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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01169

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Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors

¹ Stem Cell Regulation, Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Kawasaki, Kanagawa 216-0001, Japan
² Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan
³ Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi City, Saitama 332-0012, Japan

View larger version (106K): [in a new window]   Fig. 1. Expression of Notch genes and their ligands. (A) In the upper panel, RT-PCR shows that Notch1 and Notch2 RNA is expressed in liver throughout its development and that Jagged1 RNA is expressed in fetal liver. One-fifteenths of cDNA synthesized from 1 µg of total RNA of developing liver was used for PCR. In the lower panel, a Northern blot shows the expression of Notch1 and Notch2 RNA in the primary culture of E14.5 Dlk+ cells at 4 and 24 hours after plating; 10 µg of total RNA was used for the analysis. GAPDH expression was also examined as an internal control in both experiments. (B, C) Frozen sections of E14.5 liver were stained with rat IgG (B) and anti-Notch2 rat monoclonal Ab (C). The sections were counter-stained with hematoxyline. Notch2 is synthesized broadly in E14.5 liver (C). (D-F) Series of frozen E17.5 liver sections, incubated with rat IgG (D), anti-Notch2 Ab (E) and anti-cytokeratin 19 (CK19) Ab (F). Notch2 is abundantly synthesized in E17.5 liver (E) including ductal plates stained by anti-CK19 Ab (F). (G-I) Series of frozen P7 liver sections, incubated with (G) anti-Jagged1-, (H) anti-CK19- and (I) anti- smooth muscle actin (SMA) Abs. Jagged1 is sparsely synthesized around portal veins and the hepatic arteries (G), which are both delineated by SMA+ cells (I); CK19+ bile ducts are not in contact with Jagged1+ cells (H). (J-L) A frozen E15.5 liver section stained with anti-Jagged1 and anti-keratin Abs. Jagged1 is synthesized abundantly around portal veins (red) (J). At this stage, developing cholangiocytes consisting of keratin+ ductal plates (green) (K) are in contact with Jagged1+ cells (L). bd, bile duct; cv, central vein; dp, ductal plate; ha, hepatic artery; pv, portal vein. Bars, 50 µm.

View larger version (65K): [in a new window]   Fig. 2. NICD inhibits hepatic differentiation and induces cholangiocytic characteristics. (A-D) Albumin expression in the primary culture of Dlk+ cells. Dlk+ cells plated in gelatin-coated chamber slides were infected with pMX-GFP (A,C) and with pMX-NICD-GFP (B,D). Cells were fixed in 4% PFA, permeabilized in MeOH and stained with rabbit anti-albumin Ab. Albumin expression was visualized using rhodamine-conjugated anti-rabbit IgG Ab. Virus-infected cells showed green color by expressing GFP. Compared with the control (A), albumin expression was significantly downregulated in GFP+ cells expressing NICD 4 days after the retroviral infection (arrows in B). Furthermore, albumin was detected throughout the control culture (C), whereas it completely disappeared 7 days after infection from GFP+ cells expressing NICD (D). (E) Expression of albumin and hepatic differentiation marker genes in the primary culture. Dlk+ cells were infected with pMX-GFP, pMX-Hes1-GFP, pMX-mutantNICD and pMX-NICD-GFP, and kept for 7 days in the presence of dexamethasone (Dex) and OSM. Total RNA was extracted from each culture well and used for northern blot analysis. NICD significantly inhibited the expression of albumin, TAT and CPS in the primary culture of Dlk+ cells (NICD) compared with the control (GFP). Mutant NICD, possessing two amino acid substitutions in the fourth ankyrin repeat domain, did not affect the expression of albumin and hepatic differentiation marker genes (mutantNICD), whereas Hes1 significantly downregulated those genes (Hes1). (F) After Matrigel treatment, cholangiocyte markers such as CK19, CK7, HNF1ß and integrin ß4 were upregulated in the cells expressing NICD (NICD) compared with those in the control culture (GFP). Dlk+ cells were infected with each retroviral vector and incubated in the presence of Dex and OSM for 5 days followed by Matrigel for 5 days. The RNA expression of CK19 was detected by northern blotting, RNA expression of CK7, HNF1ß, integrin ß4 and GAPDH was determined by RT-PCR; the thermal cycle was repeated 25 times for HNF1ß and 30 times for other genes.

