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First published online December 9, 2005
doi: 10.1242/10.1242/jcs.02708

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BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells

¹ Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
² Brain Research Institute, University of Zurich, 8057 Zurich, Switzerland
³ Department of Biology, Swiss Federal Institute of Technology, 8057 Zurich, Switzerland
⁴ Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington DC, 20010, USA

View larger version (64K): [in a new window]   Fig. 1. Expression of p75NGFR, SMA and GFAP in the E14.5 rat forebrain and cranial mesenchyme. Fluorescent antibody staining of E14.5 rat embryo at the telencephalic level. Illustration at upper left orients panels A-G. Red rectangle with asterisk illustrates the cortical region dissected for all experiments. (A) p75NGFR (green) and smooth muscle -actin (red) show little or no expression in brain but high levels of p75NGFR in cranial mesenchyme and peripheral ganglia. Arrow indicates choroid plexus. (B-D) Higher magnification, showing (B) cranial mesenchyme, (C) choroid plexus (arrow) and confocal image of choroid plexus epithelium (CPe) and (D-F) mesenchyme. Notice that some CPm cells are p75NGFR+/SMA+, whereas the CPe shows fainter staining for these markers. (G) CPe shows moderate co-expression of p75NGFR (green) and GFAP (red), whereas the CPm is strongly p75NGFR+/GFAP+. Cranial mesenchyme is stongly p75NGFR+ but only weakly GFAP+. DAPI (blue) identifies all cell nuclei. Bars, 160 µm (A); 40 µm (B-C, E); 15 µm (D).

View larger version (94K): [in a new window]   Fig. 2. FGF2 and BMP2 induce CNS stem cells to neural-crest-like precursor state. (A) RT-PCR analysis of Msx1 expression in passage 1 of medium-density-plated stem cells treated with FGF2 or FGF2 and BMP2 after 3 days. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. (B) RT-PCR analysis of Snail1 and Snail2 expression under same conditions over 7 days. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. (C) Phase images of FGF2- or FGF2 and BMP2-treated E14.5 rat cortical stem cell cultures at medium density. Notice the initial extension of reticulated processes and cell flattening during BMP2 treatment. (D-G) Comparison of p75NGFR expression in passage 1 cells plated at clonal density; clones marked after 4 days FGF2 expansion (7.7±1.7 cells/clone) were further expanded ±20 ng/ml BMP2 treatment. By 5 days, p75NGFR expression is absent in (D) FGF2-expanded cells but prevalent in (E) FGF2 plus BMP2-treated cells. Percent clones containing any (F) p75NGFR+ cells and total percentage of (G) p75NGFR+ cells during FGF2 expansion without () or with () BMP2. Graphs show the mean ± s.e.m. (n=3). Bars, 80 µm (C); 40 µm (D,E).

View larger version (88K): [in a new window]   Fig. 3. FGF2 is required for BMP2-mediated EMT. Analysis of p75NGFR expression in E14.5 rat cortical explants cultured in basal medium (DMEM-F12 without N2) alone or supplemented with growth factors (see Table 1). Treatments include a 4-day first phase and a 5-day second phase. Low-magnification images show p75NGFR cytoplasmic staining (green) with DAPI+ nuclei (blue); original explants are shown on the right edge of each image. Only media containing FGF2+BMP2 (I-L, P) were able to induce neural crest precursors as measured by p75NGFR expression and migration from explant. Neither insulin nor IGF1 was required (I-K) and IGF was not sufficient (B,D,G) to induce p75NGFR+ precursors. (M,N) Substitution of EGF for FGF2 was not sufficient for neural crest induction. (O,P) A small number of p75NGFR+ cells with thin processes in three of 42 IGF1/IGF1+BMP2 treated explants (O) that differ from the flattened morphologies of FGF2+BMP2/FGF2+BMP2 treated explants (P). (Q) RT-PCR panel showing Snail2 induction only in explants cultured with both FGF2+BMP2. Abbreviations: F, FGF2; I, IGF1; E, EGF; B, BMP2. Factor concentrations listed in Materials and Methods. Bars, 80 µm (A-M); 80 µM (N-P).

View larger version (39K): [in a new window]   Fig. 4. BMP2-mediated activation of a Wnt signal and Bmp2 expression. E14.5 rat cortical stem cells were plated at 5092 cells/cm2 and exposed to 20 ng/ml BMP2 for 0, 24 or 48 hours. (A) Activated ß-catenin levels increased after 24 and 48 hours, as shown by western blotting. Activated ß-catenin control-cell lysate and -tubulin were included as references. (B-E) Immunocytochemistry, showing increased ß-catenin activation after 24 hours of BMP2 treatment; control cells show only few faintly positive cells; (B,D) DAPI staining indicates total cell nuclei. (F) RT-PCR time course analysis of medium-density cultures after exposure to FGF2 or FGF2 and BMP2. One day of BMP2 exposure was sufficient to upregulate transcription of endogenous Bmp2 mRNA. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. Bars, 20 µm.

