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First published online 7 February 2007
doi: 10.1242/dev.02806

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Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros

¹ Nephrology Division, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA.
² Renal Division, University Hospital Freiburg, Zentrale Klinische Forschung (ZKF), Breisacher Str. 66, 79106 Freiburg, Germany.

View larger version (95K): [in this window] [in a new window]   Fig. 1. Distribution of multiciliated cells and transporting epithelia containing single cilia in the pronephros. (A) Whole-mount immunofluorescence of the trunk region of a 48 hpf embryo stained with anti-acetylated tubulin reveals bright cilia bundles (arrowheads) in the pronephric lumens, as well as single ciliated cells (arrows) in a more caudal nephron segment. (B) At higher magnification, compressed bundles of cilia (arrowheads) and single cilia (arrows) can be observed in the pronephric lumen. (C) Bundles of cilia emanating from individual cells project into a distended lumen of a mechanically obstructed pronephros. Dotted lines in B and C outline the pronephric tubules. (D-F) Electron micrographs of the pronephros show isolated multiciliated cells (MCC; false-colored in red) interspersed among transporting epithelial cells (false-colored in light blue). Arrowheads show apical basal bodies. (D) MCCs are distinguished by multiple apical basal bodies, by the lack of a brush border (bb), by a small basal cell surface and by multiple apical cilia (asterisks). n, nucleus. (E) Example of an MCC with multiple apical cilia basal bodies, and bundles of cilia in the lumen. (F) Cross section of a pronephric tubule in a 7 dpf larva, showing a single MCC with multiple basal bodies, apical mitochondria (asterisks) and bundles of cilia fitting tightly into the pronephric lumen.

View larger version (105K): [in this window] [in a new window]   Fig. 2. MCCs and transporting epithelia co-exist as separate cell types in the early distal segment of the pronephric nephron. (A) Expression of the axonemal-sheath gene shippo1 in the pronephros of a 34 hpf embryo. (B) Expression of the transcription factor rfx2 in individual pronephric cells. (C) Expression of the cation transporter trpM7 in the early distal segment of the pronephric nephron. (D) Expression of the sodium-sulfate co-transporter slc13a1 in the early distal segment. (E) Double in situ hybridization of the MCC marker shippo1 (red) and slc13a1 (purple) in distinct but adjacent cells of the early distal segment in a 34 hpf embryo. (F) Double in situ hybridization of shippo1 and trpM7 in early distal segment cells. (G) Expression of GFP from the Na,K-ATPase alpha a1A4 subunit promoter is uniform in the most caudal nephron segment (arrowhead) and heterogeneous in the distal nephron. (H,I) Double staining for GFP and acetylated tubulin (red) in confocal z-series projections (H) and in histological sections (I) shows that the Na,K-ATPase alpha a1A4 promoter is not active in MCCs (arrows, arrowhead; cell bodies are outlined with dashed line).

View larger version (77K): [in this window] [in a new window]   Fig. 3. Confocal fluorescent in situ hybridization analysis of ion transporter and ciliogenic gene expression in the pronephros. (A,B) Double staining for shippo1 mRNA (in situ hybridization; green) and acetylated tubulin (immunofluorescence; red) in confocal projections reveals several examples of cilia bundles emanating from shippo1-positive cells (arrowheads) into the lumen; single cilia emanate from shippo1-negative cells (small arrows). Dotted lines outline the pronephros; posterior is to the right. (C-E) Co-expression of shippo1 (C; green) and fleer (D; red) in single cells of the pronephric tubules. (E) Merged image. (F-H) Co-expression of shippo1 (F; green) and rfx2 (G; red) in single cells of the pronephric tubules. (H) Merged image. (I-K) Expression of shippo1 (I; green) and slc13a1 (J; red) in distinct but adjacent cells of the pronephric tubules. (K) Merged image. (L-N) Expression of shippo1 (L; green) and trpM7 (M; red) in distinct but adjacent cells of the pronephros. (N) Merged image.

