Figure 7. Cystic phenotype in protonephridia is cilia-and fluid flow-dependent.

(A) Series of live images show gliding mobility in Control(RNAi) and NPHP8(RNAi) animals. Yellow dot line provides a spatial reference to illustrate progress of animal. Scale bar: 1 mm. (B) Quantification of translocation speed in indicated RNAi animals. Error bar, SD; *** p < 0.001 vs control. (C) Fluorescent overlay of ventral cilia (AcTub) with nucleus marker (DAPI) in indicated RNAi animals. Scale bar: 10 μm. (D–G) Live images show bloating phenotype in IFT88(RNAi), DNAHβ-1(RNAi), and LRRC50(RNAi) animals. Scale bar: 500 μm. (D′–G′) Fluorescent overlay of ventral cilia (AcTub) with nucleus marker (DAPI) in IFT88(RNAi), DNAHβ-1(RNAi), and LRRC50(RNAi) animals. Scale bar: 10 μm. (D′′–G′′) 3D rendering showing fluorescent overlay of AcTub staining with PT2 and PT3 marker (slc6a-13) and DT marker (CAVII-1) in Control(RNAi), IFT88(RNAi), DNAHβ-1(RNAi), and LRRC50(RNAi) animals. Scale bar: 50 μm. (D′′′–G′′′) Magnified view shows fluorescent overlay of POU2/3 with pan stem cell marker (smedwi-1) in the region surrounding photoreceptor. White arrowhead shows POU2/3⁺/smedwi-1⁺ cell. Scale bar: 25 μm. (H–I) Abnormal cilia beating in DNAHβ-1(RNAi), and LRRC50(RNAi) animals. (H) Left panel: live images show cilia beating along the lateral body edge of the planarian head region; Right panel: vector map generated by Spatiotemporal image correlation spectroscopy (STICS) analysis shows velocity magnitude and beating pattern of cilia. The brightness of the vector represents the velocity magnitude of the cilia: brighter vector, stronger ciliary beating or vice versa. (I) Quantification of ciliary velocity magnitude in indicated RNAi animals. * p < 0.05 vs control. (J) Cartoon represents working model of cyst formation in the planarian protonephridia. In normal tubule, protonephridial tubular cell turnover is maintained by integration of protonephridial progenitors, originated from the neoblasts, into the tubule. During this process, cilia-driven fluid flow is required for the maintenance of tubular geometry. Obstruction of fluid flow by disrupting cilia function leads to protonephridial cystogenesis that characterized by abnormal proliferation of protonephridial progenitors, tubular enlargement and disorganization.

DOI: http://dx.doi.org/10.7554/eLife.07405.043

Figure 7—figure supplement 1. Primary Ciliary Dyskinesia (PCD) genes in the planarian S. mediterranea.

(A) Quantification of translocation speed in indicated RNAi animals. Error bar, SD; *** p < 0.001 vs control. (B) Schematic drawing shows the structure of 9 + 2 motile cilia in planarians. Right panel: schematic representation of the expanded view of the ODA depicts several light, intermediate, and heavy chains. The planarian homologs of human PCD genes with the ODA defects indicated in this study are labeled in red (DNAHβ-1 and LRRC50). (B′) TEM image shows cross-section through a cilium of the protonephridial tubule. IDA, inner dynein arm; ODA, outer dynein arm; DHC, dynein heavy chain; LC, dynein light chain; IC, dynein intermediate chain; DC, docking complex. (C) Whole-mount expression patterns of DNAHβ-1 and LRRC50 by in situ hybridization. Scale bars: 500 μm.

DOI: http://dx.doi.org/10.7554/eLife.07405.044

Figure 7—figure supplement 2. Cystogenesis in planarian protonephridia under IFT88(RNAi) is correlated with increased proliferation.

(A) Quantification of average area of each slc6a13⁺ tubule (right) in Control(RNAi), IFT88(RNAi) and NPHP8(RNAi) animals to evaluate the severity of protonephridial cystogenesis. (B) Quantification of mitoses in Control(RNAi), IFT88(RNAi) and NPHP8(RNAi) animals. NPHP8(RNAi) animals served as a positive control in this set of experiment. The time points in the bar graph indicate the number of day after the last dsRNA introduction. (C) Quantification of average area of each slc6a13⁺ tubule (right) in Control(RNAi), IFT88(RNAi) and LRRC50(RNAi) animals to evaluate the severity of protonephridial cystogenesis. (D) Quantification of mitoses in Control(RNAi), IFT88(RNAi) and LRRC50(RNAi) animals. * p < 0.05; ** p < 0.01; *** p < 0.001 vs control.

DOI: http://dx.doi.org/10.7554/eLife.07405.045