-ACA TCG TGC ATC TGA GAC AGC-3’. Ornithine decarboxylase (odc) was used as the internal standard control. After denaturation for 10 min at 95 °C, the reactions were subjected to 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s odc was used as the internal standard control to normalize gene expression using the △△Ct method. 4.3. Probe preparation and whole-mount in situ hybridization The whole ORFs of X. tropicalisfosl1 (accession number: XM_002939331.4) and fosl2 (accession number: XM_002938419.4) were amplified using cDNA templates from st42 tadpoles. Primers used for RT-PCR are as follows: fosl1 Fw (BamH1): 5′-CGC GGA TCC ATG TAC AGA GAC TTC ACT GGA GCC TT-3’; fosl1 Re (Xhol): 5′-CCG CTC GAG CTA AAG AGT CAG TAG GCT GTT AGA ACT T-3’; fosl2 Fw: 5′-ATG TAC CAG GAT TAT CCA GGG AAT TTC GAC AG-3’; fosl2 Re: 5′-TTA CAA AGC CAG GAG GGT GGG CGA GTT C-3’. PCR fragments were then subcloned into pBlueScript II SK (+) plasmid and verified by sequencing. Plasmids were linearized and then used as templates for synthesis of digoxigenin-labeled antisense probes with T7 RNA polymerase (Roche, Indianapolis, IN). Whole-mount in situ hybridization was performed using digoxigenin-labeled antisense RNA probe and anti-digoxigenin monoclonal antibody labeled with alkaline phosphatase. Probe signals were developed using NBT/BCIP (Roche, USA) as previously described (MonsoroBurq, 2007). Vibratome sectioning was performed as previously described (Wang et al., 2011). 5. Conflicts of interest There are no conflicts of interest. Acknowledgments This work was supported by grants from the National Key R&D Program of China (2016YFE0204700), the National Natural Science Foundation of China (31802025, 81570222, 81770240, and 81270183), the Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306011), the Guangdong Science and Technology Planning Project (2014A050503043), the New Star of Pearl River on Science and Technology of Guangzhou (2014J2200002), the Top Young Talents of Guangdong Province Special Support Program (87315007), and the Fundamental Research Funds for the Central Universities (21617436), China.
4. Experimental procedures 4.1. Blast searches and phylogenetic analysis
References Percentages of identical amino acids of human Fosl proteins sequence with different species were calculated by BLAST (BLASTP) searches in the RefSeq protein database at NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi). Protein sequence alignments and phylogenetic tree were performed using the neighbor-joining method in MEGA 6.0.6 software. Protein conserved domains were retrieved by protein
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