Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ
- Stowers Institute for Medical Research, United States
- University of Utah, United States
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Howard Hughes Medical Institute, Stowers Institute for Medical Research, United States
Figures
Figure 1
Protonephridia are ultrafiltration devices in planarians.
(A) Whole-mount acetylated tubulin (AcTub) staining. Scale bars: 500 μm. Inset shows depth-coded projection of AcTub staining. Superficial structures are in blue and deeper structures are in red. …
Figure 2 with 20 supplements
Unexpected complexity of protonephridial tubules is revealed by systematic gene expression mapping of slc genes along the protonephridial tubules.
(A) Cartoon shows previous segmentation model of the protonephridial tubule and expression map of slc genes along the protonephridial tubule. (B–G) Representative images show expression domains of …
Figure 2—figure supplement 1
Solute carrier gene families in the planarian Schmidtea mediterranea.
https://doi.org/10.7554/eLife.07405.005
Figure 2—figure supplement 2
Schematic representation of phylogenetic clusters of γ- (A), δ- (B) groups of slcs and the Tim barrel- (C), IT- (D), Drug/Metabolite (E) transporter clans of slcs.
Planarian homologs are colored in red.
Figure 2—figure supplement 3
Schematic representation of phylogenetic clusters of α-groups of slcs.
Planarian homologs are colored in red.
Figure 2—figure supplement 4
Schematic representation of phylogenetic clusters of β-groups of slcs.
Planarian homologs are colored in red.
Figure 2—figure supplement 5
Schematic representation of phylogenetic clusters of Smed-slc1a (A), Smed-slc5a (B), Smed-slc22a (C), Smed-slc6a (D).
Planarian homologs are colored in red.
Figure 2—figure supplement 6
Schematic representation of phylogenetic clusters of Smed-slc4a (A), Smed-slc7a (B), Smed-slc12 (C), Smed-slc15 (D), Smed-slc20 (E), Smed-slc23 (F), Smed-slc26 (G).
Planarian homologs are colored in red.
Figure 2—figure supplement 7
Schematic representation of phylogenetic clusters of Smed-slc28 (A), Smed-slc30 (B), and Smed-slc42 (C).
Planarian homologs are colored in red.
Figure 2—figure supplement 8
Expression patterns of slc genes that belong to solute carrier families 1–6 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 9
Expression patterns of slc genes that belong to solute carrier families 7–15 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 10
Expression patterns of slc genes that belong to solute carrier families 16–23 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 11
Expression patterns of slc genes that belong to solute carrier families 24–29 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 12
Expression patterns of slc genes that belong to solute carrier families 30–38 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 13
Expression patterns of slc genes that belong to solute carrier families 40–47 in an asexual strain of the planarian S. mediterranea.
Whole-mount expression patterns of slc genes by in situ hybridization (NBT/BCIP development). Scale bars: 500 μm.
Figure 2—figure supplement 14
Expression of slc genes in the PT.
Fluorescent overlay of indicated gene (in red) (A-F) with PT2 and PT3 marker (slc6a-13), DT marker (CAVII-1) and AcTub staining. Images are maximum projections of confocal Z-sections. Scale bars: 50 …
Figure 2—figure supplement 15
Expression of slc genes in the PT1 segment of the PT.
(A) Fluorescent overlay of indicated gene (in red) with PT1 and PT2 marker (CUBN1), PT2 and PT3 marker (slc6a-13) and AcTub staining. (B–D) Fluorescent overlay of indicated gene (in red) with PT2 …
Figure 2—figure supplement 16
Expression of slc genes in the PT2 and PT3 segments of the PT.
Fluorescent overlay of indicated gene (in red) with PT2 and PT3 marker (slc6a-13), DT marker (CAVII-1) and AcTub staining. Images are maximum projections of confocal Z-sections. Scale bars: 50 μm. A …
Figure 2—figure supplement 17
Expression of slc genes in PT3 segment of the PT.
Fluorescent overlay of indicated gene (in red) (A-C) with PT2 and PT3 marker (slc6a-13), DT marker (CAVII-1) and AcTub staining. Images are maximum projections of confocal Z-sections. Scale bars: 50 …
Figure 2—figure supplement 18
Expression of slc genes in the DT.
Fluorescent overlay of indicated gene (in red) (A-G) with PT marker (slc6a-13 or CUBN1), DT marker (CAVII-1) or CD marker (slc12a-1 or slc24a-9) together with AcTub staining. Images are maximum …
Figure 2—figure supplement 19
Expression of slc genes in the collecting duct.
Fluorescent overlay of indicated gene (in red) (A-P) with PT2 and PT3 marker (slc6a-13), DT marker (CAVII-1) or CD marker (slc24a-9) together with AcTub staining. Images are maximum projections of …
Figure 2—figure supplement 20
Expression of slc genes weakly expressed in both proximal and DTs.
Fluorescent overlay of indicated gene (in red) (A-B) with PT2 and PT3 marker (slc6a-13), DT marker (CAVII-1) and AcTub staining. Images are maximum projections of confocal Z-sections. Scale bars: 50 …
Figure 3
Extensive structural and functional homology between planarian protonephridia and vertebrate nephrons.
