ABSTRACT
Heparan sulfate proteoglycans (HSPGs) control many cellular processes and have been implicated in the regulation of left-right (LR) development by as yet unknown mechanisms. Using lineage-targeted knockdowns, we found that the transmembrane HSPG Syndecan 2 (Sdc2) regulates LR patterning through cell-autonomous functions in the zebrafish ciliated organ of asymmetry, Kupffer's vesicle (KV), including regulation of cell proliferation and adhesion, cilia length and asymmetric fluid flow. Exploring downstream pathways, we found that the cell signaling ligand Fgf2 is exclusively expressed in KV cell lineages, and is dependent on Sdc2 and the transcription factor Tbx16. Strikingly, Fgf2 controls KV morphogenesis but not KV cilia length, and KV morphogenesis in sdc2 morphants can be rescued by expression of fgf2 mRNA. Through an Fgf2-independent pathway, Sdc2 and Tbx16 also control KV ciliogenesis. Our results uncover a novel Sdc2-Tbx16-Fgf2 pathway that regulates epithelial cell morphogenesis.
Subject(s)
Cilia/metabolism , Embryo, Nonmammalian/metabolism , Epithelial Cells/cytology , Fibroblast Growth Factor 2/metabolism , Syndecan-2/metabolism , T-Box Domain Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Epithelial Cells/metabolism , Fibroblast Growth Factor 2/genetics , Immunohistochemistry , In Situ Hybridization , Syndecan-2/genetics , T-Box Domain Proteins/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
An early step in establishing left-right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the left lateral plate mesoderm, activating a transcriptional response of Nodal expression in the left lateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the left LPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFß family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left-right signal from KV to the LPM.
Subject(s)
Animal Structures/embryology , Body Patterning , Mesoderm/embryology , Signal Transduction , Transforming Growth Factor beta/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animal Structures/metabolism , Animals , Body Patterning/drug effects , Body Patterning/genetics , Cilia/drug effects , Cilia/metabolism , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Immunoprecipitation , Mesoderm/drug effects , Mesoderm/metabolism , Mice , Models, Biological , Morpholinos/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transforming Growth Factor beta/genetics , Xenopus , Zebrafish Proteins/geneticsABSTRACT
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.
