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Sequential specification of neurons and glia by developmentally regulated extracellular factors

Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA

View larger version (58K): [in a new window]   Fig. 1. Extracellular signals in the cortex regulate neuronal and glial differentiation. (A,B) Dissociated E15 cortical GFP cells were cultured over rat E18 or P15 cortical slices for 5-10 days in vitro, and then processed for immunofluorescence to identify differentiated neurons and glia. Examples of E15 GFP cells growing over an E18 cortical slice after 5 days in vitro (A) or over P15 slice after 10 days in vitro (B). Cells growing over embryonic slices differentiated into MAP-2+ neurons with characteristic neuronal morphology (A), whereas cells growing on P15 slices differentiated into GFAP+ astrocytes with characteristic glial morphology (B). The white arrowhead in A identifies a neuron immunopositive for GFP (green channel) and the neuronal-specific dendritic marker MAP-2 (red channel; yellow on the merged image) while the yellow arrowhead in B identifies a glial cell immunopositive for GFP and the astrocyte specific marker GFAP. (C-F) Quantification of the number of GFP+ neurons (filled bars) and GFP+ glia (open bars) in slice overlay cultures from different developmental ages expressed as a percentage of the total number of GFP+ cells on top of the slice. The undefined population (hatched bars) were MAP-2- cells that failed to extend processes. Bar heights represent mean ± s.e.m.

View larger version (48K): [in a new window]   Fig. 2. A developmentally regulated signal can direct the fate of dividing embryonic cortical progenitor cells. Dissociated mouse E15 GFP cortical cells were cultured over rat E18, P0, or P15 cortical slices for 5 days in vitro in the presence of 10 µM BrdU to identify progenitors that were proliferating at the time of plating. (A-C) Examples of E15 GFP cells on top of E18 slices (A), P0 slices (B), and P15 slices (C) that were processed for double immunofluorescence with antibodies directed against GFP (green channel) and BrdU (red channel). E15 GFP+/BrdU+ neurons are indicated by the white arrowheads (A and B) and GFP+/BrdU+ glia by the yellow arrowhead (C). (D-F) Quantification of the differentiated fate of dividing cortical progenitor cells plated on top of E18, P0, and P15 cortical slices. Neurons, filled bars; glia, open bars. Bar heights represent mean ± s.e.m.

View larger version (61K): [in a new window]   Fig. 3. Postnatal cortical progenitor cells are restricted to a glial cell fate. Mouse P5 GFP cortical cells were isolated from the subventricular zone, dissociated, cultured over rat E18 or P15 cortical slices for 5 days in vitro, and then processed for double immunofluorescence using anti-GFP (green channel) and anti-MAP-2 (A,B) or anti-BrdU antibodies (C,D; red channel). (A,B) Examples of GFP+/MAP-2- glia growing on top of an E18 slice (A) and a P15 slice (B). (C,D) Examples of GFP+/BrdU+ glia growing on top of an E18 slice (C) and a P15 slice (D). (E,F) Quantification of GFP cells that differentiated into neurons (filled bars) or glia (open bars) when cultured over E18 or P15 slices. Bar heights represent mean ± s.e.m. Note that although P5 GFP cells differentiate into glia in the presence of E18 or P15 slices, the cells acquire different morphologies in the two cases, suggesting that developmentally regulated signals may also play a role in specifying glial morphology. The glia that differentiate on top of E18 slices are always multipolar, while those that differentiate on top of P15 slices have a flattened morphology typical of type 2 astrocytes.

View larger version (45K): [in a new window]   Fig. 4. A diffusible factor present in postnatal cortical slices can induce glial fates. (A) Diagrammatic representation of the assay used to evaluate the influence of cortex-derived diffusible factors on cell fate specification. Dissociated E15 cortical GFP cells were diluted 1:1000 with wild-type E15 cortical cells and plated on glass coverslips underneath membrane inserts containing either rat E18 or P15 cortical slices. After 5 DIV individual clones of GFP+ cells were classified as neuronal (B) or glial (C) by immunofluorescence with anti-GFP (green channel) and anti-MAP-2 (red channel) antibodies. (D) Quantification of the percentage of GFP+ clones that contained only neurons (filled bars), glia (open bars), and both neurons and glia (mixed clones; hatched bars) under indicated experimental conditions. Asterisks indicate statistically significant differences (P<0.05) between the experimental condition and control (medium) for the indicated clonal type. (E) Quantification of the average number of total GFP+ clones, the average number of neuronal, glial and mixed clones, and the average cell number in either neuronal, glial, or mixed clones under indicated experimental conditions (± s.e.m.). (F) Time-lapse images of the development of a GFP-positive clone between 4 and 7 DIV.

View larger version (65K): [in a new window]   Fig. 5. FGF-2 induces a cell fate switch in clonal cultures and slice overlay assays. (A) Dissociated E15 cortical GFP cells were diluted 1:1000 with wild-type E15 cortical cells and plated on glass coverslips in medium supplemented with 10 ng/ml CNTF, 10 ng/ml PDGF, 10 ng/ml EGF, or 10 ng/ml FGF2. The percentage of clones that were composed only of neurons (filled bars), only of glia (open bars), and both neurons and glia (mixed clones; hatched bars) are shown as a percentage of the total number of GFP+ clones. Asterisks indicate statistically significant differences (P<0.05) between the experimental condition and control (medium) for the indicated clonal type. (B) Quantification of the number of neurons (filled bars) and glia (open bars) over E18 slices expressed as a percentage of the total number of GFP+ cells on top of the slice under indicated experimental conditions. Bar heights represent mean ± s.e.m. (C,D) Dissociated E15 GFP cells were cultured over rat E18 cortical slices for 5 DIV in the absence (C) or presence (D) of 50 ng/ml of recombinant FGF2, and then processed for double immunofluorescence with anti-GFP (green channel) and anti-MAP-2 (red channel) antibodies. The GFP cells differentiate into neurons (C: white arrowhead) under normal conditions, and into glia (D: yellow arrowhead) in the presence of FGF2.

View larger version (57K): [in a new window]   Fig. 6. A signal in postnatal cortical slices and CNTF induce terminal astrocytic differentiation. (A,B) Dissociated E15 GFP cells were cultured over rat E18 or P15 cortical slices for 10 DIV. Examples of E15 GFP+/GFAP+ cells on top of E18 slices (A), and P15 slices (B) that were processed for double immunofluorescence with antibodies directed against GFP (green channel) and GFAP (red channel). The yellow arrowheads show GFP+/GFAP+ astrocytes. (C) Quantification of the differentiated fate of GFP+/MAP-2- cells cultured for 10 days over E18 and P15 cortical slices. Asterisks indicate statistically significant differences (P<0.05) in the percentage of GFP+/GFAP+ cells over P15 slices compared to E18 slices. (D-F) Dissociated E15 GFP cells were cultured over rat E18 cortical slices for 10 DIV in the absence (D) or presence (E) of 50 ng/ml of recombinant CNTF, and then processed for double immunofluorescence with anti-GFP (green channel) and anti-GFAP (red channel) antibodies. (F) Quantification of the differentiated fate of GFP+/MAP-2- cells cultured for 10 days over E18 and P15 cortical slices in the presence of indicated growth factors, expressed as a percentage of the total number of GFP+ cells on top of the slice. Asterisks indicate statistically significant differences (P<0.05) in the percentage of GFP+/GFAP+ cells over E18 slices in the presence of CNTF compared to control (medium) conditions.

View larger version (25K): [in a new window]   Fig. 7. Model of possible regulation of cell fate decisions and glial differentiation by extracellular factors in the developing cerebral cortex.