Abstract
DLK1 and DLK2 are transmembrane proteins belonging to the EGF-like repeat-containing family that function as non-canonical NOTCH inhibitory ligands. DLK1 is usually downregulated after embryo development and its distribution in some adult and embryonic tissues has been described. However, the expression and role of DLK2 in embryo and adult tissues remains unclear. To better understand the relevance of both proteins during embryo development, we analyzed the expression pattern of DLK1 and DLK2 in 16.5-day-old mouse embryos (E16.5) and evaluated the possible relationship between these two proteins in embryo tissues and cell types. We found that DLK1 and DLK2 proteins exhibited a broad distribution pattern, which was detected in developing mouse organs from each of the three germ layers: ectoderm (brain, salivary glands), mesoderm (skeletal muscle, vertebral column, kidney, cartilage), and endoderm (thymus, lung, pancreas, intestine, liver). The expression pattern of DLK1 and DLK2 indicates that both proteins could play a synergistic role during cell differentiation. This study provides additional information for understanding temporal and site-specific effects of DLK1 and DLK2 during embryo morphogenesis and cell differentiation.
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16 May 2019
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References
Abdallah BM, Jensen CH, Gutierrez G, Leslie RG, Jensen TG, Kassem M (2004) Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1. J Bone Miner Res 19:841–852. https://doi.org/10.1359/JBMR.040118
Andersen DC, Petersson SJ, Jorgensen LH, Bollen P, Jensen PB, Teisner B, Schroeder HD, Jensen CH (2009) Characterization of DLK1+ cells emerging during skeletal muscle remodeling in response to myositis, myopathies, and acute injury. Stem Cells 27:898–908. https://doi.org/10.1634/stemcells.2008-0826
Andersen DC, Laborda J, Baladron V, Kassem M, Sheikh SP, Jensen CH (2013) Dual role of delta-like 1 homolog (DLK1) in skeletal muscle development and adult muscle regeneration. Development 140:3743–3753. https://doi.org/10.1242/dev.095810
Ansell PJ, Zhou Y, Schjeide BM, Kerner A, Zhao J, Zhang X, Klibanski A (2007) Regulation of growth hormone expression by Delta-like protein 1 (Dlk1). Mol Cell Endocrinol 271:55–63 (pii:S0303-7207(07)00151-7)
Appelbe OK, Yevtodiyenko A, Muniz-Talavera H, Schmidt JV (2013) Conditional deletions refine the embryonic requirement for Dlk1. Mech Dev 130:143–159. https://doi.org/10.1016/j.mod.2012.09.010
Baladrón V, Ruiz-Hidalgo MJ, Nueda ML, Díaz-Guerra MJ, García-Ramírez JJ, Bonvini E, Gubina E, Laborda J (2005) dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats. Exp Cell Res 303(2):343–359. https://doi.org/10.1016/j.yexcr.2004.10.001
Bamman MM, Shipp JR, Jiang J, Gower BA, Hunter GR, Goodman A, McLafferty CL Jr, Urban RJ (2001) Mechanical load increases muscle IGF-I and androgen receptor mRNA concentrations in humans. Am J Physiol Endocrinol Metab 280:E383–E390
Chen L, Qanie D, Jafari A, Taipaleenmaki H, Jensen CH, Saamanen AM, Sanz ML, Laborda J, Abdallah BM, Kassem M (2011) Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway. J Biol Chem 286:32140–32149. https://doi.org/10.1074/jbc.M111.230110
Cheung LY, Rizzoti K, Lovell-Badge R, Le Tissier PR (2013) Pituitary phenotypes of mice lacking the notch signalling ligand delta-like 1 homologue. J Neuroendocrinol 25:391–401. https://doi.org/10.1111/jne.12010
da Rocha ST, Charalambous M, Lin SP, Gutteridge I, Ito Y, Gray D, Dean W, Ferguson-Smith AC (2009) Gene dosage effects of the imprinted delta-like homologue 1 (dlk1/pref1) in development: implications for the evolution of imprinting. PLoS Genet 5:e1000392. https://doi.org/10.1371/journal.pgen.1000392
