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First published online 11 November 2008
doi: 10.1242/jcs.024802

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Myc increases self-renewal in neural progenitor cells through Miz-1

¹ Developmental Biology, Institute of Biomedicine, Haartmaninkatu 8, PO Box 63, 00014 University of Helsinki, Finland
² Institute for Molecular Biology and Tumor Research (IMT), University of Marburg, Emil-Mannkopff-Str.2, 35033 Marburg, Germany
³ HUCH Laboratory Diagnostics, Helsinki University Central Hospital, Finland

View larger version (29K): [in this window] [in a new window]   Fig. 1. Myc expression in NPCs. (A) MycER and the mutated Myc deficient in Miz-1 binding (MycV394D) were transduced to NPCs with retroviruses and the expression was analyzed by western blotting with an anti-Myc antibody. The 100 kDa transgene is not seen in the control NPCs. The endogenous 64 kDa Myc is only poorly visible in the control neurosphere lysates but increased in the Myc NPCs. The MycV394D mutant protein is of the same size as the endogenous mouse Myc. (B) 91.4% (s.d.=5.0, n=4) of the transduced NPCs express Myc as shown by flow cytometry. (C) RT-PCR shows the expression of the endogenous Myc in the mouse neurospheres (NPCs). (D) A sagittal section of an embryonic E16 mouse brain shows Myc immunoreactive cells in the lateral ventricle wall area.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Myc overexpression increases proliferation and self-renewal in NPCs. (A) Overexpression of Myc increases proliferation as shown by the proportion of the BrdU-positive NPCs in neurosphere cultures. During 24 hours of exposure to BrdU, 28.9% of the control NPCs (s.d.=5.6, n=4) were in cell cycle whereas Myc overexpression increased the rate to 76.7% (s.d.=5.3, n=5, Student's t-test **P=7.2E-06). (B) Myc increased the total cell number 70% in 1 week (from 3448, s.d.=274, n=3 to 5810, s.d.=1004, n=3, Student's t-test **P=0.00149). (C) Self-renewal assay set up and result in which Myc increases the proportion of self-renewing NPCs by fivefold from 4.2% (s.d.=2.2, n=13) in controls to 24.0% (s.d.=8.7, n=10, Student's t-test **P=0.000104) in the Myc-overexpressing neurospheres. The ability of single cells to form new neurospheres was measured by microscopy after 7 days of culture in the EGF- and FGF-containing stem cell medium. (D) Flow cytometry shows quantitatively that Myc overexpression increases the expression of the neural progenitor markers nestin (74.3%, s.d.=11.3, n=10) and Bmi-1 (93.3%, s.d.=4.1, n=11) as compared with the control NPCs (nestin 44.2%, s.d.=13.9, n=5 and Bmi-1 84.5%, s.d.=6.2, n=7, Student's t-test for nestin **P=0.0043 and for Bmi-1 **P=0.0085). (E) Even if the self-renewing proportion of the heterogenous neurosphere cell population is increased, Myc overexpression does not affect the expression of differentiation markers GFAP and TUJ-1 and thus the proportion of differentiated cells in the neurosphere (control TUJ-1 3.7%, s.d.=4.3, n=6; GFAP 15.2%, s.d.=7.9, n=6 and Myc TUJ-1 8.7%, s.d.=4.5, n=10; GFAP 15.8%, s.d.=12, n=9).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Myc overexpression does not block differentiation but delays the cell cycle exit of neural cells. (A) After 4 days in 2% FCS both Myc-overexpressing and control cells expressed the differentiation markers GFAP and TUJ-1 although the morphology of the Myc NPCs varies. (B) The expression of the NPC markers Bmi-1 and nestin decrease dramatically during differentiation of NPCs. (C) Double staining shows that GFAP-positive Myc-overexpressing cells, with the morphology of differentiated cells, are not also positive for the progenitor marker Bmi-1. (D) Myc delays the cell cycle exit and increases the number of cells in S phase during differentiation-promoting conditions (2% FCS) for 3 days as demonstrated by BrdU labelling. During the first day 10.8% of the control cells (s.d.=7.2, n=5) and 32.8% of the Myc cells (s.d.=11.7, n=5) were still proliferating. During the second day, 2.1% (s.d.=2.1, n=5) of the control cells and 6.5% (s.d.=6.6, n=5) of the Myc cells had not exited the cell cycle. By day 3 almost none of the control cells (0.0%, s.d.=0.14, n=6) but 4.4% of the Myc-overexpressing NPCs (s.d.=3.9, n=4) were still proliferating (Student's t-test *P=0.041).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Myc maintains self-renewal ability of differentiating NPCs. (A) Illustration of the experimental set-up for the re-sphering assay. (B) A light microscopy image of a `re-sphering' Myc neurosphere in 2% FCS after 4 weeks of culture. (C) During differentiation-promoting culture conditions Myc overexpression induced maintenance of self-renewal, seen as re-sphering, in 1-2% of the 76,800 plated cells (800 cells per well in 150 µl in a 96-well culture plate in each experiment) during differentiation in 2% FCS (Myc 1 week 0.0%, s.d.=0.05, n=4; Myc 2 weeks 1.5%, s.d.=1.4, n=7; Myc 3 weeks 1.3%, s.d.=1.3, n=7; Myc 4 weeks 1.5%, s.d.=1.3, n=7). Thus, re-sphering was visible after 2 weeks in culture. The number of formed neurospheres did not increase with time (when kept in 2% FCS). All the control cells differentiated and did not ever form neurospheres in differentiation-promoting conditions (contr 1–4 weeks 0%, s.d.=0, n=7, 2 weeks Student's t-test *P=0.028; 3 weeks *P=0.043; 4 weeks *P=0.020).

