DLK1 is a somato-dendritic protein expressed in hypothalamic arginine-vasopressin and oxytocin neurons

Delta-Like 1 Homolog, Dlk1, is a paternally imprinted gene encoding a transmembrane protein involved in the differentiation of several cell types. After birth, Dlk1 expression decreases substantially in all tissues except endocrine glands. Dlk1 deletion in mice results in pre-natal and post-natal growth deficiency, mild obesity, facial abnormalities, and abnormal skeletal development, suggesting involvement of Dlk1 in perinatal survival, normal growth and homeostasis of fat deposition. A neuroendocrine function has also been suggested for DLK1 but never characterised. To evaluate the neuroendocrine function of DLK1, we first characterised Dlk1 expression in mouse hypothalamus and then studied post-natal variations of the hypothalamic expression. Western Blot analysis of adult mouse hypothalamus protein extracts showed that Dlk1 was expressed almost exclusively as a soluble protein produced by cleavage of the extracellular domain. Immunohistochemistry showed neuronal DLK1 expression in the suprachiasmatic (SCN), supraoptic (SON), paraventricular (PVN), arcuate (ARC), dorsomedial (DMN) and lateral hypothalamic (LH) nuclei. DLK1 was expressed in the dendrites and perikarya of arginine-vasopressin neurons in PVN, SCN and SON and in oxytocin neurons in PVN and SON. These findings suggest a role for DLK1 in the post-natal development of hypothalamic functions, most notably those regulated by the arginine-vasopressin and oxytocin systems.     

The atypical mammalian ligand Delta-like homologue 1 (Dlk1) can regulate Notch signalling in Drosophila

Background

Mammalian Delta-like 1 (Dlk-1) protein shares homology with Notch ligands but lacks a critical receptor-binding domain. Thus it is unclear whether it is able to interact with Notch in vivo. Unlike mammals, Drosophila have a single Notch receptor allowing a simple in vivo assay for mammalian Dlk1 function.

Results

Here we show that membrane-bound DLK1 can regulate Notch leading to altered cellular distribution of Notch itself and inhibiting expression of Notch target genes. The resulting adult phenotypes are indicative of reduced Notch function and are enhanced by Notch mutations, confirming that DLK1 action is antagonistic. In addition, cells expressing an alternative Dlk1 isoform exhibit alterations in cell size, functions previously not attributed to Notch suggesting that DLK1 might also act via an alternative target.

Conclusion

Our results demonstrate that DLK1 can regulate the Notch receptor despite its atypical structure.     

Alternative splicing of delta-like 1 homolog (DLK1) in the pig and human

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be present in mouse and cattle. Surprisingly, DLK1-A was undetected in various tissues from fetal and postnatal pigs. Instead, DLK1-C2 was the most abundant isoform while DLK1-B was expressed to a lesser extent. In fractionated adipose tissue, pDLK1 was most highly expressed in the stromal-vascular cell fraction. In addition, total pDLK1 was highly expressed in fetal adipose tissue but dramatically decreased postnatally. Our data suggests that expression of DLK1-B and -C2 isoforms is sufficient for normal pig development. Furthermore, human and pig samples showed no alterations in species-specific splicing, but expression levels decreased with age, suggesting that regulation of expression, not splicing, is the most likely mechanism controlling the biological function of DLK1.     

Conditional deletions refine the embryonic requirement for Dlk1

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing β-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1^Sul-pat), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1^flox) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic β-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1^Sul-pat null mice. Dlk1 expression is therefore not essential for the normal development of β-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1^flox mice will continue to provide an important tool for further research into the function of Dlk1.     

dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.     

MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes

Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes.In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1^M and soluble DLK1^S are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1^M protein while it increases the amount of DLK1^S supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1^S.Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.     