Davis E, Jensen CH, Schroder HD, Farnir F, Shay-Hadfield T, Kliem A, Cockett N, Georges M, Charlier C (2004) Ectopic expression of DLK1 protein in skeletal muscle of padumnal heterozygotes causes the callipyge phenotype. Curr Biol 14:1858–1862 (pii:S0960-9822(04)00757-2)
Falix FA, Aronson DC, Lamers WH, Gaemers IC (2012) Possible roles of DLK1 in the Notch pathway during development and disease. Biochim Biophys Acta 822(6):988–995. https://doi.org/10.1016/j.bbadis.2012.02.003
Ferron SR, Charalambous M, Radford E, McEwen K, Wildner H, Hind E, Morante-Redolat JM, Laborda J, Guillemot F, Bauer SR, Farinas I, Ferguson-Smith AC (2011) Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis. Nature 475:381–385. https://doi.org/10.1038/nature10229
Floridon C, Jensen CH, Thorsen P, Nielsen O, Sunde L, Westergaard JG, Thomsen SG, Teisner B (2000) Does fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials? A study of FA1 in embryonic, fetal, and placental tissue and in maternal circulation. Differentiation 66:49–59 (pii:S0301-4681(09)60574-0)
Garcia-Gallastegui P, Ibarretxe G, Garcia-Ramirez JJ, Baladron V, Aurrekoetxea M, Nueda ML, Naranjo AI, Santaolalla F, Sanchez-del Rey A, Laborda J, Unda F (2014) DLK1 regulates branching morphogenesis and parasympathetic innervation of salivary glands through inhibition of NOTCH signalling. Biol Cell 106:237–253. https://doi.org/10.1111/boc.201300086
Garcia-Gallastegui P, Luzuriaga J, Aurrekoetxea M, Baladron V, Ruiz-Hidalgo MJ, Garcia-Ramirez JJ, Laborda J, Unda F, Ibarretxe G (2016) Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice. Cell Tissue Res 364:513–525. https://doi.org/10.1007/s00441-015-2344-z
Giachino C, Taylor V (2014) Notching up neural stem cell homogeneity in homeostasis and disease. Front Neurosci 8:32. https://doi.org/10.3389/fnins.2014.00032
Gordon J, Manley NR (2011) Mechanisms of thymus organogenesis and morphogenesis. Development 138:3865–3878. https://doi.org/10.1242/dev.059998
Gubina E, Ruiz-Hidalgo MJ, Baladron V, Laborda J (1999) Assignment of DLK1 to human chromosome band 14q32 by in situ hybridization. Cytogenet Cell Genet 84:206–207 (pii:15259)
Hermida C, Garces C, de Oya M, Cano B, Martinez-Costa OH, Rivero S, Garcia-Ramirez JJ, Laborda J, Aragon JJ (2008) The serum levels of the EGF-like homeotic protein dlk1 correlate with different metabolic parameters in two hormonally different children populations in Spain. Clin Endocrinol (Oxf) 69:216–224. https://doi.org/10.1111/j.1365-2265.2008.03170.x
Imayoshi I, Sakamoto M, Yamaguchi M, Mori K, Kageyama R (2010) Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 30:3489–3498. https://doi.org/10.1523/JNEUROSCI.4987-09.2010
Jensen CH, Krogh TN, Hojrup P, Clausen PP, Skjodt K, Larsson LI, Enghild JJ, Teisner B (1994) Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. Eur J Biochem 225:83–92
Kim Y, Lin Q, Zelterman D, Yun Z (2009) Hypoxia-regulated delta-like 1 homologue enhances cancer cell stemness and tumorigenicity. Cancer Res 69:9271–9280. https://doi.org/10.1158/0008-5472.CAN-09-1605
Lebkuechner I, Wilhelmsson U, Mollerstrom E, Pekna M, Pekny M (2015) Heterogeneity of Notch signaling in astrocytes and the effects of GFAP and vimentin deficiency. J Neurochem 135:234–248. https://doi.org/10.1111/jnc.13213
Lee D, Yoon SH, Lee HJ, Jo KW, Park BC, Kim IS, Choi Y, Lim JC, Park YW (2016) Human soluble delta-like 1 homolog exerts antitumor effects in vitro and in vivo. Biochem Biophys Res Commun 475:209–215. https://doi.org/10.1016/j.bbrc.2016.05.076
Lindstrom M, Pedrosa-Domellof F, Thornell LE (2010) Satellite cell heterogeneity with respect to expression of MyoD, myogenin, Dlk1 and c-Met in human skeletal muscle: application to a cohort of power lifters and sedentary men. Histochem Cell Biol 134:371–385. https://doi.org/10.1007/s00418-010-0743-5
