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First published online 28 October 2008
doi: 10.1242/jcs.035493

Journal of Cell Science 121, 3815-3823 (2008)
Published by The Company of Biologists 2008
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The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation

Danqiong Sun*, Hui Li* and Anna Zolkiewska{ddagger}

Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA


Figure 1
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  		Fig. 1. Notch activity in myogenic cells during differentiation in vitro. Primary mouse myoblasts or C2C12 cells were incubated in growth medium until they were 90-100% confluent (GM, Day 0) and then they were transferred to differentiation medium (DM, Day 1 to Day 3). (A) The levels of active Notch-1 (NICD), Pax7, MyoD, myogenin, p21 and tubulin were determined by western blotting. NICD was detected using epitope-specific antibody against {gamma}-secretase-cleaved Notch-1. (B) The amount of NICD in A was quantified by gel densitometry and normalized to the amount of tubulin (black bars); the amount of NICD at Day 0 is set as 1. Percentage of BrdU-positive cells was determined after 3-hour-pulse BrdU labeling (gray bars). Experiments in A and B were repeated three times with similar results; representative experiments for primary myoblasts and C2C12 are shown. (C) C2C12 cells incubated for 3 days in differentiation medium were subjected to partial trypsinization to separate myotubes (Mt, integrin {alpha}7A-positive) from reserve cells (RC, Pax7-positive). The amount of NICD in Mt and RC fractions and in total cell lysate was determined by western blotting.

 

Figure 2
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  		Fig. 2. Notch activity is essential for the maintenance of Pax7-positive cells during myogenic differentiation in vitro. (A) Primary myoblasts, incubated in growth medium until 90-100% confluent, were transferred to differentiation medium and were incubated for 1 day in the presence of DMSO or 5 µM GM6001, a metalloproteinase inhibitor (left), or in the presence of DMSO or 1 µM DAPT, a {gamma}-secretase inhibitor (right). The levels of NICD, Pax7, Pax3, MyoD, myogenin and p21 were determined by western blotting, tubulin is a gel-loading control. A representative experiment out of three is shown. (B) Primary myoblasts incubated for 1 day in differentiation medium in the presence of DMSO, 5 µM GM6001 or 1 µM DAPT were stained with mouse anti-Pax7 and goat anti-desmin antibodies, and then with Rhodamine-Red-X-conjugated anti-mouse IgG and AMCA-conjugated anti-goat IgG antibodies. (C) Confluent C2C12 cells were incubated for 1 day in differentiation medium in the presence of DMSO or 5 µM DAPT and the levels of NICD, Pax7, Pax3, MyoD, myogenin, p21 and tubulin were analyzed by western blotting.

 

Figure 3
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  		Fig. 3. Notch stimulation expands Pax7-positive cells during myogenic differentiation in vitro. (A) Primary myoblasts were infected with retroviruses containing constitutively active mouse Notch-1 (caNotch-AP) or with control retroviruses (c-AP). One day after infection, cells were transferred to differentiation medium and, 1 day later, cells were fixed, co-stained with mouse anti-Pax7 and rabbit anti-cleaved Notch-1 (caNotch-AP-infected cells) or anti-Flag antibodies (c-AP-infected cells) and analyzed by immunofluorescence microscopy. The relative number of Pax7-positive cells among NICD-negative and NICD-positive cells (or Flag-negative and Flag-positive cells) on the same slide was calculated (mean ± s.e.m.; n=3; at least 200 Pax7-positive cells were counted in each determination). (B) Primary myoblasts or C2C12 cells (~70% confluent) were co-cultured for 1 day with CHO cells stably transfected with mouse DLL1 (CHO-DLL1) or with empty vector (CHO-V). The levels of NICD, Pax7, MyoD, myogenin, p21 and tubulin were analyzed by western blotting.

 

Figure 4
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  		Fig. 4. Proteolytic processing of DLL1 in reserve cells. (A) Schematic diagram of the sequential cleavage of DLL1. The full-length DLL1 (DLL1FL, 90 kDa) is cleaved by an ADAM. The transmembrane and intracellular domain fragment (DLL1TMIC, 29 kDa) is then cleaved by {gamma}-secretase and the intracellular domain (DLL1IC, 26 kDa) is released. The {gamma}-secretase-mediated cleavage is inhibited by DAPT, the antibody recognition site is located in the intracellular domain of DLL1. (B) C2C12 cells incubated in differentiation medium for 3 days were separated into myotubes and reserve cells, as in Fig. 1, lyzed, and immunoblotted with anti-DLL1 antibody (left panel). Arrows indicate DLL1FL, DLL1TMIC and DLL1IC, respectively; asterisk indicates a nonspecific band (left panels). The position of DLL1FL corresponds to the position of the radioactive 90 kDa DLL1FL band detected in the immunoprecipitate from [35S]-labeled C2C12 cells (top right panel). DLL1TMIC and DLL1IC contain ~10 times less cysteine and methionine residues than the full-length DLL1 and give weak signals in autoradiograms. The identities of DLL1TMIC and DLL1IC are confirmed by the relative increase in the abundance of DLL1TMIC and decrease in the abundance of DLL1IC after treatment of C2C12 cells with DAPT (bottom right panel).

