Delta-like 1 homolog (DLK1) is a noncanonical ligand in the Delta-Notch signalling pathway. Although Dlk1 mRNA is abundantly present embryonically and declines rapidly just before birth, Dlk1 knockouts display a relatively mild phenotype. To assess whether this mild phenotype was due to posttranscriptional regulation, we studied the expression of DLK1 protein in mouse embryos and found abundant expression in liver, lung, muscle, vertebrae, pancreas, pituitary, and adrenal gland(s). DLK1 expression was absent in heart, stomach, intestine, kidney, epidermis, and central nervous system. DLK1 protein expression, therefore, correlates well with the reported Dlk1 mRNA expression pattern, which shows that its expression is mainly regulated at the pretranslational level. The comparison of the reported expression patterns of Notch mRNA and those of DLK1 in organs where lineage commitment and branching morphogenesis are important developmental processes suggests that DLK1 is a ligand that prevents premature Notch-dependent differentiation, possibly by competing with canonical ligands. 1. Introduction DLK1, also known as preadipocyte factor 1 (Pref-1), is a transmembrane EGF-like protein consisting of an N-terminal signal sequence, six EGF-like repeats, a short juxta-membrane region containing a TACE-mediated cleavage site, a transmembrane domain, and a short C-terminal cytoplasmic tail [1, 2]. DLK1 is a noncanonical member of the evolutionarily conserved Delta-Notch signalling pathway, which is involved in stem-cell decisions during development [3]. Although DLK1 lacks the Delta-Serrate-Lag2 (DSL) domain for binding with the EGF-like repeats of Notch receptors, which all canonical Notch ligands possess [4, 5], specific interaction of DLK1 with the NOTCH1 receptor was demonstrated with the yeast two-hybrid system and Notch1 signalling was inhibited by Dlk1 [6, 7]. Furthermore, in Drosophila, Dlk1 was shown to regulate the function of the Notch receptor, resulting in an altered cellular distribution of Notch itself and inhibition of expression of Notch target genes [8]. DLK1 function has been studied most in the murine preadipocyte cell line 3T3L1, which expresses both the transmembrane (55？kDa) and soluble (50？kDa) form of the DLK1 protein. Soluble DLK1 acts as an inhibitor of adipogenesis, preventing the differentiation of murine preadipocytes into mature adipocytes [1, 9, 10]. However, recent data show that DLK1 is also able to promote adipogenesis of mesenchymal stem cells [4]. Other proposed roles for DLK1 have been in maturation along the chromaffin lineage
References
[1]
C. M. Smas, L. Chen, and H. S. Sul, “Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation,” Molecular and Cellular Biology, vol. 17, no. 2, pp. 977–988, 1997.
[2]
Y. Wang and H. S. Sul, “Ectodomain shedding of preadipocyte factor 1 (Pref-1) by tumor necrosis factor alpha converting enzyme (TACE) and inhibition of adipocyte differentiation,” Molecular and Cellular Biology, vol. 26, no. 14, pp. 5421–5435, 2006.
[3]
R. Kopan and M. X. Ilagan, “The canonical Notch signalling pathway: unfolding the activation mechanism,” Cell, vol. 137, no. 2, pp. 216–233, 2009.
[4]
S. Artavanis-Tsakonas, K. Matsuno, and M. E. Fortini, “Notch signalling,” Science, vol. 268, no. 5208, pp. 225–232, 1995.
[5]
H. von Boehmer, “Coming to grips with Notch,” Journal of Experimental Medicine, vol. 194, no. 7, pp. F43–F46, 2001.
[6]
V. Baladron, M. J. Ruiz-Hidalgo, M. L. Nueda, et al., “dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats,” Experimental Cell Research, vol. 303, no. 2, pp. 343–359, 2005.
[7]
M. L. Nueda, V. Baladron, B. Sanchez-Solana, M. A. Ballesteros, and J. Laborda, “The EGF-like protein dlk1 inhibits Notch signalling and potentiates adipogenesis of mesenchymal cells,” Journal of Molecular Biology, vol. 367, no. 5, pp. 1281–1293.
[8]
S. J. Bray, S. Takada, E. Harrison, S. C. Shen, and A. C. Ferguson-Smith, “The atypical mammalian ligand Delta-like homologue 1 (dlk1) can regulate Notch signalling in Drosophila,” BMC Developmental Biology, vol. 8, p. 11, 2008.
