|
Fig. 4
V2s neurons depend on Notch signalling. (A) Mean number of cells expressing sox1a and gata2a mRNA at 24 hpf (black) and 30 hpf (white) determined by FISH (Fig. S5A-B″). Cells expressing sox1a and gata2a mRNA decreased by about 48% from 24 (black, n=71) to 30 hpf (white, n=34), whereas sox1a:eGFP+ V2 cells co-expressed nkx1.2lb mRNA (Fig. S5C-D″) at similar levels at both stages. (B-E‴) Examples of co-expression of vsx1:GFP, gata2a and sox1amRNA. (B-B‴) A pair of vsx1:GFP+ cells with one cell being vsx1:GFP+;gata2a+;sox1a+ (arrows) extending axons at 24 hpf. Committed V2a cells express only vsx1:GFP (arrowheads). (C-C‴) Example of a vsx1:GFP+ cell co-expressing gata2a (+) at 24 hpf. (D-D‴) A V2s cell that is still vsx1:GFP+ and expresses sox1a mRNA (x) at 26 hpf. (E-E‴) sox1a is co-expressed with gata2a in one cell of a V2a/V2b,s pair (arrows) at 22 hpf. Arrowheads indicate vsx1:GFP+ V2a neurons. (F) Disruption of Notch signalling in sox1a:eGFP embryos from 16 to 24 hpf and assessment of neurotransmitter type at 30 hpf shows a 2.3-fold increase in sox1a:eGFP+ neurons expressing the GABAergic marker gad1b. In contrast, blocking of Notch signalling leads to a 2.3-fold decrease in sox1a:eGFP+;slc6a5+ neurons (DMSO-treated control, white; LY treated, black; for original data, see Fig. S5E-H″). (G-J) sox1a precursor cells divide largely before 24 hpf. sox1a:eGFP+ (green) embryos were treated with EdU (magenta) during two different time windows before processing with EdU click-chemistry at 30 hpf (G). sox1a:eGFP embryos treated from 16 to 24 hpf (H-H″) or from 24 to 30 hpf (I-I″) showing a five-somite spinal cord segment at 30 hpf (sox1a:eGFP+; EdU+ cells, arrows; sox1a:eGFP+; EdU− cells, +). (J) Percentages of EdU+and EdU−; sox1a:eGFP+ cells. Most cells divided before 24 hpf. (A,F) Counts of six to nine embryos from two independent experiments in the V2 domain of the spinal cord above the yolk extension over a five-somite distance. Dorsal is upwards; anterior is leftwards. Data are mean±s.e.m. (A,F) or mean±s.d. (J). Statistical significance was assessed using the unpaired two-tailed Student's t-test. **P≤0.01. Scale bars: 25 µm.