Fig. 7

Nuclear-localized Rest rescues FBMN migration in Rest-deficient but not Pk1b-deficient embryos. (A) Schematic of Rest protein domains with the added NLS sequence PKKKRKV at the C-terminus (Rest-NLS). This construct was tagged with mCherry and cloned into both pCS2+ and Tol2 vectors. (B–D) Expression of mCherry-tagged Rest-NLS (injected mRNA) in blastula stage embryos (5 hpf), compared with expression of Rest protein expression using immunohistochemistry. Rest-NLS is correctly localized to the nucleus while endogenous Rest is expressed in the cytoplasm and nucleus (Kok et al., 2012). (E–H) Expression of mCherry-Rest-NLS specifically in FBMNs using Tol2 transgenesis (as described in Fig. 2) in Tg(islet1:GFP);MZrest^sbu29/sbu29 mutant embryos. Embryos were classed into four groups based on the number of mCherry+ FBMNs present: zero (controls, E), 1–10, 11–20, and 21–30. (F–H) Increasing the number of FBMNs expressing Rest-NLS rescues FBMN migration to r6. (E′–H′) Counts of Tg(islet1:GFP) FBMNs (green) in r4 (black), r5 (checkered) and r6 (white). (F′′–H′′) Counts of mCherry+ FBMNs (red/yellow) in each group of mCherry-expressing embryos. mCherry+ FBMNs are more likely to reside in r6, suggesting their ability to “pull along” mutant FBMNs lacking the construct to complete migration. Split channel images confirmed that mCherry+ cells were also GFP+ as expected. (I–K) Rest-NLS expression within Pk1b mutant FBMNs using Tol2 transgenesis does not rescue migration out of r4.