Figure 2

Six1 mediates the developmental transition of trunk sensory neurons during Xenopus development. (A-K) Earlier expression of Six1 reduces the number of Rohon-Beard (RB) cells and promotes differentiation of extramedullary sensory neurons in Xenopus. (A-E) The combinations of injected mRNAs and treatment with dexamethasone (Dex) are indicated at the top. Arrowheads: enhanced GFP (EGFP)-positive RB cells, which are marked with both Isl1/2 (magenta) and Tlx3 (green) in the spinal cord (sc) at St. 25/26 (A-D). Arrows: EGFP-, Isl1/2- and Tlx3-triple-positive cells outside sc at St. 33/34 (E). (F) Quantification of RB cells and (G) percentage of EGFP-positive RB cells in 250 μm of the thoracic level at St. 25/26. White bars: total number of RB cells, green bars: EGFP-positive cells. (H) EGFP- and cleaved CASP3 (cCasp3)-double-positive cells (arrowheads) are noted in Six1-GR + Dex. (I) In Six1-GR + Dex embryos, Isl1/2- and EGFP-double positive cells (arrow) are located outside sc. Laminin (green): sc outline, TOPRO3 (magenta): nuclei. (J) Quantification of Isl1/2- and Tlx3-double positive cells outside sc. White bars: total number of cells, green bars: EGFP-positive cells. (K) Schematic representation of the results. Activation of Six1 reduced the number of RB cells (pink) in sc and enhanced the differentiation of DRG neuron-like cells (blue) outside sc. (L-N) siRNA mediated knockdown of Six1 increased the number of RB cells. (L) Top: schematic representation of electroporation of Xenopus embryo. Bottom: merged picture of epifluorescence and bright field in obliquely dorsal view of the trunk at St. 45. Fluorescein isothiocyanate-labeled control small interfering RNA (siRNA) persists in the dorsal sc. (M) si1, si2 and si3: siRNA targeted sequences in six1; mut: three silent mutations, each corresponding to siRNA targeted regions in mutated six1. (N) Number of Isl1/2-positive RB cells in the trunk (level in somite (so) pairs 1 to 9) at St. 45/46. Data are mean ± standard error of the mean. *p <0.01; **p<0.001. Dashed lines demarcate the position of sc and notochord (nc). Scale bars: 100 μm (L) and 25 μm (A-K,M,N).