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FGF signalling during embryo development regulates cilia length in diverse epithelia.
Neugebauer JM
,
Amack JD
,
Peterson AG
,
Bisgrove BW
,
Yost HJ
.
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Cilia are cell surface organelles found on most epithelia in vertebrates. Specialized groups of cilia have critical roles in embryonic development, including left-right axis formation. Recently, cilia have been implicated as recipients of cell-cell signalling. However, little is known about cell-cell signalling pathways that control the length of cilia. Here we provide several lines of evidence showing that fibroblast growth factor (FGF) signalling regulates cilia length and function in diverse epithelia during zebrafish and Xenopus development. Morpholino knockdown of FGF receptor 1 (Fgfr1) in zebrafish cell-autonomously reduces cilia length in Kupffer's vesicle and perturbs directional fluid flow required for left-right patterning of the embryo. Expression of a dominant-negative FGF receptor (DN-Fgfr1), treatment with SU5402 (a pharmacological inhibitor of FGF signalling) or genetic and morpholino reduction of redundant FGF ligands Fgf8 and Fgf24 reproduces this cilia length phenotype. Knockdown of Fgfr1 also results in shorter tethering cilia in the otic vesicle and shorter motile cilia in the pronephric ducts. In Xenopus, expression of a dn-fgfr1 results in shorter monocilia in the gastrocoel roof plate that control left-right patterning and in shorter multicilia in external mucociliary epithelium. Together, these results indicate a fundamental and highly conserved role for FGF signalling in the regulation of cilia length in multiple tissues. Abrogation of Fgfr1 signalling downregulates expression of two ciliogenic transcription factors, foxj1 and rfx2, and of the intraflagellar transport gene ift88 (also known as polaris), indicating that FGF signalling mediates cilia length through an Fgf8/Fgf24-Fgfr1-intraflagellar transport pathway. We propose that a subset of developmental defects and diseases ascribed to FGF signalling are due in part to loss of cilia function.
Figure 2. FGF signaling controls cilia length and directional fluid flow in Kupffer’s Vesicle(a-b) Confocal images of 10 SS embryos, KV labeled with antibodies against aPKC (red) and acetylated tubulin (green). Control and fgfr1 morphants had similar KV structure, but cilia were shorter in fgfr1 morphants (compare insets in a and b). (c) Cilia lengths were significantly different (p< 2.88e-06) in fgfr1 morphants (688 cilia; 18 embryos) versus Control morphants (437 cilia; 9 embryos). Cilia length was similar in WT uninjected (533 cilia; 10 embryos) and Control morphant (p< 0.93), cilia numbers per KV were similar in Control and fgfr1 morphants (p< 0.26). Cilia length defects in fgfr1 morphants were rescued by Xenopus FGFR1 (xFGFR1) mRNA (p< 4.70e-05; 807 cilia; 21 embryos). Injection of xFGFR1 mRNA alone had no affect on cilia length (p<0.73; 526 cilia, 14 embryos). (d) Embryos treated with SU5402 during shield stage (248 cilia; 12 embryos) had shorter cilia compared to DMSO control embryos (p<3.26e-06; 686 cilia; 15 embryos). (e) Cilia were shorter in transgenic DN-FGFR embryos that were heat shocked at 60% epiboly (656 cilia; 19 embryos) compared to heat shocked non-transgenic siblings (p<6.94e-03; 375 cilia; 10 embryos) and non-heat-shocked siblings (p<6.99e-03; 910 cilia; 16 embryos). (f) There was no difference in cilia length (p<0.28) in fgf24 morphants (455 cilia; 10 embryos) versus Control morphants (481 cilia; 10 embryos). However, cilia were shorter when both FGF8 and FGF24 ligands were diminished (fgf24 MO in ace mutants; 12 embryos; 244 cilia), compared to single ligand knockdown (FGF8/ace mutants: p< 1.39e-04; 10 embryos; 480 cilia; fgf24 MO in ace sibs: p< 3.44e-04; 15 embryos; 643 cilia) and WT ace siblings (p<3.63e-07; 13 embryos; 626 cilia). (g-h) DIC images of bead-injected KVs in Control and fgfr1 morphants injected with fluorescent beads. (i-j) Bead paths tracked by Metamorph software. Directional KV fluid flow was absent in fgfr1 morphants (j; p< 6.4e-15; 44 beads, 9 embryos) compared to counterclockwise flow in Control morphants (i; 39 beads, 8 embryos). Error bars are standard error of the mean (s.e.m).
Figure 3. Cilia length in pronephric ducts, otic vesicles, gastrocoel roof plate epithelia and mucociliary epithelia is controlled by FGF signaling(a-b) Pronephric duct cilia were shorter and disorganized in fgfr1 morphants (p<4.24×10-4; 528 cilia; 10 embryos,) compared to WT (517 cilia; 10 embryos) 26 SS embryos. (c-d) Otic vesicle tethering cilia (arrows, and inset) were shorter (p< 1.10e-07) in fgfr1 morphants (325 cilia; 10 embryos) compared to WT embryos (322 cilia; 8 embryos) at 24 hpf. (g-j, m). GRP cilia in Xenopus embryos were normal length in cells expressing GFP alone (green cells in g, outlined in h; 316 cilia 18 embryos, p<0.11), neighboring cells (outside boundaries in h; 653, 18 embryos), and cells neighboring DN-FGFR+GFP expression (outside boundaries in j; 652 cilia 15 embryos, p<0.99). In contrast, GRP cilia were shorter in cells expressing DN-FGFR+GFP (i, inside boundaries in j; 155 cilia, 15 embryos) compared to neighboring cells (p<6.1e-03) and cells expressing GFP alone (p<2.7e-03). (k, l) Z plane rendering of mucociliary epithelia (scale bar 20 um), showing shorter cilia in cells expressing DN-FGFR+GFP (13 cells, 7 embryos) compared to controls expressing GFP alone (14 cells, 4 embryos). (n) Multicilia area is reduced in cells expressing DN-FGFR+GFP (p<0.019). Error bars are s.e.m.
Figure 4. FGF signaling controls ciliogenic genes in DFC/KV cells(a, b) sox17 expression in DFC/KV (and endoderm cells in a different focal plane) in 90% epiboly embryos was normal in fgfr1 morphants and WT embryos. (c, d) Expression of dnah9 in 95 % epiboly embryos was normal in fgfr1 morphants and WT embryos. (e, f) In contrast, foxJ1 was down-regulated in fgfr1 morphants versus WT embryos at 90% epiboly. (g, h) Similarly, polaris was down-regulated in fgfr1 morphants versus WT embryos at tailbud stage. (i) Comparison of percentage of embryos with WT expression levels of each gene indicated. (j) Proposed mechanism by which FGF signaling controls length of motile cilia: FGF ligands bind to FGFR1 activating downstream transcription factors (TF) including foxj1 and rfx2, these TF activate IFT genes (e.g. polaris) to maintain motile cilia length on epithelial cells.
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