Figure 1.
The expression of Notch1 in the commissural neurons.
Double immunostaining of Notch1 (green) with Pax7 (red, A–C), TAG1 (red, D–F) and DCC (red, G–I) was performed on the sections from HH20 chick embryos to examine the expression of endogenous Notch signaling in the commissural precursors and neurons, respectively. The dashed line on the schematic diagram (top left) indicates the level of transverse sections. The dashed box on the diagram (top right) shows the region presented in (A–I). Insets in (C), (F) and (I) show the high magnification views of the boxed areas in their respective panels. Arrows in insets indicate the double-labeled cells and arrowhead shows the nuclear location of Notch1 immunoreactivity. 4V, fourth ventricle; d, diencephalon; fp, floor plate; m, mesencephalon; r1, rhombomere 1; rp, roof plate; t, telencephalon. Scale bars: 200 µm for (A–I); 50 µm for insets in (C), (F) and (I).
Figure 2.
The expression of Notch target gene Hes5 and NICD in chick embryos after Notch transgene delivery.
The rostral hindbrains of HH10-11 chick embryos were in ovo electroporated with the expression vectors indicated. The schematic diagram (Top left) depicts the electroporation procedure. The dashed line on the whole-mount embryo (bottom left) shows the level of transverse sections presented in (A–F). (A–F) HH10-11 hindbrains were electroporated and Hes5-hybridized at HH22-23. (A) In the control HH22-23 hindbrain electroporated with empty vector alone, Hes5 is expressed normally in the ventricular zone. Mis-expression of NICD (B), VP16 (C) or Hes5 (F) upregulates Hes5 expression on the electroporated side (left hemisphere, arrows), compared to that on the contralateral side (right hemisphere). (D) Expressing R218H slightly downregulates Hes5 expression while Hes1 expression (E) does not affect Hes5 expression on the electroporated side. (G–I) Chicken hindbrains were electroporated with NICD construct at HH10-11 and double-immunostained with GFP (G, green) and NICD (H, red) at HH22-23. Inset in (I) is the high magnification of the boxed areas. Arrows in insets refer to GFP/NICD double-positive cells and arrowheads refer to GFP single-positive cells. Scale bars: 200 µm for (A–I); 25 µm for the inset in (I).
Figure 3.
Modulation of Notch signaling affects axon outgrowth.
(A–A'') A representative section from control embryos shows a large number of commissural axons crossing the floor plate midline in the rostral hindbrain. After growing circumferentially towards the ventromedial region, the axons turn and project along the longitudinal axis. (B–B'') Mis-expression of NICD results in a drastic decrease in the number of commissural axons crossing the midline. Only a very few axons are observed in the ventrolateral region of the contralateral rostral hindbrain. (C–C'') VP16 expression reduces the number of commissural axons midline crossing, and the projection patterns in the contralateral side do not appear affected. (D–D'') The expression of R218H seems also to lead to a reduction in the number of commissural axons crossing the midline while the axons that cross the midline maintain a linear, circumferential trajectory into the ventromedial region of the contralateral side. (E–E'') Hes1 mis-expression has a similar effect on axon outgrowth as that of NICD. (F–F'') Hes5 mis-expression does not affect commissural axon outgrowth. (G–G'') Hes1 knockdown by siRNA (siHes1) significantly increases the number of axons crossing midline and projecting along the circumferential number as compared with Hes1 mis-expression. (H–H'') Hes5 knockdown by siRNA (siHes5) seems not to affect axon outgrowth. (I–I'') Hes1 knockdown rescues the phenotype of axonal growth inhibition induced by NICD. (J–J'') Hes5 knockdown does not have effects on NICD-induced axonal phenotype. Arrowheads in all panels indicate axons crossing the midline, and arrows indicate axons extending in the longitudinal plane. All insets show the high magnification views of the boxed areas in their respective panels. Scale bars: 200 µm for (A–J); 50 µm for (A'–J', A''–J''). (K) The panel shows the method to normalize relative fluorescence intensity (RFI) of GFP+ axons. Red and green dash lines refer to the regions for quantitation in the electroporated (E.P.) and contralateral (C.L.) side, respectively. (L) Quantitation of GFP+ axons crossed to the contralateral hemisphere. *, p<0.05; **, p<0.01, compared to the control; #, p<0.05, compared to Hes1; Δ, p<0.05, compared to NICD. (M–O) GFP immunostaining was performed on whole-mount filets electroporated with empty vector (M), NICD (N) and 218H (O). In control filets (M), a number of GFP+ axons cross the midline and change their trajectories along the anterior-posterior (A–P) axis (small arrowheads). Forced expressing NICD (N) significantly reduces the number of GFP+ axons. Mis-expressing R218H (O) enhances GFP+ axons to grow on the electroplated side (arrows), and project alone circumferential (arrowheads), but not A–P axis. Dash lines indicate the midline in the floor plate. Large arrowheads refer to the axons across the midline.
Figure 4.
Forced Notch signaling does not affect commissural axons to cross the midline.
Double immunostaining for GFP and TAG1 was performed after electroporation of empty and NICD vectors. (A–C) In the control, GFP+ (A) and TAG1+ (B) axons cross the ventral midline normally. GFP/TAG1 double-labeled axons are observed at the floor plate (C, arrowheads in inset). (D–F) NICD mis-expression decreases the number of GFP+ axons in the midline (D) and TAG1+ axons in the electroporated side (E, arrow) compared with those in the control (arrow in B). Although GFP+ axons toward across the midline are greatly reduced in number, the remainders are still GFP/TAG1 double-positive (F, arrowheads in inset). Insets in (C) and (F) show the high magnification views of the boxed areas in their respective panels. Scale bars: 200 µm for (A, B, D, E); 100 µm for (C, F); 50 µm for the insets in (C, F). (G) Quantitation of the ventral areas spanned by TAG1+ axons in the rostral hindbrain. Compared to those on the contralateral (C.L.) side, the expression of NICD, VP16 or Hes1 decreases the area ratios of TAG1+ axons on the electroporated (E.P.) side. R218H or Hes5 expression does not affect TAG1+ axons. *, p<0.05.
Figure 5.
Modulation of Notch signaling does not affect cell fate determination of commissural neurons.
After electroporation with the respective constructs, the transverse sections were immunostained with Tuj1 (A, D, G, J, M), TAG1 (B, E, H, K, N) or Pax7 (C, F, I, L, O). Mis-expression of NICD (A–C), VP16 (D–F), R218H (G–I), Hes1 (J–L) or Hes5 (M–O) appears not to affect the expression of Tuj1, TAG1 or Pax7 on the electroporated (C.L.) side, as compared to that on the contralateral (E.P.) side. Scale bar: 100 µm.