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FGF signalling is required for differentiation-induced cytoskeletal reorganisation and formation of actin-based processes by podocytes

Gary Davidson^1,*, Rosanna Dono^1,2 and Rolf Zeller^1,2,

¹ EMBL, Meyerhofstrasse1, D-69117 Heidelberg, Germany
² Department of Developmental Biology, Utrecht University, Padualaan 8, NL-3584CH Utrecht, The Netherlands
^* Present address: Division of Molecular Embryology, DKFZ, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

View larger version (109K): [in a new window]   Fig. 1. Upregulation of FGF2 protein levels in podocytes during post-mitotic differentiation. (A) Distribution of FGF2 proteins in differentiating glomeruli in a mesonephric kidney (isolated from a stage 21/22 chicken embryo). (B) Detection of BrdU-positive proliferating cells in a section adjacent to the one shown in A. Arrows in B point to groups of nonproliferating podocytes expressing high levels of FGF2 proteins (arrowheads in A). (C) Conditionally immortalised MPC cells express only low levels of FGF2 proteins during proliferation (permissive conditions). (D) High levels of nuclear FGF2 proteins accumulate in post-mitotic and differentiated MPC cells (grown for 7 days under nonpermissive conditions). (E) Expression of all three FGF2 protein isoforms is upregulated in differentiated MPC cells. SYN, synaptopodin, a marker for differentiated podocytes. (F) Cellular fractionation of proliferating and differentiated MPC cells reveals preferential nuclear accumulation of the two large FGF2 isoforms (21.5 and 22 kDa) in differentiated MPC cells (right panel). By contrast, the 18 kDa FGF2 isoform is detected in all three fractions. Cyto, cytoplasm; Diff, differentiated cells; JUN, c-Jun as a marker for the nuclear fraction; note downregulation in differentiated cells. Mem, membrane; Nuc, nucleus; Prol, proliferating cells; TUB, -tubulin as a marker for the cytoplasm; note upregulation in differentiated cells. Equal amounts of protein were used for all samples shown in panels E and F.

View larger version (166K): [in a new window]   Fig. 2. Post-mitotic differentiation of Fgf2 mutant MPC cells is severely altered. (A) Wild-type MPC cells grown under permissive conditions. (B) Fgf2 mutant MPC cells grown under permissive conditions. Note that these cells tend to grow more as aggregates than their wild-type counterparts. (C) Differentiated wild-type MPC cells (grown for 7 days under nonpermissive conditions). Inset shows a representative branched process of a differentiated wild-type MPC cell. (D) Differentiated Fgf2 mutant MPC cells. Inset shows the disruption of process formation (compare with inset in C). Diff, differentiated cells; Prol, proliferating cells.

View larger version (46K): [in a new window]   Fig. 3. General alteration of FGF signalling in Fgf2 mutant MPC cells. Semi-quantitative RT-PCR analysis of the expression of several Fgfs (Fgf2, Fgf1, Fgf7 and Fgf10) and their receptors (Fgfr1, Fgfr2) in wild-type (+/+) and Fgf2 mutant (-/-) MPC cells and adult kidney cortex tissue (enriched in glomeruli). Note that transcription of the Fgf7 and Fgf10 ligands is upregulated similar to Fgf2 in differentiated wild-type MPC cells grown for 7 days under nonpermissive conditions. Their expression is severely downregulated (Fgf7) or absent (Fgf10, Fgf2) in Fgf2 mutant MPC cells. By contrast, Fgf1 expression is upregulated in Fgf2 mutant MPC cells. No such changes are observed when comparing wild-type to Fgf2 mutant adult kidney cortex tissue. Expression of Fgf receptor isoform IIIc (Fgfr1(IIIc)) remains unchanged, but the Fgfr2(IIIc) isoform is downregulated in Fgf2 mutant MPC cells. By contrast, expression of both Fgfr1(IIIb) and Fgfr2(IIIb) isoforms is upregulated. No changes in Fgf receptor isoform expression levels are observed in adult Fgf2 mutant kidney cortex tissue. Glyceraldehyde-3-phosphate dehydrogenase (Gpdh) was used to normalise RNA content of samples. Diff, differentiated cells; Prol, proliferating cells.

View larger version (53K): [in a new window]   Fig. 4. Mutant MPC cells maintain podocyte lineage but display differentiation defects affecting actin cytoskeletal reorganisation. (A,B) Comparison of the actin cytoskeleton (green) and focal adhesion contacts (paxillin; red) in differentiated wild-type (+/+) and mutant (-/-) MPC cells. Note that the actin stress fibers in wild-type MPC cells extend to the cell periphery (orange arrowheads in A). By contrast, a belt of disorganised actin fibers forms in the perinuclear region in mutant MPC cells (B). Actin fibers still terminate in focal adhesions, but fail to reach the cell periphery (indicated by white arrowheads). Note: the cytoplasm of the cell shown extends beyond the right margin of the panel. (C,D) Distribution of the actin-associated protein synaptopodin (SYN, green) in wild-type and mutant MPC cells. Although synaptopodin marks actin stress fibers in differentiated wild-type MPC cells (C), only low levels of are detected in mutant MPC cells (D). Nuclei in panels A to D are counterstained by DAPI (blue). (E) Analysis of markers for podocyte lineage by semi-quantitative RT-PCR confirms maintenance of podocyte lineage in mutant MPC cells. Syn: Expression of the podocyte differentiation marker synaptopodin is downregulated in mutant (-/-) MPC cells (compare also with D). WT-1: Expression of Wilms’ tumour antigen-1 is also downregulated in mutant MPC cells. (33): RT-PCR with 33 cycles; (40): RT-PCR with 40 cycles. By contrast, Podocalyxin (Pdx) expression is not affected, whereas Pod-1 is upregulated in proliferating, mutant MPC cells. Gpdh: Glyceraldehyde-3-phosphate dehydrogenase was used to normalise RNA content of samples. Diff, differentiated cells; Prol, proliferating cells.

View larger version (47K): [in a new window]   Fig. 5. Upregulation of epithelial and loss of mesenchymal characteristics in differentiated mutant MPC cells. (A,B) Distribution of the intermediate filament protein vimentin in wild-type (+/+) and mutant (-/-) differentiated MPC cells. Note that high levels of vimentin protein are expressed by wild-type MPC cells (A) and localised in cellular processes (inset in A). Mutant MPC cells express much lower levels or no vimentin (B). (C,D) Redistribution of the tight junction protein ZO-1 to cellular processes in differentiated wild-type MPC cells (see, for example, C, arrowhead). This redistribution is disrupted in MPC mutant cells (D). Note the rather homogenous ZO-1 distribution in cell membranes of mutant MPC cells. (E) Semi-quantitative RT-PCR analysis of several markers for mesenchymal and/or epithelial cell states. Vim: Expression of the intermediate filament protein vimentin is disrupted in MPC mutant cells (compare with B). ZO-1: Only one ZO-1 isoform is expressed by wild-type MPC cells, whereas both ZO-1 isoforms (± motif) are expressed by mutant MPC cells. DscI2: Expression of the epithelial desmosomal cadherin protein desmocollin type 2 is downregulated in wild-type MPC cells, but is maintained in mutant MPC cells. Slug: Expression of slug is disrupted in mutant MPC cells, whereas it is upregulated in wild-type MPC cells. Gpdh: Glyceraldehyde-3-phosphate dehydrogenase was used to normalise RNA content of samples.