Figure 4. ndl-3, ptk7, and wntP-2 are expressed in a graded fashion in domains along the anteroposterior axis.

(A) In situ hybridizations showing body-wide graded expression of ptk7 centered in the trunk, wntP-2 expression in a gradient from the posterior and ndl-3 expression in a graded fashion from the anterior. (B) Quantitation of colorimetric in situ hybridization staining across the body axis. 4–6 planarians stained as in (A) were imaged on a dissecting microscope, the images were inverted and then analyzed for position-specific staining intensity along a lateral domain depicted in the diagram (dotted line with arrow showing directionality). To compare animals of different lengths, position was normalized to length of this domain and signal intensity was normalized such that the minimum and maximum values across each animal were 0 and 1, respectively, and average intensity at each region was determined for animals stained with each probe treatment computed followed by obtaining average intensity, with bars showing standard deviations. (C) Triple FISH showing expression of ndl-3 (red), ptk7 (blue), and wntP-2 (green) mRNA. Panels are maximum projections from a stack of seven 1-micron thick confocal images taken at 40x along the body axis at the regions represented in the cartoon, then adjusted for brightness and contrast uniformly for each channel across the image series. m, mouth. Bars, 100 microns. (D–E) Quantification of FISH signal intensity for cells identified in images shown in (C). 3-color images were segmented by merging all three channels to define a set of cells in each region with wntP-2, ndl-3 and/or ptk7 expression and this mask used to measure mean FISH signal intensity for each cell. (D) Scatter and box plots showing expression of ndl-3 highest in the anterior, expression of ptk7 highest in the trunk and tail, and highest wntP-2 expression in the posterior. (E) Plots comparing pairwise FISH signal intensity between the indicated genes across eight body axis regions (R1-R8) as in (C). Note the existence of cells expressing both ndl-3 and wntP-2 (R3-R5), ptk7 and ndl-3 (R3-R4), and wntP-2 and ptk7 (R5-R7). Bars, 100 (C) or 200 (A) microns.

DOI: http://dx.doi.org/10.7554/eLife.12850.011

Figure 4—figure supplement 1. Distribution of ptk7, wntP-2 and ndl-3 expression states across the body axis.

(A) Mean fluorescence intensity of cells from all regions in Figure 4C were combined and density histograms plotted to determine a cutoff (dotted line=55 in arbitrary units) for higher versus lower expression of ptk7, wntP-2, and ndl-3. Cells within each region shown in Figure 4C were assigned membership in one of eight classes defined by high ('hi') or low (unstated) expression of each of the three genes (1: wntP-2^hi only, 2: ptk7^hiwntP-2^hi, 3: ptk7^hi only, 4: ptk7^hiwntP-2^hindl-3^hi, 5: ptk7^hindl-3^hi, 6: wntP-2^hindl-3^hi, 7: ndl-3^hi, 8: all 3 genes low). Class membership was plotted as a fraction of all cells measured in each region (B) and as a scatterplot of all cells examined (C). By these criteria, several classes of cells expressing combinations of ptk7, wntP-2, and ndl-3 exist and are distributed in domains along the body axis. The tail tip has the highest frequency of wntP-2+ only cells, whereas anterior tail and trunk regions have a comparatively greater fraction of wntP-2+ptk7+ cells. The above analysis was repeated for a range of high/low expression cutoffs between 30 and 75, resulting in similar the same trends, with lower thresholds resulting in fewer cells assigned as ptk7^lowntP-2^londl3^loand more cells as ptk7^hiwntP-2^hindl-3^hi.

DOI: http://dx.doi.org/10.7554/eLife.12850.012