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First published online 5 January 2005
doi: 10.1242/dev.01592

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CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain

Genes and Development Research Group, Hotchkiss Brain Institute, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada

View larger version (82K): [in a new window]   Fig. 1. CNTF/LIF/gp130 receptor signaling promotes the self-renewal/expansion of FGF-responsive neural stem cells (NSCs) from the ventricular zone (VZ) of the ventral forebrain. E9.5 ventral neuroepithelial cells expressed the FGF receptors FGFR1 (A) and FGFR2 (B). CNTFR expression (C) was not observed; however, LIFRß (D) was expressed. At E11.5, FGFR1 (E), FGFR2 (F), CNTFR (G) and LIFRß (H) were expressed within the LGE VZ. Double-labeling of the E14.5 LGE for FGFR2 (I) and Ki67 (J), as well as LIFRß (K) and Ki67 (L), revealed receptor expression by VZ precursors, not SVZ precursors (arrows in J,L indicate examples of co-expressing cells). (M) The total number of cells generated in pass 1 FGF-2 neurosphere cultures doubled in the presence of either CNTF (F+C) (**P=0.009; n=5) or LIF (F+L) (*P=0.026; n=5). (N) The total number of neurospheres generated in pass 1 FGF-2 neurosphere cultures was not changed by the addition of either CNTF or LIF (n=3). (O,P) The number of neurospheres that were 200 µm or greater in diameter was doubled by the addition of either CNTF (**P=0.0003) or LIF (**P=0.005; results are normalized to FGF control; n=3). (Q) CNTF (*P=0.03) and LIF (*P=0.04) significantly increased the percentage of cells that expressed the undifferentiated cell marker nestin in pass 1 FGF2 neurosphere cultures (n=3). (R) The number of secondary neurospheres formed in FGF-2 alone per 2000 cells plated was significantly increased by either CNTF (*P=0.02) or LIF (*P=0.02; results are normalized to FGF control; n=3). Scale bars: in A, 50 µm for A-H; in I, 50 µm (25 µm in enlarged images) for I-L; in O, 100 µm.

View larger version (90K): [in a new window]   Fig. 2. CNTF/LIF/gp130 receptor signaling does not promote spinal cord neural stem cell (NSC) self-renewal/expansion in vitro. FGFR2 (A) was robustly expressed in the E11.5 spinal cord VZ. By contrast, the expression of CNTFR (B) was largely restricted to the mantle zone, demonstrated by ß-tubulin III staining (C). By E14.5, the FGFR2-expressing VZ precursor population (D) was reduced. CNTFR (E) continued to be expressed in the mantle zone, indicated by ß-tubulin III (F), though low levels were present in the VZ. (G) CNTF- (**P=0.003) or LIF- (**P=0.007) treated E14.5 spinal cord neurospheres generated significantly fewer secondary neurospheres compared with FGF2 alone (results are normalized to FGF2 control; n=4). (H) CNTF (**P=0.007) or LIF (**P=0.0002) treatment significantly increased the number of ß-tubulin III neurons generated by E14.5 spinal cord NSCs (n=4). (I) Neither CNTF nor LIF significantly increased the number of ß-tubulin III-expressing neurons generated by ventral forebrain NSCs. Scale bars: in A, 50 µm for A-C; in D, 100 µm for D-F.