 

Figure 5
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  		Fig. 5. Soluble, catalytically inactive extracellular domain of ADAM12 inhibits DLL1 processing and stimulates Notch signaling in myoblasts. (A) Coomassie-blue-stained SDS-PAGE gel showing the recombinant, soluble, extracellular domain of mouse ADAM12 containing the E349Q mutation in the catalytic site (protein X), expressed in Drosophila S2 cells and purified from culture medium. The molecular size markers (kDa) are shown on the right. (B) Inhibition of DLL1 cleavage by protein X. COS-7 cells transfected to express DLL1 were incubated for 24 hours in the absence or presence of 2 µM protein X (left panel). Cell extracts from DLL1-transfected and empty vector (V)-transfected cells were analyzed by western blotting using antibody against the cytoplasmic domain of DLL1 (right panel). DLL1FL and DLL1TMIC are indicated with the arrows, positions of the molecular size markers are on the right. The extent of DLL1 cleavage was calculated as the ratio of band intensities of DLL1TMIC and DLL1FL (mean ± s.e.m., n=3). (C) The effect of protein X on Notch signaling. (a) Primary myoblasts were incubated for 1 day in differentiation medium without protein X or with 2 µM protein X, in the absence or presence of 1 µM DAPT (D). The level of active Notch, NICD, was analyzed by western blotting using an epitope-specific antibody, tubulin is a gel-loading control. The amount of NICD was quantified by densitometry and normalized to the amount of tubulin (mean ± s.e.m., n=3). (b) Primary myoblasts or C2C12 cells were transfected with a CBF1-luciferase reporter and pRL-TK vector, 24 hours after transfection cells were transferred to differentiation medium (Day 0) and incubated for additional 24 hours (Day 1), in the absence (gray bars) or presence (black bars) of 2 µM protein X. The relative firefly and Renilla luciferase activities were measured using the Dual-Luciferase reporter assay. Fold of CBF1 activation over the level at Day 0, in the absence of protein X, was calculated. The data represent the means ± s.e.m. from three measurements, the experiment was repeated twice with similar results.

 

Figure 6
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  		Fig. 6. The effect of protein X on the myogenic progression and Pax7 expression. (A) Primary myoblasts were incubated for 1 day in differentiation medium without protein X or with 2 µM protein X, in the absence or presence of 1 µM DAPT (D). Levels of Pax7, MyoD, myogenin, p21, and tubulin expression were examined by western blotting, band intensities were quantified by densitometry and are plotted on the right. Data represent mean values ± s.e.m. from three different experiments. (B) C2C12 cells were incubated for 1 day in differentiation medium without or with 2 µM protein X, and the levels of Pax7, MyoD, myogenin, p21 and tubulin expression were examined as in A. (C) Primary myoblasts incubated for 1 day in differentiation medium without or with 2 µM protein X were co-stained with rabbit anti-MyoD and mouse anti-Pax7 antibodies and with AMCA-conjugated anti-rabbit IgG and Rhodamine-Red-X-conjugated anti-mouse IgG antibodies. Two representative images are shown. The number of Pax7+/MyoD+, Pax7+/MyoD– and Pax7–/MyoD+ cells was counted in 15 different microscopic fields, 300-400 cells were analyzed for each experimental condition (without and with protein X). The data show mean values of cells counted on three different slides, error bars represent s.e.m. (*P<0.05). The experiment was repeated twice with similar results.

 

Figure 7
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  		Fig. 7. Proposed model of modulation of Notch activity by DLL1 shedding during myogenic differentiation. The Notch pathway is active in proliferating Pax7+/MyoD+ cells derived from Pax7+ quiescent satellite cells. Upon exit from the cell cycle, Notch signaling is downregulated in Pax7+/MyoD+ cells that later become Pax7–/MyoD+, progress into differentiation, and eventually fuse to give rise to myofibers. The level of Notch activity (shown as the yellow gradient) is maintained (or upregulated) in Pax7+/MyoD– cells that replenish the pool of satellite cells. The balance between Pax7+/MyoD– and Pax7+/MyoD+ cells is maintained by DLL1 shedding by ADAM proteases in a stochastic and cell-density-dependent manner. DLL1 shedding in a pool of cells leads to ligand depletion and downregulation of Notch signaling in neighboring cells, maintenance of MyoD expression, and eventually loss of Pax7 expression. Cells in which DLL1 cleavage takes place acquire higher level of Notch activity than their neighbors, leading to downregulation of MyoD and sustained Pax7 expression.

 

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© The Company of Biologists Ltd 2008