View larger version (112K): [in this window] [in a new window]   Fig. 4. jagged 2 expression in multiciliated cells. (A,B) Whole-mount in situ hybridization showing expression of jagged 2 (arrowheads) at the 18-somite stage in cells of the intermediate mesoderm (A) and in individual cells of the pronephros at 42 hpf (B). (C) Cross section of the pronephros double stained for acetylated tubulin (HRP:DAB immunohistochemistry) and jagged 2 (in situ hybridization) shows that isolated jagged 2-positive cells possess apical cilia bundles (arrow). Dashed line outlines the cross section of the pronephric tubule. (D) Double in situ hybridization with the MCC marker shippo1 (red) and jagged 2 (blue) demonstrates jagged 2/shippo1 co-expression in isolated single cells. (E-G) Confocal optical section of the pronephros in situ hybridized for shippo1 mRNA (E; green) and jagged 2 (F; red) confirm jagged 2 expression in shippo1-positive MCCs. (G) Merged image. Dotted lines outline the pronephros.

View larger version (139K): [in this window] [in a new window]   Fig. 5. jagged 2 loss of function transfates pronephric cells to MCCs. Three markers of MCCs - shippo1 (A-C), flr (D-F) and rfx2 (G-I) - show expanded expression in the pronephros of jagged 2 morphants at 34 hpf. Control invert morpholino (A,D,G) had no effect on MCC-marker expression, whereas ATG blocking (J2atgMO; B,E,H) and exon 20 mis-splicing morpholinos (J2ex20MO; C,F,I) resulted in uniform expression of MCC markers. (J) MCCs exist as single cells (arrowheads; flr-expressing cells) in the wild-type pronephros in cross section. (K,L) Expressions of shippo1 (K) and flr (L) expand to include all cells in tubule cross sections in J2exon20 morphant embryos. (M-O) slc13a1 expression seen in the control (M) is lost in J2atg (N) and J2exon20 (O) morphants. Similarly, the observed expression of trpM7 in control morpholino-injected embryos (P) is lost in J2exon20 morphants (Q). Control jagged 2 morpholino does not alter the single-cell jagged 2 mRNA expression pattern (R), whereas combined injection of both jagged 2 atg and exon 20 antisense morpholinos (U) result in expanded jagged 2 mRNA expression. (S) Na,K-ATPase:GFP transgenic zebrafish express GFP in the pronephros distal segments (arrowheads). (T) GFP expression is lost specifically in the distal segment of jagged 2 exon 20 morphant embryos (arrowheads).

View larger version (117K): [in this window] [in a new window]   Fig. 6. Notch3 mediates Jagged 2 signaling in the pronephros. (A) Expression of notch3 in the pronephros (arrowheads). (B) Expression of notch3 in the proximal pronephric tubules (arrowhead). (C,D) shippo1 expression in single cells of control embryos at 34 hpf (C) is expanded by notch3 exon 27-morpholino injection (D). (E,F) Similarly, expression of flr that is observed in control embryos (E) is expanded in notch3 exon 27 morphants (F). (G,I) Transporting-cell markers slc13a1 (G) and trpM7 (I) are greatly reduced in notch3 exon 27 morphants (H and J, respectively).

View larger version (123K): [in this window] [in a new window]   Fig. 7. Transfating of pronephric cells in the mind bomb mutant. (A,B) shippo1 expression in control wild-type embryos is restricted to single cells (A), whereas, in mind bomb homozygotes (B), pronephric distal segment cells (ds) are uniformly positive and there is an increase in shippo1-positive cells in the proximal segment (ps). (C,D) Wild-type jagged 2 expression (C) is also expanded in mind bomb homozygotes (D). (E,G) trpM7 (E) and slc13a1 (G) expression is almost completely lost in mind bomb homozygotes (F and H, respectively).