View larger version (75K): [in a new window]   Fig. 3. Promotion of hepatic differentiation by disrupting Notch signaling. (A) Downregulation of Notch2 gene expression by siRNA. E14.5 Dlk+ cells were transfected with siRNA specific for Notch2 mRNA using oligofectamin 18 hours after plating. The mRNA levels of Notch2 and those of TAT and CPS were examined 4 days (Notch2) and 6 days (TAT and CPS) after lipofection by northern blotting. GAPDH expression was also examined as internal control. siRNA reduced mRNA levels of Notch2 by to about 60% of that in the control culture. Hepatic differentiation markers TAT and CPS were upregulated in the presence of siRNA. (B) Inhibition of presenilin. The -secretase inhibitor L685,458 in DMSO was added to the primary culture of E14.5 Dlk+ cells. After 7 days of incubation, total RNA was extracted from each well and used for northern blot analysis to detect marker gene expression. Hepatic differentiation markers TAT and CPS were upregulated in the primary culture of Dlk+ cells. GAPDH expression was examined as a control. (C,D) Morphological examination of the cultures showed that addition of the presenilin inhibitor induced the formation of clusters, containing cells with condensed cytosol and clear round-shape nuclei (D) when compared with the addition of DMSO (C).

View larger version (38K): [in a new window]   Fig. 4. NICD controls the RNA expression of liver-enriched transcription factors. (A-D) Primary cultures of Dlk+ cells were infected with pMX-GFP (GFP), pMX-mutantNICD-GFP (mutant NICD), pMX-NICD-GFP (NICD) and pMX-Hes1-GFP (HES1), kept for 4 days and then used for cDNA synthesis. Quantitative RT-PCR was performed to analyze mRNA levels of HNF1 (A), HNF1ß (B), HNF4 (C), and HNF6 (D) 4 days after infection. Mutant NICD had no effect on the expression of these transcription factors. NICD downregulated HNF1 and HNF4 expression, whereas HNF1ß expression was upregulated and the expression of HNF6 was not significantly affected. Hes1, slightly upregulated the expression of HNF1 and downregulated the one of HNF4. All signals were normalized to the GAPDH levels and to the relative amounts of mRNA (mRNA level in the control culture=1). Analysis was repeated using three sets of samples prepared from three independent cultures. The mRNA levels plotted in the graphs are the average of the three measurements. (E) HNF4 protein expression was examined by immunostaining. Dlk+ cells plated in gelatin-coated chamber slides were infected with pMX-GFP (1) or pMX-NICD-GFP (2). After 4 days of incubation, cells were fixed in 4% PFA, permeabilized in MeOH and stained with rabbit anti-mouse HNF4 Ab. Expression of HNF4 was visualized with rhodamine-conjugated anti-rabbit IgG Ab (red). HNF4 expression was downregulated in GFP+ cells expressing NICD (green in 1) when compared with the control (2). (F) Expression of C/EBP mRNA. Primary cultures of E14.5 Dlk+ cells were infected with viral vectors encoding GFP, Hes1, mutant NICD or NICD. The RNA expression of C/EBP was examined 4 days after infection by northern blot (upper panel). GAPDH expression was also examined as internal control (middle panel). The lower graph shows the level of C/EBP normalized to the GAPDH level, indicating NICD reduced the C/EBP mRNA level to about 60% of that in the control culture (GFP), whereas mutant NICD and Hes1 had no effect. The signals of C/EBP and GAPDH were quantified using the NIH image program.