Hepatic stellate cell-derived delta-like homolog 1 (DLK1) protein in liver regeneration

Hepatic stellate cells (HSCs) undergo myofibroblastic activation in liver fibrosis and regeneration. This phenotypic switch is mechanistically similar to dedifferentiation of adipocytes as such the necdin-Wnt pathway causes epigenetic repression of the master adipogenic gene Pparγ, to activate HSCs. Now we report that delta-like 1 homolog (DLK1) is expressed selectively in HSCs in the adult rodent liver and induced in liver fibrosis and regeneration. Dlk1 knockdown in activated HSCs, causes suppression of necdin and Wnt, epigenetic derepression of Pparγ, and morphologic and functional reversal to quiescent cells. Hepatic Dlk1 expression is induced 40-fold at 24 h after partial hepatectomy (PH) in mice. HSCs and hepatocytes (HCs) isolated from the regenerating liver show Dlk1 induction in both cell types. In HC and HSC co-culture, increased proliferation and Dlk1 expression by HCs from PH are abrogated with anti-DLK1 antibody (Ab). Dlk1 and Wnt10b expression by Sham HCs are increased by co-culture with PH HSCs, and these effects are abolished with anti-DLK Ab. A tail vein injection of anti-DLK1 Ab at 6 h after PH reduces early HC proliferation and liver growth, accompanied by decreased Wnt10b, nonphosphorylated β-catenin, p-β-catenin (Ser-552), cyclins (cyclin D and cyclin A), cyclin-dependent kinases (CDK4, and CDK1/2), p-ERK1/2, and p-AKT. In the mouse developing liver, HSC precursors and HSCs express high levels of Dlk1, concomitant with Dlk1 expression by hepatoblasts. These results suggest novel roles of HSC-derived DLK1 in activating HSCs via epigenetic Pparγ repression and participating in liver regeneration and development in a manner involving the mesenchymal-epithelial interaction.     

Localizing transcriptional regulatory elements at the mouse Dlk1 locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression.     

Possible roles of DLK1 in the Notch pathway during development and disease

The Delta-Notch pathway is an evolutionarily conserved signaling pathway which controls a broad range of developmental processes including cell fate determination, terminal differentiation and proliferation. In mammals, four Notch receptors (NOTCH1-4) and five activating canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3 and DLL4) have been described. The precise function of noncanonical Notch ligands remains unclear. Delta-like 1 homolog (DLK1), the best studied noncanonical Notch ligand, has been shown to act as an inhibitor of Notch signaling in vitro, but its function in vivo is poorly understood. In this review we summarize Notch signaling during development and highlight recent studies in DLK1expression that reveal new insights into its function.     

哺乳动物印记域DLK1-DIO3的研究进展

DLK1-DIO3印记域定位于人14号染色体、小鼠12号染色体及绵羊18号染色体远端, 在真哺乳亚纲动物中印记保守。该印记域包含3个编码蛋白的父系表达基因Dlk1、Rtl1和Dio3以及若干大小不同的母系表达印记非编码RNA, 如miRNAs、snoRNAs 和大型非编码RNA Gtl2等。人和小鼠该印记域内印记基因剂量的改变将导致严重的表型异常甚至胚胎致死, 暗示正常的发育需要域内印记基因的正常表达。文章重点论述了哺乳动物DLK1-DIO3印记域的印记调控机制和域内印记基因及其功能的研究进展。     

Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness

DLK1 is a maternally imprinted, paternally expressed gene coding for the transmembrane protein Delta-like homologue 1 (DLK1), a non-canonical NOTCH ligand with well-described roles during development, and tumor-supportive functions in several aggressive cancer forms. Here, we review the many functions of DLK1 as a regulator of stem cell pools and tissue differentiation in tissues such as brain, muscle, and liver. Furthermore, we review recent evidence supporting roles for DLK1 in the maintenance of aggressive stem cell characteristics of tumor cells, specifically focusing on central nervous system tumors, neuroblastoma, and hepatocellular carcinoma. We discuss NOTCH -dependent as well as NOTCH-independent functions of DLK1, and focus particularly on the complex pattern of DLK1 expression and cleavage that is finely regulated from a spatial and temporal perspective. Progress in recent years suggest differential functions of extracellular, soluble DLK1 as a paracrine stem cell niche-secreted factor, and has revealed a role for the intracellular domain of DLK1 in cell signaling and tumor stemness. A better understanding of DLK1 regulation and signaling may enable therapeutic targeting of cancer stemness by interfering with DLK1 release and/or intracellular signaling.     

The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.