View larger version (38K): [in this window] [in a new window]   Fig. 5. The phenomenon of re-sphering is density dependent and the re-sphered cells resemble the primary Myc cells. (A) In 96-well plates (in 150 µl of 2% FCS per 25 mm2 well, one plate per experiment), no re-sphering was seen if less than 200 Myc NPCs were plated per well. Also, the percentage of re-sphering did not increase by adding of more cells after 400 cells per well or by time. The numbers shown represent averages of the three counted time points. (40–100 cells per well 0%, n=5; 200–300 cells per well 0.3%, s.d.=0.4, n=4; 400–600 cells per well 2.9%, s.d.=1.9, n=3; 700–900 cells per well 1.5%, s.d.=1.5, n=6; 4000 cells per well 2.2%, n=1). (B) A western blot of primary and re-sphered Myc neurosphere populations shows that the level of Myc expression was approximately the same in both populations and thus was not increased in the re-sphered population. Staining for nucleolin verifies the same result. Staining for β-actin shows that the amount of the cell lysate per lane is similar. (C) When re-sphered single Myc neurospheres after 4 weeks of differentiation in FCS were changed back to FGF and EGF and expanded into a NPC population, the proportion of self-renewing cells increased from the initial 22.5% to 29% but the difference is not statistically significant (s.d.=9,7, n=10, Student's t-test P=0,1). (D) The re-sphered Myc cells proliferated fast but the increase in cell number is not statistically significant (re-sphered Myc 8100, s.d.=638, n=3, Student's t-test P=0.0531). (E) Staining and FACS analysis show no significant change in Bmi-1- or nestin-positive cells. (Re-sphered Myc nestin 83.1%, s.d.=2.2, n=3 and Bmi-1 83.9%, s.d.=3.5, n=3.) (F) Re-sphered Myc NPCs can differentiate. Redifferentiated re-sphered Myc NPCs again attached to the culture well and expressed neuronal and astrocyte markers TUJ-1 and GFAP after 4 days culture in 2% FCS.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Myc-induced effects on self-renewal are mediated through Miz-1 binding. (A) Western blot of the control, WT Myc and mutated MycV394D neurosphere lysates shows that the level of Myc expression is approximately the same in both Myc populations. Staining for nucleolin verifies the same result. Staining for β-actin shows that the amount of the cell lysate per lane is similar. (B) Overexpression of the Myc mutant, deficient in Miz-1 binding (MycV394D), increased proliferation of NPCs to the same level as wild-type Myc, as measured by BrdU labelling (mutant 86.4%, s.d.=27.2, n=5; Myc 76.7%, s.d.=5.3, n=5; control 28.9%, s.d.=5.6, n=4; Student's t-test for mutants vs control, **P=0.008). (C) The total number of cells was counted during 7 days of stem cell culture (mutant 6510, s.d.=1250, n=3, Student's t-test for mutants vs control, *P=0.0457). (D) Self-renewal is not increased by Myc overexpression if Miz-1 binding is inhibited. Self-renewal was measured by the ability of single cells to form new neurospheres in EGF- and FGF-containing stem cell culture medium. (MycV394D 9.8%, s.d.=8.2, n=9; wt Myc 24.0%, s.d.=8.7, n=10; control 4.2%, s.d.=2.2, n=13; Student's t-test for mutants vs Myc **P=0.0021). (E) Overexpression of Myc does not induce maintenance of self-renewal if Miz-1 binding is inhibited, as measured by the ability of cells to form neurospheres in conditions (2% FCS) that promote differentiation in the re-sphering assay (mycV394D mutant 1-4 weeks 0%, s.d.=0, n=5; control 1-4 weeks 0%, s.d.=0, n=7; Myc 1 week 0.0%, s.d.=0.05, n=4; Myc 2 weeks 1.5%, s.d.=1.4, n=7; Myc 3 weeks 1.3%, s.d.=1.3, n=7; Myc 4 weeks 1%, s.d.=1.3, n=7; Student's t-test for mutants vs Myc 2 weeks Student's t-test *P=0.028; 3 weeks *P=0.043; 4 weeks *P=0.020).

View larger version (37K): [in this window] [in a new window]   Fig. 7. Myc may cause polyploidy in NPCs but it does not increase apoptosis. (A) Tetraploid Myc NPCs were found in one of the three transduced NPC progeny (Myc II) as shown by propidium iodide (PI) staining and flow cytometry. A higher rate of proliferation caused by Myc is seen as an increase in the percentages of cells in S and G2-M phases. The different amounts of debris shown can result from a number of factors: in addition to natural cell death, the mechanical dissociation of neurospheres into single cells causes debris and the amount varies somewhat between experiments. (B) Apoptosis is not changed by Myc overexpression in NPCs as analysed by Annexin staining and flow cytometry. Almost no apoptosis could be detected in NPCs (Contr 0.02%, n=5, s.d.=0.04 and Myc 0.18%, n=6, s.d.=0.35 Student's t-test P=0.31). Induced apoptosis in HU-VEC cells was used as a technical positive control for the Annexin staining.

View larger version (57K): [in this window] [in a new window]   Fig. 8. Myc and Miz-1 mRNA expressions are both localized in the same areas of the brain that are known to contain neural progenitors. A coloured `heat map' from blue (low) to red (high) represents the intensity of the expression. The in situ hybridization images were obtained from the Allen Brain Atlas [Internet], Seattle, WA: Allen Institute for Brain Science. © 2006. Available from: http://www.brain-map.org. The red line in the sketches below the images illustrates the plane of the section in the mouse brain. (A,B) Myc (C-Myc) and miz-1 mRNA expression in the hippocampus is especially concentrated to the dentate gyrus (white arrow) shown in coronal sections. (C) A sagittal section shows miz-1 mRNA expression in the lateral ventricle zone (red arrow).

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