SummaryKupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut Research article Development 1248However, genes implicated in ciliated node cell function are also expressed in non-node cells, and mutations of these genes give rise to a variety of laterality phenotypes and more pleiotropic phenotypes, making it impossible to exclude other mechanisms for LR development during early embryogenesis (for reviews, see Tabin and Vogan, 2003;Wagner and Yost, 2000;Yost, 2003). Furthermore, it is not known whether nodal flow is a murine-specific mechanism. Cilia formation and expression of lrd homologues have recently been observed in structures analogous to the node in chick, frog (Xenopus laevis) and zebrafish embryos (Essner et al., 2002), but the existence of motile cilia and nodal flow has not been demonstrated in non-murine embryos. Further confounding the issue, there are molecular asymmetries that precede the appearance of lrd expression and monocilia in Xenopus Levin et al., 2002) and perhaps in chick (Levin et al., 1995;Stern et al., 1995). This raises the issue of whether ciliated cells in other vertebrate embryos generate fluid flow and have a conserved function for ciliogenesis genes in LR development. In zebrafish, ciliated cells arise in the tailbud at the end of gastrulation (Essner et al., 2002) in a transient spherical organ called Kupffer's vesicle (KV). KV, first described in 1868 (Kupffer, 1868), is a conserved structure among teleost fishes. Electron microscopy studies in the bait fish Fundulus heteroclitus have shown that a single cilium (i.e. monocilium) protrudes from each cell lining KV into the lumen (Brummett and Dumont, 1978). In zebrafish, KV is formed from a group of approximately two-dozen cells, known as dorsal forerunner cells (DFCs), that migrate at the leading edge of the embryonic shield (the zebrafish equivalent of the mouse node) during gastrulation. In contrast to other cells in this region, DFCs do not involute during gastrulation, but remain at the leading edge of epibolic movements. At the end of gastrulation, DFCs migrate deep into the embryo and organize to form KV (Cooper and D'Amico, 1996; D'Amico and Cooper, 1997;Melby et al., 1996). During subsequent somite stages, KV is found ventral to the forming notochord in the tailbud and adjacent to the yolk cell. Although KV was first described well over 100 years ago, it remains unknown whether DFCs and KV are mesodermal or endodermal in origin, and it is unclear what role they play during development, leading to the categorization of KV as an embryonic 'organ of ambiguity ' (Warga and Stainier, 2002).Recently, using a novel technique to knockdown gene expression specifically in DFCs, we reported the first evidence that DFCs and/or KV function in LR patterning (Amack and Yost, 2004). Here, we show that cilia that arise inside KV are motile and generate a consistent counterclockwise fluid flow. A combination of laser ablations, embryological manipul...
Cilia are cell surface organelles found on most epithelia in vertebrates. Specialized groups of cilia play critical roles in embryonic development, including left-right (LR) axis formation. Recently, cilia have been implicated as recipients of cell-cell signaling1, 2. However, little is known about cell-cell signaling pathways that control the length of cilia3. Here we provide several lines of evidence showing that fibroblast growth factor (FGF) signaling regulates cilia length and function in diverse epithelia during zebrafish and Xenopus development. Morpholino (MO) knockdown of FGF receptor 1 (FGFR1) in zebrafish cell-autonomously reduces cilia length in Kupffer’s vesicle (KV) and perturbs directional fluid flow required for LR patterning of the embryo. Expression of a dominant-negative FGFR (DN-FGFR), treatment with SU5402, a pharmacological inhibitor of FGF signaling, 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-FGFR results in shorter monocilia in the gastrocoel roof plate (GRP) that control LR patterning4 and in shorter multicilia in external mucociliary epithelium. Together, these results suggest a fundamental and highly conserved role for FGF signaling in the regulation of cilia length in multiple tissues. Abrogation of FGFR1 signaling down-regulates expression of two ciliogenic transcription factors, foxj1 and rfx2, and the intraflagellar transport (IFT) gene, polaris, suggesting that FGF signaling mediates cilia length through an FGF8/FGF24 - FGFR1- IFT pathway. We propose that a subset of developmental defects and diseases ascribed to FGF signaling are due in part to loss of cilia function.