Mirshekar-Syahkal B, Haak E, Kimber GM, van Leusden K, Harvey K, O’Rourke J, Laborda J, Bauer SR, de Bruijn MF, Ferguson-Smith AC, Dzierzak E, Ottersbach K (2013) Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells. Haematologica 98:163–171. https://doi.org/10.3324/haematol.2012.070789
Moore KA, Pytowski B, Witte L, Hicklin D, Lemischka IR (1997) Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs. Proc Natl Acad Sci USA 94:4011–4016
Muller D, Cherukuri P, Henningfeld K, Poh CH, Wittler L, Grote P, Schluter O, Schmidt J, Laborda J, Bauer SR, Brownstone RM, Marquardt T (2014) Dlk1 promotes a fast motor neuron biophysical signature required for peak force execution. Science 343:1264–1266. https://doi.org/10.1126/science.1246448
Nakakura T, Sato M, Suzuki M, Hatano O, Takemori H, Taniguchi Y, Minoshima Y, Tanaka S (2009) The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development. Histochem Cell Biol 131:141–153. https://doi.org/10.1007/s00418-008-0494-8
Nueda ML, Baladron V, Garcia-Ramirez JJ, Sanchez-Solana B, Ruvira MD, Rivero S, Ballesteros MA, Monsalve EM, Diaz-Guerra MJ, Ruiz-Hidalgo MJ, Laborda J (2007a) The novel gene EGFL9/Dlk2, highly homologous to Dlk1, functions as a modulator of adipogenesis. J Mol Biol 367:1270–1280 (pii:S0022-2836(06)01346-5)
Nueda ML, Baladron V, Sanchez-Solana B, Ballesteros MA, Laborda J (2007b) The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells. J Mol Biol 367:1281–1293 (pii:S0022-2836(06)01415-X)
Nueda ML, Garcia-Ramirez JJ, Laborda J, Baladron V (2008) dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells. J Mol Biol 379:428–442. https://doi.org/10.1016/j.jmb.2008.03.070
Nueda ML, Naranjo AI, Baladron V, Laborda J (2017) Different expression levels of DLK1 inversely modulate the oncogenic potential of human MDA-MB-231 breast cancer cells through inhibition of NOTCH1 signaling. FASEB J 31:3484–3496. https://doi.org/10.1096/fj.201601341RRR
Pawson T, Nash P (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300:445–452. https://doi.org/10.1126/science.1083653
Raghunandan R, Ruiz-Hidalgo M, Jia Y, Ettinger R, Rudikoff E, Riggins P, Farnsworth R, Tesfaye A, Laborda J, Bauer SR (2008) Dlk1 influences differentiation and function of B lymphocytes. Stem Cells Dev 17:495–507. https://doi.org/10.1089/scd.2007.0102
Rivero S, Diaz-Guerra MJ, Monsalve EM, Laborda J, Garcia-Ramirez JJ (2012) DLK2 is a transcriptional target of KLF4 in the early stages of adipogenesis. J Mol Biol 417:36–50. https://doi.org/10.1016/j.jmb.2012.01.035
Sanchez-Solana B, Nueda ML, Ruvira MD, Ruiz-Hidalgo MJ, Monsalve EM, Rivero S, Garcia-Ramirez JJ, Diaz-Guerra MJ, Baladron V, Laborda J (2011) The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other’s activities. Biochim Biophys Acta 1813:1153–1164. https://doi.org/10.1016/j.bbamcr.2011.03.004
Schaeren-Wiemers N, Gerfin-Moser A (1993) A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100:431–440
Smas CM, Sul HS (1993) Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell 73:725–734 (pii:0092-8674(93)90252-L)
Smas CM, Green D, Sul HS (1994) Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1. Biochemistry 33:9257–9265
Tanimizu N, Nishikawa M, Saito H, Tsujimura T, Miyajima A (2003) Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci 116:1775–1786
Taylor MK, Yeager K, Morrison SJ (2007) Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems. Development 134:2435–2447 (pii:dev.005520)
Traustadottir GA, Jensen CH, Thomassen M, Beck HC, Mortensen SB, Laborda J, Baladron V, Sheikh SP, Andersen DC (2016) Evidence of non-canonical NOTCH signaling: delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals. Cell Signal 28:246–254. https://doi.org/10.1016/j.cellsig.2016.01.003