View larger version (126K): [in a new window]   Fig. 3. Gross morphological changes in the forebrains of E12.5 Lifr-/- embryos relative to control littermates are suggestive of defects related to growth. At rostral regions of the forebrain, the LGE (arrow) in Lifr-/- embryos (B) was smaller relative to Lifr+/+/Lifr+/- (A) littermates. At more caudal regions, decreased thickness of the cerebral wall was observed (arrowhead), as well as decreased size of the LGE and MGE (arrows) in Lifr-/- (D,F) relative to Lifr+/+/Lifr+/- embryos (C,D). At the most caudal regions analyzed (G,H), a reduced caudal ganglionic eminence (arrow) was clearly observed (56% penetrant; Lifr-/-n=9; Lifr+/+/Lifr+/- n=9). Measurements of the area of the LGE revealed a 22% decrease in size in Lifr-/- embryos (I,J; paired t-test **P=0.008; n=5). A concurrent decrease in the mantle zone was also observed, as shown by ß-tubulin III staining (K,L). The number of dying cells in the LGE of Lifr-/- embryos indicated by TUNEL labeling was normalized to the area of the LGE (M; 0.5±0.1 cells/unit area) and no difference was observed relative to Lifr+/+/Lifr+/- embryos (N; 0.5±0.1 cells/unit area; n=4; arrows indicate positive cells; stars indicate examples of autoflorescence). The number of BrdU+ precursors in Lifr+/+/Lifr+/- (O; 865±41) was significantly higher than Lifr-/- embryos (P; 608±15; paired t-test **P=0.009; n=5). Scale bar: in A, 100 µm for A-H; in I, 100 µm for I,J; in K, 50 µm for K,L; in M, 50 µm for M-P.

View larger version (62K): [in a new window]   Fig. 4. A gradient of VZ precursor differentiation is defined in the LGE by the expression of GSH2, MASH1 and DLX2. At E9.5, GSH2 (A) was expressed by precursors within the LGE (P1 precursors). (B) MASH1 was expressed in a small population of VZ precursors, and all MASH1+ cells co-expressed GSH2 in the primordial LGE (P2 precursors; C). (D) A few DLX2+ cells were present at E9.5, primarily near the pial surface of the neuroepithelium. All DLX2+ cells co-expressed MASH1 at this stage (E,F; presumptive GSH2+MASH1+DLX2-expressing P3 precursors). At E12.5, GSH2 expression appeared within the VZ (G), while MASH1 expression was relatively low in the VZ, but increased towards the SVZ (H). GSH2 and MASH1 double labeling revealed a molecular gradient from GSH2 (P1 precursors) to GSH2/MASH1 (P2 and P3 precursors) and MASH1 alone (presumptive P4 precursors) expressing precursors (I). DLX2 was also expressed in a VZ to SVZ gradient (J). MASH1 expression demonstrated a similar gradient of expression (K) and double-labeling (L), suggesting that farther from the ventricular surface precursors increasingly expressed MASH1, MASH1/DLX2 (P3 and P4 precursors) and (finally) DLX2 alone (P5 precursors). CNTFR was expressed by a subpopulation of GSH2+ cells close to the ventricular surface (M; arrow indicates example of a double-positive cell). LIFRß expression was similar to CNTFR, and very few MASH1+ cells co-expressed LIFRß (N; arrow indicates a double-positive cell). FGFR2 and MASH1 double labeling also revealed only a very small population of co-expressing cells (O; arrow indicates double-labeled cell). (P) The proposed gradient of VZ precursor differentiation and a potential role for CNTF/LIF/gp130 signaling in maintaining the P1 precursor population within the gradient (see Results and Discussion for details). Scale bar: 50 µm and 100 µm in enlarged images.

View larger version (116K): [in a new window]   Fig. 5. The absence of LIFRß signaling during development results in the precocious differentiation of E12.5 VZ precursors in the lateral ganglionic eminence (LGE). The number of GSH2+ cells within the LGE VZ was significantly decreased in the Lifr-/- embryos (A; 911±23) relative to Lifr+/+/Lifr+/- littermates (B; 1324±44; paired t-test **P=0.0006; n=4). By contrast, the number of MASH1+ cells was significantly increased in the Lifr-/- embryos (C; 802±30) relative to littermate controls (D; 504±22; paired t-test **P=0.0007; n=3). Double-labeling with GSH2 and MASH1 revealed more GSH2-MASH1+ precursors in the VZ of the Lifr-/- littermates (E,F). Increased DLX2 expression was observed within the VZ of Lifr-/- embryos (G,H). Double-labeling with MASH1 and DLX2 revealed an increase in the population of MASH1+DLX2+ cells (P4 precursors) in the VZ of Lifr-/- embryos (I,J). DLX1 expression was increased within the VZ of Lifr-/- (L) compared with Lifr+/+/Lifr+/- embryos (K). GAD65 expression was restricted to the SVZ and mantle zone regions in wild-type embryos (M), but ectopically expressed within the VZ of Lifr-/- embryos (N; arrows indicate GAD65 staining). The number of surface pHH3+ cells (VZ precursors) was reduced by 40% in the LGE of the Lifr-/- embryos compared with littermate Lifr+/+/Lifr+/- embryos (O,P; paired t-test **P=0.0005; n=4). The number of non-surface pHH3+ cells (SVZ precursors) was not significantly different between the Lifr-/- and Lifr+/+/Lifr+/- littermates. For all experiments, n3. Scale bars: in A, 100 µm for A-D,G,H,K-N; in E, 50 µm for E,F,I,J; in M, 100 µm for M-P.

