Her9 controls the fate and behavior of boundary cells. (A) Scheme depicting the experimental design. Tg[BCP:Gal4] embryos at the one-cell stage were injected with Cre protein and the UAS:her9DN-zebrabow2.0 or the UAS:her9FL-zebrabow2.0 constructs to conditionally modulate Her9 in the boundary cells. Transgenes contain the YFP, mTurquoise, and tdTomato genes, and tdTomato-labeled clones co-express either with her9DN or her9FL in the context of the non-red, wild-type clones. For color analysis, hindbrains were imaged at 36?hpf and 48?hpf. The clones were then analyzed for color, cell fate, and cell division mode. (B,C) Boundary cell fate upon her9 loss (LOF) or gain (GOF) of function at 48?hpf. Violin plots show the percentage of neurons (B) or progenitors (C) in boundary control clones and upon her9DN or her9FL expression, respectively. (B) 43.4±33% of neurons in control clones (median=45%) versus 78.9±33.7% in her9DN clones (median=100%); **P=0.003. (C) 53.8±26.7% of progenitors in control clones (median=50%) versus 92.9±18.9% in her9FL clones (median=100%); **P=0.008. (D) Boundary cell division mode in her9 LOF at 48?hpf. Violin plots show the percentage of PP (left), PN (middle), and NN (right) divisions [PP: 57.4±27.8% in control clones (median=50%), 28.6±37.7% in her9DN clones (median=0%), **P=0.007; PN: 37±25.5% in control clones (median=45%), 21.4±32.1% in her9DN clones (median=0%), P=0.06; NN: 5.6±23.6% in control clones (median=0%), 50±47.5% in her9DN clones (median=50%), **P=0.001]. (E) Boundary cell division mode in her9 GOF at 48?hpf. Violin plots show the percentage of PP (left), PN (middle), and NN (right) divisions (PP: 45.5±30.7% in control clones (median=33.3%), 90.5±25.2% in her9FL clones (median=100%), **P=0.009; PN: 54.6±30.7% in control clones (median=66.7%), 9.5±25.2% in her9FL clones (median=0%), **P=0.009; NN: 0% in control clones (median=0%), 0% in her9FL clones (median=0%), P>0.9). Violin plots display the median in black. (F,G) Analysis of clonal cell growth upon her9 LOF and GOF at 36 and 48?hpf. Plots show the number of cells per clone in each condition (mean±s.d.): LOF: 3.7±1.2 in control versus 2.7±1.1 in her9DN clones at 36?hpf, **P=0.003; 4.3±1.3 in control versus 3.2±1.4 in her9DN clones at 48?hpf, *P=0.016. GOF: 4.1±1.4 in control versus 3.5±0.8 in her9FL clones at 36?hpf, P=0.187; 4.9±1.3 in control versus 3.1±1.1 in her9FL clones at 48?hpf, **P=0.008. 36?hpf: n=21 control clones, n=20 her9DN clones (12 embryos); 48?hpf: n=18 control clones, n=19 her9DN clones (15 embryos). 36?hpf: n=17 control clones, n=17 her9FL clones (13 embryos); 48?hpf: n=11 control clones, n=7 her9FL clones (15 embryos). Mann?Whitney test was performed in all comparisons. Images show transverse projections of control, her9DN, or her9FL boundary clones with dorsal to the top. Dotted lines demarcate the ventricular zone (VZ) and the mantle zone (MZ) as depicted in A. White circles in the images indicate cell centers. BCP, boundary cell population; hpf, hours post-fertilization; PP, progenitor-progenitor division; PN, progenitor-neuron division; NN, neuron-neuron division; ns, not significant. Scale bars: 20?µm.
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