[9]
C. M. Smas and H. S. Sul, “Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation,” Cell, vol. 73, no. 4, pp. 725–734, 1993.
[10]
C. M. Smas and H. S. Sul, “Molecular mechanisms of adipocyte differentiation and inhibitory action of pref-1,” Critical Reviews in Eukaryotic Gene Expression, vol. 7, no. 4, pp. 281–298, 1997.
[11]
V. Limpt van, A. J. Chan, P. G. Van Sluis, H. N. Caron, C. J. Van Noesel, and R. Versteeg, “High delta-like 1 expression in a subset of neuroblastoma cell lines corresponds to a differentiated chromaffin cell type,” International Journal of Cancer, vol. 105, no. 1, pp. 61–69, 2003.
[12]
K. A. Moore, B. Pytowski, L. Witte, D. Hicklin, and I. R. Lemischka, “Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 8, pp. 4011–4016, 1997.
[13]
E. Davis, C. H. Jensen, H. D. Schroder, et al., “Ectopic expression of dlk1 protein in skeletal muscle of padumnal heterozygotes causes the callipyge phenotype,” Current Biology, vol. 14, no. 20, pp. 1858–1862, 2004.
[14]
N. Tanimizu, M. Nishikawa, H. Saito, T. Tsujimura, and A. Miyajima, “Isolation of hepatoblasts based on the expression of Dlk/Pref-1,” Journal of Cell Science, vol. 116, part 9, pp. 1775–1786, 2003.
[15]
N. Tanimizu, T. Tsujimura, K. Takahide, T. Kodama, K. Nakamura, and A. Miyajima, “Expression of Dlk/Pref-1 defines a subpopulation in the oval cell compartment of rat liver,” Gene Expression Patterns, vol. 5, no. 2, pp. 209–218, 2004.
[16]
C. H. Jensen, B. Teisner, P. Hojrup, et al., “Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2,” Human Reproduction, vol. 8, no. 4, pp. 635–641, 1993.
[17]
C. Floridon, C. H. Jensen, P. Thorsen, et al., “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, vol. 66, no. 1, pp. 49–59, 2000.
[18]
A. Yevtodiyenko and J. V. Schmidt, “dlk1 expression marks developing endothelium and sites of branching morphogenesis in the mouse embryo and placenta,” Developmental Dynamics, vol. 235, no. 4, pp. 1115–1123, 2006.
[19]
M. Andersen, C. H. Jensen, R. K. Stoving, et al., “Fetal antigen 1 in healthy adults and patients with pituitary disease: relation to physiological, pathological, and pharmacological GH levels,” Journal of Clinical Endocrinology & Metabolism, vol. 86, no. 11, pp. 5465–5470, 2001.
[20]
D. Tornehave, C. H. Jensen, B. Teisner, and L. I. Larsson, “FA1 immunoreactivity in endocrine tumours and during development of the human fetal pancreas, negative correlation with glucagon expression,” Histochemistry and Cell Biology, vol. 106, no. 6, pp. 535–542, 1996.
[21]
J. B. Larsen, C. H. Jensen, H. D. Schroder, B. Teisner, P. Bjerre, and C. Hagen, “Fetal antigen 1 and growth hormone in pituitary somatotroph cells,” The Lancet, vol. 347, no. 8995, p. 191, 1996.
[22]
Y. S. Moon, C. M. Smas, K. Lee, et al., “Mice lacking paternally expressed Pref-1/dlk1 display growth retardation and accelerated adiposity,” Molecular and Cellular Biology, vol. 22, no. 15, pp. 5585–5592, 2002.
[23]
R. Rugh, Vertebrate Embryology (The Dynamics of Development), Harcourt, Brace & World, New York, NY, USA, 1964.
[24]
F. Geisler, F. Nagl, P. K. Mazur, et al., “Liver-specific inactivation of Notch2, but not Notch1, compromises intrahepatic bile duct development in mice,” Hepatology, vol. 48, no. 2, pp. 607–616, 2008.
[25]
F. Lemaigre and K. S. Zaret, “Liver development update: new embryo models, cell lineage control, and morphogenesis,” Current Opinion in Genetics & Development, vol. 14, no. 5, pp. 582–590, 2004.