View larger version (76K): [in a new window]   Fig. 6. The choroid plexus is the likely source of CNTF and LIF that acts upon VZ precursors in the LGE of the developing forebrain. (A,B) Immunostaining for CNTF in the E14.5 forebrain revealed strong expression by the choroid plexus (CP; arrow in B) and at the base of the choroid plexus (large arrowhead in B). Immunoreactive cells were also observed in the mantle zone (small arrowhead in B). Similar staining was not observed in control sections labeled with goat IgG (A). (C,D) LIF immunoreactivity was observed in the choroid plexus (arrow in D), but not other regions of the E14.5 ventral forebrain. Similar staining was not observed in control sections labeled with rabbit IgG (C). (E) The expression of both CNTF and LIF in the E14.5 choroid plexus was confirmed by RT-PCR analysis (n=3). (F) These findings suggest a model in which the choroid plexus secretes CNTF and LIF, establishing a gradient of VZ to SVZ precursor cell differentiation from P1 to P5 precursors within the LGE germinal zone. GE, ganglionic eminence; T, thalamus; LGE, lateral ganglionic eminence; CTX, cortex. Scale bar: 100 µm.

View larger version (64K): [in a new window]   Fig. 7. CNTF is sufficient to promote the formation and maintenance of the VZ precursor differentiation gradient in the LGE. (A) Schematic overview of the E14.5 explant culture experiment (see Results for details). (B-D) The number of mitotically active precursors, indicated by pHH3 expression, in the LGE of CNTF-treated explants (C,D; 234±27) was significantly increased relative to basal media explants (B,D; 132±19; paired t-test **P=0.009; n=5). (E-G) GSH2 expression in normal E14.5 embryos (E) was expressed throughout the VZ. The number of GSH2+ cells was 51% higher in the VZ of the CNTF treated explants (G) relative to the basal media condition (F; paired t-test *P=0.03; n=4). (H-J) A gradient of increasing MASH1 expression from VZ to SVZ was present in normal E14.5 embryos (H). This MASH1 gradient was absent in the basal media-treated explants, in which MASH1 expression was localized in the VZ region (I; n=5). The MASH1 VZ to SVZ gradient was restored in CNTF-treated explants (J; n=5). (K-M) A gradient of increasing DLX1 expression from VZ to SVZ was observed in normal E14.5 embryos (K). The DLX1 gradient was absent in basal media-treated explants (L; n=5) and this defect was rescued in the CNTF-treated explants (M; n=5). A NOTCH1 gradient was present at E14.5 where the highest expressing cells were at the ventricular surface (N). The gradient was lost and replaced with clustered NOTCH1+ cells in basal media explants (O), but restored in CNTF-treated explants (P; n=5). MASH1 expression was normally excluded from the highest NOTCH1-expressing cells in the gradient at the ventricular surface (Q). CNTF treatment maintained high NOTCH1- and MASH1-expressing cells as largely separate populations in opposing gradients (R,S). Arrows indicate regions of robust immunoreactivity. Scale bars: in B, 100 µm for B,C; in E, 50 µm for E-M; in N, 50 µm for N-S.