Tremblay KD, Zaret KS (2005) Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Dev Biol 280:87–99 (pii:S0012-1606(05)00004-7)
Tucker AS (2007) Salivary gland development. Semin Cell Dev Biol 18:237–244 (pii:S1084-9521(07)00032-8)
Waddell JN, Zhang P, Wen Y, Gupta SK, Yevtodiyenko A, Schmidt JV, Bidwell CA, Kumar A, Kuang S (2010) Dlk1 is necessary for proper skeletal muscle development and regeneration. PLoS One 5:e15055. https://doi.org/10.1371/journal.pone.0015055
Wang Y, Sul HS (2006) Ectodomain shedding of preadipocyte factor 1 (Pref-1) by tumor necrosis factor alpha converting enzyme (TACE) and inhibition of adipocyte differentiation. Mol Cell Biol 26:5421–5435
Wang J, Shaner N, Mittal B, Zhou Q, Chen J, Sanger JM, Sanger JW (2005) Dynamics of Z-band based proteins in developing skeletal muscle cells. Cell Motil Cytoskelet 61:34–48. https://doi.org/10.1002/cm.20063
Wang Y, Zhao L, Smas C, Sul HS (2010) Pref-1 interacts with fibronectin to inhibit adipocyte differentiation. Mol Cell Biol 30:3480–3492. https://doi.org/10.1128/MCB.00057-10
Warburton D, El-Hashash A, Carraro G, Tiozzo C, Sala F, Rogers O, De Langhe S, Kemp PJ, Riccardi D, Torday J, Bellusci S, Shi W, Lubkin SR, Jesudason E (2010) Lung organogenesis. Curr Top Dev Biol 90:73–153
Weidenbusch M, Rodler S, Song S, Romoli S, Marschner JA, Kraft F, Holderied A, Kumar S, Mulay SR, Honarpisheh M, Kumar Devarapu S, Lech M, Anders HJ (2017) Gene expression profiling of the Notch-AhR-IL22 axis at homeostasis and in response to tissue injury. Biosci Rep. https://doi.org/10.1042/BSR20170099
Xu W, Wang Y, Zhao H, Fan B, Guo K, Cai M, Zhang S (2018) Delta-like 2 negatively regulates chondrogenic differentiation. J Cell Physiol 233(9):6574–6582. https://doi.org/10.1002/jcp.26244
Yanai H, Nakamura K, Hijioka S, Kamei A, Ikari T, Ishikawa Y, Shinozaki E, Mizunuma N, Hatake K, Miyajima A (2010) Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J Biochem 148:85–92. https://doi.org/10.1093/jb/mvq034
Yevtodiyenko A, Schmidt JV (2006) Dlk1 expression marks developing endothelium and sites of branching morphogenesis in the mouse embryo and placenta. Dev Dyn 235:1115–1123. https://doi.org/10.1002/dvdy.20705
Acknowledgements
This work was funded with project grants from the University of the Basque Country UPV/EHU (GIU16/66) and a grant from the Consejería de Educación, Cultura y Deportes of Castilla-La Mancha (PPII-2014-018-P), supported with FEDER funds. P.G. received a fellowship from the University of the Basque Country UPV/ EHU. Technical and human support provided by SGIker (UPV/EHU, MINECO, GV/EJ, ERDF, and ESF) is gratefully acknowledged.
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Supplementary Fig. 1
. (a) In situ hybridization with sense riboprobe of E16.5 mouse embryo sections. Scale bar: 2 mm. (b) Negative control of the immunohistochemistry, sections of E16.5 mouse embryo incubated with the preserum of immunized rabbits and then with anti-rabbit antibody. Scale bar 1 mm. (TIF 5249 KB)
Supplementary Fig. 2
. Antibody validation. 293T cell transfected with pLNCX2-cmyc empty vector (lanes 1 and 3) or with pLNCX2-Dlk2-cmyc (lanes 2 and 4). 50 mg of cell extracts were electrophoresed and blotted with anti-c-myc antibody (SIGMA clone 9E10, lanes 1 and 2) or anti-Dlk2 antibodies (lanes 3 and 4). Identical 50 kDa band protein was detected by both antibodies. (TIF 113 KB)
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Garcia-Gallastegi, P., Ruiz-García, A., Ibarretxe, G. et al. Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis. Histochem Cell Biol 152, 47–60 (2019). https://doi.org/10.1007/s00418-019-01778-4
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DOI: https://doi.org/10.1007/s00418-019-01778-4