(A) Tables summarize expression domains of selected slc genes in planarian protonephridia and rodent metanephros. Cartoons showing segmental organization of planarian protonephridia and rodent …
Figure 4 with 3 supplements
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 …
Figure 4—figure supplement 1
Slit-diaphragm components in the planarian S. mediterranea.
(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 …
Figure 4—figure supplement 2
NPHS1-6 is not required for flame cell viability during normal homeostasis, as well as regeneration.
(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 …
Figure 4—figure supplement 3
NPHS1-6 is required for de novo formation of filtration diaphragm during regeneneration.
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.
Figure 5 with 3 supplements
Down-regulation of nephrocystin members leads to the formation of cyst-like structure in protonephridia.
(A) Protonephridial defects in NPHP5(RNAi), NPHP6(RNAi) and NPHP8(RNAi) animals. Top panel: live images show edema in intact RNAi animals. Scale bars: 500 μm; middle panel: monochrome showing AcTub …
Figure 5—figure supplement 1
Nephrocystins in the planarian S. mediterranea.
(A) Homology analysis of planarian nephrocystins. Domains predicted by SMART for planarian and human proteins. Best reciprocal BLAST hits in human, C. elegans, and fly refseq protein database. (B) …
Figure 5—figure supplement 2
Abnormal tubular enlargement in NPHP8(RNAi) animals.
(A) Fluorescent overlay of lumen marker with PT2 and PT3 marker slc6a-13 and nuclei (DAPI) in Control(RNAi) and NPHP8(RNAi) animals. Scale bars: 25 μm. (A) Fluorescent overlay of PT marker (slc6a-13)…
Figure 5—figure supplement 3
Ultrastructure of the PT in NPHP(RNAi) animals.
TEM images showing cross-section through a tubule of protonephridia in indicated RNAi animals. Inset in red box shows abnormally localized basal body in indicated RNAi animals. Inset in green box …
Figure 6 with 3 supplements
Cystogenesis in planarian protonephridia results from direct proliferation of protonephridia progenitors and requires the presence of stem cells.
(A) BrdU pulse-chase experiment shows the presence of diving protonephridial progenitors in the proximity of protonephridial tubule in Control(RNAi) and NPHP8(RNAi) animals. Yellow arrowhead …
Figure 6—figure supplement 1
Increase of protonephridial progenitors during cystogenesis in planarian protonephridia.
(A) Left: Whole-mount expression patterns of POU2/3 by in situ hybridization. Scale bars: 500 μm. Right: Fluorescent overlay of POU2/3 with DT marker (CAVII-1) and AcTub. Scale bar: 50 μm. (B) Left: …
Figure 6—figure supplement 2
Gut and brain progenitors in NPHP8(RNAi) animals.
(A, B) Left panel: whole-mount expression patterns of pax6A (A) and HNF4 (B) by in situ hybridization. Scale bars: 500 μm; Right panel: fluorescent overlay of (A) pax6A and (B) HNF4 with pan stem …
Figure 6—figure supplement 3
The severity of cystic phenotype in protonephridia depends on proliferation rate and requires the presence of stem cells.
(A) Quantification of mitoses in Control(RNAi) and NPHP8(RNAi) animals. Experimental paradigm is described in Figure 5F. The time point in the bar graph indicates the number of the day after the …
Figure 7 with 2 supplements
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) …
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 …
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 …
Videos
Video 1
Protonephridial collecting duct opens to the dorsal epithelia.
Serial TEM images showing the protonephridial collecting duct connected to the dorsal epithelia.
Video 2
Flame cell morphology in Control(RNAi) animal.
Serial TEM images showing flame cell in Control(RNAi) animal.
Video 3
Flame cell morphology in NPHS1-6(RNAi) animal.
Serial TEM images showing flame cell in NPHS1-6(RNAi) animal.
Video 4
3D rendering of normal protonephridial tubule in Control(RNAi) animal.
https://doi.org/10.7554/eLife.07405.037
Video 5
3D rendering of cystic-like protonephridial tubule in NPHP8(RNAi) animal.
https://doi.org/10.7554/eLife.07405.038
Video 6
Cilia beating in Control(RNAi) animal.
Serial images show beating of the cilia along the lateral body edge of the planarian head region in Control(RNAi) animal.
Video 7
Cilia beating in DNAHβ-1(RNAi) animal.
Serial images show beating of the cilia along the lateral body edge of the planarian head region in DNAHβ-1(RNAi) animal.
Video 8
Cilia beating in LRRC50(RNAi) animal.
Serial images show beating of the cilia along the lateral body edge of the planarian head region in LRRC50(RNAi) animal.
Additional files
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Supplementary file 1
Summary information of the planarian homologs of slc genes.
- https://doi.org/10.7554/eLife.07405.049
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Supplementary file 2
Expression domains of slc genes in the planarian protonephridia.
- https://doi.org/10.7554/eLife.07405.050
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Supplementary file 3
Expression domains of slc genes in the rodent metanephros.
- https://doi.org/10.7554/eLife.07405.051
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Supplementary file 4
Summary information of the planarian homologs of human kidney disease genes.
- https://doi.org/10.7554/eLife.07405.052