View larger version (91K): [in this window] [in a new window]   Fig. 8. Inhibition of gamma-secretase and the timing of Notch signaling in pronephric development. All whole-mount in situ hybridization was performed at 34 hpf. (A-C) The wild-type pattern of shippo1 expression (A) is dramatically expanded when gamma-secretase is inhibited with 100 µM DAPT starting at 9.5 hpf (B) but not when DAPT is added later, at 24 hpf (C). (D-F) Conversely, the number of shippo1-positive cells seen in control embryos (D) is dramatically reduced in the pronephros of embryos induced to ectopically express the Notch1a intracellular domain under the control of HS-GAL4 induction (nicd hs) at either 8 hpf (E) or 9 hpf (F). (G-I) trpM7 expression is lost when DAPT is added at 9.5 hpf (H), but not when it is added later, at 24 hpf (I). (J-L) slc13a1 expression is lost when DAPT is added at 9.5 hpf (K), and also when DAPT is added later, at 24 hpf (L).

View larger version (18K): [in this window] [in a new window]   Fig. 9. Notch signaling acts in a short time-window early in pronephric development. shippo1-positive multiciliated cells were counted at 34 hpf (in situ hybridization) in individual tubules. (A) Quantification of MCCs per pronephric tubule in experiments adding DAPT at progressively later times. (B) Quantification of MCCs per pronephric tubule in embryos induced to express Notch1a ICD by heat shock at 8 hpf and 9 hpf. See text for details.

View larger version (96K): [in this window] [in a new window]   Fig. 10. Enhanced ciliogenesis and double bubble-mutant rescue by inhibition of Jagged/Notch signaling. (A) Wild-type double bubble (dbb) heterozygotes viewed in confocal projections show alternating thick acetylated tubulin-positive bundles of cilia (arrowheads) in pronephric lumens (individual tubules are marked with asterisks in A-D). (B) jagged 2 exon 20-morpholino injection results in enhanced ciliogenesis, seen here in a single pronephric tubule with a slightly expanded lumen. (C) A single dbb-mutant pronephric lumen showing loss or shortening of cilia bundles (arrowheads), and a grossly dilated lumen (*). (D) dbb-mutant homozygotes injected with jagged 2 exon 20 morpholino recover normal tubule morphology (paired tubules marked with asterisks) and show a dramatic recovery of ciliogenesis. (E) dbb-mutant homozygote at 2.5 dpf showing bilateral cyst formation in the proximal pronephros (arrow). (F) dbb-mutant homozygote injected with jagged 2 exon 20 morpholino at the one-cell stage, showing the absence of cyst formation (arrow; mutant rescue). (G) Histological section of dbb-homozygote pronephros showing a cystic pronephric tubule (*), dilated pronephric tubules (arrow) and edema (#). (H) Histological section of a dbb homozygote injected with jagged 2 exon 20 morpholino at the one-cell stage, showing complete absence of cystic pathology and edema (arrow, wild-type-appearing pronephric tubules; *, normal glomerular structure). (I) Control DMSO-treated dbb-mutant embryo showing cystic proximal pronephros (*). (J) DAPT treatment (100 µM started at 9.5 hpf) of dbb homozygotes eliminates cystic distension of the pronephros (*, glomerulus; arrow, pronephric tubule). In these experiments, dbb homozygotes were identified independently of cyst formation by the characteristic mutant ventral axis curvature in 25% of embryos from heterozygote pair matings.

View larger version (16K): [in this window] [in a new window]   Fig. 11. A model of Jagged/Notch regulation of cell-type specification in the pronephros. Early in pronephric cell differentiation (8-10 hpf), cells expressing high levels of Jagged 2 interact locally with Notch3-expressing cells to inhibit the expression of rfx2 and jagged 2. Jagged 2/Notch3 signaling requires the E3 ligase mind bomb, and initiates regulated intramembranous proteolysis (RIP) of Notch3 and the liberation of the transcriptionally active Notch3 intracellular domain (N(3)ICD). N(3)ICD activates expression of unknown repressors (?), which downregulate rfx2 (and downstream cilia genes) and jagged 2. Later in development (assayed here at 34-56 hpf), a subset of cells becomes multiciliated and N(3)ICD-mediated transcriptional regulation in neighboring cells directly or indirectly regulates the expression of ion channel genes and allows these cells to acquire a transporting-epithelial-cell fate.