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. 2015 Jun 9;4:e07405. doi: 10.7554/eLife.07405

Figure 4. Vertebrate slit-diaphragm components are expressed in planarian flame cells and are required for the maintenance of their filtration diaphragm.

(A) Whole-mount expression patterns of indicated marker genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm. (B) Fluorescent overlay of indicated gene (red) with flame cell marker EGFR-5 and AcTub staining. Images are maximum projections of confocal Z-sections. Scale bars: 50 μm. (C) Live images show edema in intact NPHS1-6(RNAi) and NEPH-3(RNAi) animals. Scale bars: 500 μm. (D) TEM images show cross-section through a flame cell in intact Control(RNAi), NPHS1-6(RNAi) and NEPH-3(RNAi) animals. Inset shows a high magnification of the filtration diaphragm. Scale bar: 1 μm. (E, F) Ultrafiltration assay assesses the ultrafiltration capacity in NPHS1-6(RNAi) animals. (E) Representative images show dextran uptake in the animals that co-injected with 10 kDa and 500 kDa fluorescently labeled dextran. Scale bar: 50 μm. (F) Quantification of small and large dextran uptake.

DOI: http://dx.doi.org/10.7554/eLife.07405.027

Figure 4.

Figure 4—figure supplement 1. Slit-diaphragm components in the planarian S. mediterranea.

Figure 4—figure supplement 1.

(A) Cartoon showing the glomerular filtration barrier. Top: A schematic view of the podocyte. The podocyte wraps around the capillary wall on the outer surface of the glomerular basement membrane with its extended inter-digitating foot processes. Podocyte foot processes are then bridged by a slit diaphragm. Middle: A close-up view of the glomerular filtration barrier consisting of three components: porous endothelium, glomerular basement membrane, and podocyte foot processes with the interposed slit diaphragm. The endothelial pores are not bridged by a diaphragm. Bottom: Schematic drawing of the molecular equipment of slit diaphragm. NPHS1 undergoes homophilic interaction on neighboring podocyte foot processes. The intercellular junction also contains the adhesion molecule NEPH-1. (B) Homology analysis of the planarian homologs of NPHS1 and NEPH. Domains predicted by SMART for planarian and human proteins. Best reciprocal BLAST hits in human, C. elegans, and fly refseq protein database. (C) Whole-mount expression patterns of NPHS1 and (D) NEPH by in situ hybridization. Scale bars: 500 μm.
DOI: http://dx.doi.org/10.7554/eLife.07405.028
Figure 4—figure supplement 2. NPHS1-6 is not required for flame cell viability during normal homeostasis, as well as regeneration.

Figure 4—figure supplement 2.

(A, B) Fluorescent overlay of flame cell markers (CXCRL and EGFR-5) with AcTub staining in intact (A) and regenerating (B) Control(RNAi) and NPHS1-6(RNAi). Images are maximum projections of confocal Z-sections. Scale bars: 50 μm.
DOI: http://dx.doi.org/10.7554/eLife.07405.029
Figure 4—figure supplement 3. NPHS1-6 is required for de novo formation of filtration diaphragm during regeneneration.

Figure 4—figure supplement 3.

TEM images showing cross-section through a flame cell in regenerating Control(RNAi) and NPHS1-6(RNAi) animals. Inset shows high magnification of filtration diaphragm. Scale bar: 1 μm.
DOI: http://dx.doi.org/10.7554/eLife.07405.030