Figure 2. Expression of if-1 and cali in neuropil cells is dependent on Hh signaling.

(A) Double FISH for if-1 (green) and cali (magenta) in wild-type animals. Cells co-expressing both markers are located in the cell body-sparse neuropil of the cephalic ganglia and ventral nerve cords. The cell body-rich cortical region is labeled by DAPI (blue). Yellow letters indicate regions detailed in E–G. (B) Double FISH for if-1 and cali in cephalic ganglia neuropil. (C) Double FISH for if-1/cali (magenta) and ptc (green) indicates co-expression of the genes. Probes for if-1 and cali were combined into a single channel (denoted if-1/cali) to improve coverage and signal intensity. 97.8 ± 2.1% of if-1⁺/cali⁺ cells in the neuropil and 100% of if-1⁺/cali⁺ cells outside the neuropil expressed ptc. (D) Double FISH for if-1/cali (magenta) and hh (green) indicates lack of co-expression. (E–G) Single if-1⁺/cali⁺ cells in the (E) cephalic ganglion neuropil, (F) ventral nerve cord, and (G) head rim. (H) Double FISH for if-1 (green) and cali (magenta) in animals following inhibition of a control gene, hh, or ptc. White dotted line delineates the edge of animal. (I) Quantification of the results from (H), with distribution of if-1⁺ only cells (green), cali⁺ only cells (magenta), and if-1⁺/cali⁺ cells (white). Within the neuropil, cells expressing one or both markers are present at 2135.6 ± 265.8 cells/mm² in control(RNAi) conditions (n = 5 animals), 169.3 ± 118.6 cells/mm² in hh(RNAi) conditions (n = 4 animals), and 3354.0 ± 249.5 cells/mm² in ptc(RNAi) conditions (n = 5 animals). Differences were significant in both hh RNAi and ptc RNAi (**p<0.001, two-tailed t test). In the head not including the neuropil region, cells expressing one or both markers are present at 64.4 ± 16.6 cells/mm² in control(RNAi) conditions (n = 5 animals), 1.5 ± 2.9 cells/mm² in hh(RNAi) conditions (n = 4 animals), and 465.4 ± 68.7 cells/mm² in ptc(RNAi) conditions (n = 5 animals). Differences were significant in both hh RNAi and ptc RNAi (**p<0.001, two-tailed t test). Anterior up, ventral side shown for A–D, H. Scale bars: 100 um for A–D, H; 10 um for E–G.

DOI: http://dx.doi.org/10.7554/eLife.16996.010

Figure 2—figure supplement 1. if-1⁺/cali⁺ cells are found in multiple regions.

(A) The neuropil of the planarian CNS revealed by DAPI and FISH for pc2. The neuropil is the cell body-sparse region surrounded by neurons and extends from the cephalic ganglia through the ventral nerve cords (left). Neuropil boundaries (yellow dashed line) in cephalic ganglia images (center) were based on the borders between DAPI-dense regions and DAPI-sparse regions. Neuropil boundaries can also be resolved in this fashion in the ventral nerve cords (right). Cells within the CNS not entirely surrounded by other cells (i.e. adjacent to the cell body-sparse area) were considered part of the neuropil. (B–C) Double FISH for if-1 (magenta) and cali (green) in animals following inhibition of a control gene or ptc. The images show detail of (B) the tail region between the ventral nerve cords and (C) the head rim region. (D) FISH for if-1/cali (green) in d6 anterior blastemas following inhibition of a control gene, hh, or ptc. Images of anterior blastemas show accumulation of if-1⁺/cali⁺ cells during regeneration. Images of the pharyngeal region show presentation of hh or ptc phenotype. Nuclei labeled with DAPI (blue). Anterior up, ventral side shown for all. Scale bars: 100 um for A and D; 50 um for B and C.

DOI: http://dx.doi.org/10.7554/eLife.16996.012

Figure 2—figure supplement 2. if-1 and cali expression following inhibition of gli transcription factors.

FISH for if-1/cali (magenta) in animals following inhibition of a control gene, gli-1, gli-2, or gli-3. Nuclei labeled with DAPI (blue). Anterior up, ventral side shown for all. Scale bars: 100 um for all.

DOI: http://dx.doi.org/10.7554/eLife.16996.013