The heart, brain, and gut develop essential left-right (LR) asymmetries. Specialized groups of ciliated cells have been implicated in LR patterning in mouse, chick, frog, and zebrafish embryos. In zebrafish, these ciliated cells are found in Kupffer's vesicle (KV) and are progeny of dorsal forerunner cells (DFCs). However, there is no direct evidence in any vertebrate that the genes involved in LR development are specifically required in ciliated cells. By using a novel method in zebrafish, we knocked down the function of no tail (ntl, homologous to mouse brachyury) in DFCs without affecting its expression in other cells in the embryo. We find that the Ntl transcription factor functions cell autonomously in DFCs to regulate KV morphogenesis and LR determination. This is the first evidence that loss-of-gene function exclusively in ciliated cells perturbs vertebrate LR patterning. Our results demonstrate that the ciliated KV, a transient embryonic organ of previously unknown function, is involved in the earliest known step in zebrafish LR development, suggesting that a ciliary-based mechanism establishes the LR axis in all vertebrate embryos.
Successful embryogenesis requires proper sorting and compartmentalization of different cell types. Mechanical interactions between cells help govern these processes. In the past, physics-based theories have guided in vitro studies of cell sorting and tissue surface tension. Recent experiments have challenged this approach, indicating that adhesive molecules also act as signaling molecules that initiate local reorganization of actomyosin and demonstrating that cells at the boundary of a colony of initially identical cells become "mechanically polarized." Extending physical models to account for mechanical polarization helps solve a long-standing paradox about magnitudes of tissue surface tensions and potentially explains discrepancies between recent in vivo and in vitro cell-sorting experiments. New experiments are needed to further explore the connection between mechanical polarization and tissue boundary formation in vivo.
The brain, heart and gastro-intestinal tract develop distinct left-right (LR) asymmetries. Asymmetric cilia-dependent fluid flow in the embryonic node in mouse, Kupffer's vesicle in zebrafish, notochordal plate in rabbit and gastrocoel roof plate in frog appears to be a conserved mechanism that directs LR asymmetric gene expression and establishes the orientation of organ asymmetry. However, the cellular processes and genetic pathways that control the formation of these essential ciliated structures are unknown. In zebrafish, migratory dorsal forerunner cells (DFCs) give rise to Kupffer's vesicle (KV), a ciliated epithelial sheet that forms a lumen and generates fluid flow. Using the epithelial marker atypical Protein Kinase C (aPKC) and other markers to analyze DFCs and KV cells, we describe a multi-step process by which DFCs form a functional KV. Using mutants and morpholinos, we show that two T-box transcription factors-No tail (Ntl)/Brachyury and Tbx16/Spadetail-cooperatively regulate an early step of DFC mesenchyme to epithelial transition (MET) and KV cell specification. Subsequently, each transcription factor independently controls a distinct step in KV formation: Tbx16 regulates apical clustering of KV cells and Ntl is necessary for KV lumen formation. By targeting morpholinos to DFCs, we show that these cell autonomous functions in KV morphogenesis are necessary for LR patterning throughout the embryo.
Cilia-generated fluid flow in an ‘organ of asymmetry’ is critical for establishing the left–right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry—called Kupffer’s vesicle (KV)—ciliated cells are asymmetrically positioned along the anterior–posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as ‘KV remodeling’ that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left–right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left–right patterning of the embryo.
Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signaling cascades that regulate cilia formation remain incompletely understood. Here we report that prostaglandin signaling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants display ciliogenesis defects, and lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme Cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates cAMP-mediated signaling cascade, are required for cilia formation and elongation. Importantly, PGE2 signaling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signaling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.
The transition zone (TZ) is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules (MTs) are heavily cross-linked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the TZ, but factors that directly recognize axonemal MTs to specify TZ assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identified an axoneme-associated protein, CEP162, tethered specifically at centriole distal ends to promote TZ assembly. CEP162 interacts with core TZ components, and mediates their association with MTs. Loss of CEP162 arrests ciliogenesis at the stage of TZ assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme, and ectopically assemble TZ components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein “pre-tethered” at centriole distal ends prior to ciliogenesis to promote and restrict TZ formation specifically at the cilia base.
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