V0v cells co-express evx1 and evx2. a Schematic of Evx2 showing exon boundaries (dotted lines), homeobox-domain (pink) and location of evx2 sa140 mutation (red arrow). b Examples of genotyping WT, heterozygous and homozygous evx2 sa140 mutant embryos using gel electrophoresis (see methods). The two fragments from the mutant allele restriction product run at the same position on the gel. c, e-h lateral views, dorsal up, anterior left, of spinal cord at 24 h (f-h) or 27 h (c & e). c Evx2 immunohistochemistry on WT (top panel) and homozygous evx2 sa140 mutant (bottom panel) embryos. Stars indicate Evx2-expressing cells. Mutant embryos have no Evx2 expression. d Schematic showing Shuffle-LAGAN analysis of evx1 genomic region with zebrafish sequence as baseline compared to orthologous regions in mouse and human genomes. Conserved coding sequences are indicated in blue, arrow indicates 5'-3' orientation. CNEs in 3’ region are indicated in pink. The region amplified to create transgenic lines is indicated with red dotted lines. e-h Double staining for (e) EGFP (green) and Evx2 (red) in Tg(evx1:EGFP) SU1 embryos, (f) EGFP (green) and evx1 (red) in Tg(evx1:EGFP) SU1 embryos, (g) evx1 (red) and evx2 (green) in WT embryos, (h) EGFP (green) and dbx1a (red) in Tg(evx1:EGFP) SU1 embryos (i and iii) and EGFP (green) and dbx1b (red) in Tg(evx1:EGFP) SU1 embryos (ii and iv). In e-g and h_iii - h_iv, merged and single channel views are provided. White crosses indicate cells that only express evx1. In (f) these probably represent cells that have just started to express evx1 as there is a delay in expression of EGFP. White stars in h indicate double-labelled cells. Three wider panels at bottom of h (iii and iv) are magnified single-confocal-plane views of white dotted rectangle regions in panels h_i and h_ii respectively. Thin panel on RHS in each case shows a cross-section projection (slice) created in Image J confirming that GFP expression is lateral to dbx expression. Scale bar: 50 μm (c & e-h)
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