View larger version (91K): [in a new window]   Fig. 5. BMP-treated CNS stem cells efficiently differentiate into smooth muscle and co-express Sox9, SMMHC1+2 and calponin. (A) Differentiation paradigm 1 (for panels B-F,M) and paradigm 2 (panels G-L,N-V). Cortical stem cells were seeded at low density after first passage. (B-F) Cells differentiated by (B) FGF2-withdrawal or (C) 8 ng/ml TGFß1 co-treatment are all SMA–. By contrast, 20 ng/ml of BMP2, BMP 4 or BMP 7 efficiently generate SMA+ cells. (M) Quantitation of B-F. Notice that the passage 5 experiment varies from paradigm 1 by using a 7-day co-treatment and 7-day withdrawal. (G-L) Dose-response assay showing no or few SMA+ cells in (G) FGF2-expanded or (H,I) low-dose BMP2 co-treated cells. (J) Percentage of SMA+ cells increases at 10 ng/ml BMP2 but yields immature-looking cells, and plateaus to almost 100% at 20 ng/ml BMP2, even during continued mitogenic expansion (K,L, quantitation in N). (O) Initial transient exposure to 20 ng/ml BMP2 during FGF2 expansion is sufficient to induce SMA+ differentiation by 8 days. (P-V) Cells co-treated with 10 ng/ml FGF2 (F) and 20 ng/ml BMP2 (B) nearly all co-express SMA and Sox9 (Q-R), SMMHC (S-T) and calponin (U-V). Graphs show mean ± s.e.m. (n=3). Bars 40 µm (B-L); 20 µm (Q-V).

View larger version (72K): [in a new window]   Fig. 6. BMP-treated CNS stem cells differentiate to non-myelinating, non-CNS glia. (A-F) Generation of glia cells using paradigm 1 after high-density plating. (A-C) Control cultures generate immature GFAP+ (green) astrocytes (A), whereas BMP2 co-treatment yields distinctively flattened cells, most co-expressing GFAP (green) and p75NGFR (red), consistent with a non-myelinating Schwann cell phenotype (B, quantitation in C). (D-F) Control cultures generate only small numbers of GalC+ CNS oligodendrocytes and no GalC+/p75NGFR+ cells (D), wheras BMP2 co-treatment yields morphologically distinct GalC+ (red) and 75NGFR+ (green) co-expressing cells (D, quantitation in F). (G-I) Peripherin+ neurons could be generated in acute cultures only by adding retinoic acid during a 3-day FGF2/BMP2 co-treatment, followed by BDNF, GDNF, NGF and HRG during a 7-day mitogen withdrawal. Peripherin+ cells had long and sometimes branched processes (G, quantitation in I); 92% of peripherin+ cells co-expressed Brn3a (H), consistent with a peripheral neuron identity. Graphs show mean ± s.e.m. (n=3-4). Bars, 20 µm (A,B); 10 µm (D,E); 80 µm (G), 10 µm (H).

View larger version (46K): [in a new window]   Fig. 7. FGF2 and BMP2 induce adult rat SVZ stem cells to dorsalized precursors. Adult SVZ stem cells were expanded in FGF2 with noggin prior to passage. (A) RT-PCR time course analysis of Snail1 and Snail2 expression in passage 1, during FGF2 ± BMP2 exposure. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) GAPDH samples as loading controls. (B-E) Comparison of p75NGFR expression after low-density plating; clones marked after 5 days expansion (116±54 cells/clone) were further expanded with FGF2 ± BMP2. Expression of p75NGFR is absent in (B) control cultures but prevalent in (C) BMP2 co-treated cells. Percentage of clones containing (D) at least one p75NGFR+ cell and total percentage of (E) p75NGFR+ cells during FGF2 expansion without () or with () BMP2. Graphs show mean ± s.e.m. (n=2). Bars 20 µm.

View larger version (66K): [in a new window]   Fig. 8. BMP-treated adult SVZ stem cells preferentially differentiate into non-CNS glia. Passaged adult SVZ stem cells were plated at low density, FGF2-expanded for 3 days and then treated with FGF2 ± BMP2 for 7 days. (A) Control cultures generate no SMA+ cells; (B) BMP2 co-treatment yields flattened SMA+ cells; (C) Percent SMA+ cells is decreased in cells from older embyos and adults. (D) Control cultures generate thin, immature GFAP+ cells; (E) BMP2 co-treatment yields flattened GFAP+ cells; (F) Percent GFAP+ cells increases as a function of animal age (F). (G-I) FGF2-expanded cultures and cultures where FGF2 was withdrawn generate infrequent GalC+ cells with immature CNS oligodendrocyte morphologies and do not contain p75NGFR+ cells (G), whereas BMP2 co-treatment yields flattened p75NGFR+ cells, some of which co-express GalC (H). The percentage of FGF2-BMP2-generated GalC+/p75NGFR+ cells increases as a function of animal age (I). All graphs show mean ± s.e.m., n=3-4. Bar, 20 µm.

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