[26]
K. M. Loomes, D. B. Taichman, C. L. Glover, et al., “Characterization of Notch receptor expression in the developing mammalian heart and liver,” American Journal of Medical Genetics, vol. 112, no. 2, pp. 181–189, 2002.
[27]
K. M. Loomes, P. Russo, M. Ryan, et al., “Bile duct proliferation in liver-specific Jag1 conditional knockout mice: effects of gene dosage,” Hepatology, vol. 45, no. 2, pp. 323–330, 2007.
[28]
J. Lozier, B. McCright, and T. Gridley, “Notch signalling regulates bile duct morphogenesis in mice,” PLoS ONE, vol. 3, no. 3, p. e1851, 2008.
[29]
Y. Zong, A. Panikkar, J. Xu, et al., “Notch signalling controls liver development by regulating biliary differentiation,” Development, vol. 136, no. 10, pp. 1727–1739, 2009.
[30]
R. Williams, U. Lendahl, and M. Lardelli, “Complementary and combinatorial patterns of Notch gene family expression during early mouse development,” Mechanisms of Development, vol. 53, no. 3, pp. 357–368, 1995.
[31]
C. E. Lindsell, J. Boulter, G. diSibio, A. Gossler, and G. Weinmaster, “Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development,” Molecular and Cellular Neuroscience, vol. 8, no. 1, pp. 14–27, 1996.
[32]
V. Limpt van, A. Chan, H. Caron, et al., “SAGE analysis of neuroblastoma reveals a high expression of the human homologue of the Drosophila Delta gene,” Medical and Pediatric Oncology, vol. 35, no. 6, pp. 554–558, 2000.
[33]
H. Z. Sheng and H. Westphal, “Early steps in pituitary organogenesis,” Trends in Genetics, vol. 15, no. 6, pp. 236–240, 1999.
[34]
X. Zhu, J. Zhang, J. Tollkuhn, et al., “Sustained Notch signalling in progenitors is required for sequential emergence of distinct cell lineages during organogenesis,” Genes & Development, vol. 20, no. 19, pp. 2739–2753, 2006.
[35]
L. B. Goldberg, P. K. Aujla, and L. T. Raetzman, “Persistent expression of activated Notch inhibits corticotrope and melanotrope differentiation and results in dysfunction of the HPA axis,” Developmental Biology, vol. 358, no. 1, pp. 23–32, 2011.
[36]
R. A. Puertas-Avendano, M. J. Gonzalez-Gomez, M. D. Ruvira, et al., “Role of the non-canonical Notch ligand delta-like protein 1 in hormone-producing cells of the adult male mouse pituitary,” Journal of Neuroendocrinology, vol. 23, no. 9, pp. 849–859, 2011.
[37]
A. Horowitz and M. Simons, “Branching morphogenesis,” Circulation Research, vol. 103, no. 8, pp. 784–795, 2008.
[38]
P. N. Tsao, F. Chen, K. I. Izvolsky, et al., “Gamma-secretase activation of Notch signalling regulates the balance of proximal and distal fates in progenitor cells of the developing lung,” Journal of Biological Chemistry, vol. 283, no. 43, pp. 29532–29544, 2008.
[39]
J. S. Guseh, S. A. Bores, B. Z. Stanger, et al., “Notch signalling promotes airway mucous metaplasia and inhibits alveolar development,” Development, vol. 136, no. 10, pp. 1751–1759, 2009.
[40]
E. Lammert, J. Brown, and D. A. Melton, “Notch gene expression during pancreatic organogenesis,” Mechanisms of Development, vol. 94, no. 1-2, pp. 199–203, 2000.
[41]
A. Apelqvist, H. Li, L. Sommer, et al., “Notch signalling controls pancreatic cell differentiation,” Nature, vol. 400, no. 6747, pp. 877–881, 1999.
[42]
J. Jensen, E. E. Pedersen, P. Galante, et al., “Control of endodermal endocrine development by Hes-1,” Nature Genetics, vol. 24, no. 1, pp. 36–44, 2000.
[43]
J. Hald, J. P. Hjorth, M. S. German, O. D. Madsen, P. Serup, and J. Jensen, “Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development,” Developmental Biology, vol. 260, no. 2, pp. 426–437, 2003.
