Search

The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.

Pontin (Ruvbl1) and Reptin (Ruvbl2) are closely related AAA ATPases. They are components of the Ruvbl1-Rvubl2-Tah1-Pih1 (R2TP) complexes that function as co-chaperones for the assembly of multiple macromolecular protein complexes. Here, we show that Pontin is essential for cilia motility in both zebrafish and mouse and that Pontin and Reptin function cooperatively in this process. pontin zebrafish mutants display phenotypes tightly associated with cilia defects and cilia motility is lost in a number of ciliated tissues along with a reduction in the number of both outer and inner dynein arms (ODAs and IDAs). Pontin protein is enriched in cytosolic puncta in ciliated cells in zebrafish embryos. In addition, Pontin is essential for the stabilization of axonemal dynein intermediate chain 1 (DNAI1) and DNAI2, the first appreciated step in axonemal dynein arm assembly. Strikingly, multiple dynein arm assembly factors show structural similarities to either Tah1 or Pih1, the other two components of the R2TP complex. Based on these results, we propose that Pontin and Reptin function to facilitate dynein arm assembly in cytosolic foci enriched with R2TP-like complexes.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptin hi2394 mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptin hi2394 mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound “ciliabrevin” due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug. © 2011 Wiley-Liss, Inc.

The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.

One of the most widespread cellular organelles in nature is cilium, which is found in many unicellular and multicellular organisms. Formerly thought to be a mostly vestigial organelle, the cilium has been discovered in the past several decades to play critical motile and sensory roles involved in normal organogenesis during development. The role of cilia has also been implicated in an ever increasing array of seemingly unrelated human diseases, including blindness, kidney cysts, neural tube defects and obesity. In this article we review some of the recent developments in research on cilia, and how defects in ciliogenesis and function can give rise to developmental disorders and disease.

The cell surface organelle called the cilium is essential for preventing kidney cyst formation and for establishing left-right asymmetry of the vertebrate body plan. Recent advances suggest that the cilium functions as a sensory organelle in vertebrate cells for multiple signaling pathways such as the hedgehog and the Wnt pathways. Prompted by kidney cyst formation in tuberous sclerosis complex (TSC) patients and rodent models, we investigated the role of the cilium in the TSC-target of rapamycin (TOR) pathway using zebrafish. TSC1 and TSC2 genes are causal for TSC, and their protein products form a complex in the TOR pathway that integrates environmental signals to regulate cell growth, proliferation and survival. Two TSC1 homologs were identified in zebrafish, which we refer to as tsc1a and tsc1b. Morpholino knockdown of tsc1a led to a ciliary phenotype including kidney cyst formation and left-right asymmetry defects. Tsc1a was observed to localize to the Golgi, but morpholinos against it, nonetheless, acted synthetically with ciliary genes in producing kidney cysts. Consistent with a role of the cilium in the same pathway as Tsc genes, the TOR pathway is aberrantly activated in ciliary mutants, resembling the effect of tsc1a knockdown. Moreover, kidney cyst formation in ciliary mutants was blocked by the Tor inhibitor, rapamycin. Surprisingly, we observed elongation of cilia in tsc1a knockdown animals. Together, these data suggest a signaling network between the cilium and the TOR pathway in that ciliary signals can feed into the TOR pathway and that Tsc1a regulates the length of the cilium itself.

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2 , one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

Once obscure, the cilium has come into the spotlight during the past decade. It is now clear that aside from generating locomotion by motile cilia, both motile and immotile cilia serve as signaling platforms for the cell. Through both motility and sensory functions, cilia play critical roles in development, homeostasis, and disease. To date, the cilium proteome contains more than 1,000 different proteins, and human genetics is identifying new ciliopathy genes at an increasing pace. Although assigning a function to immotile cilia was a challenge not so long ago, the myriad of signaling pathways, proteins, and biological processes associated with the cilium have now created a new obstacle: how to distill all these interactions into specific themes and mechanisms that may explain how the organelle serves to maintain organism homeostasis. Here, we review the basics of cilia biology, novel functions associated with cilia, and recent advances in cilia genetics, and on the basis of this framework, we further discuss the meaning and significance of ciliary connections.

The target-of-rapamycin (TOR) pathway is a major regulator of cellular growth, division, and metabolism by coupling growth and nutrient cues to fundamental processes such as translation and transcription. In vertebrate models, disruptions affecting components of the TOR pathway result in cilia size abnormalities and ciliopathic phenotypes such as renal cysts. However, the mechanism by which the TOR pathway influenced cilia length and function was unknown. Recent work from zebrafish, in conjunction with the green alga Chlamydomonas, demonstrates that TOR complex 1 (TORC1)-mediated cilia length control is achieved through translational regulation of ciliary precursors. Further, in zebrafish, aberrant TORC1 signaling leads to left-right asymmetric patterning defects due to inefficient ciliary motility and fluid flow generation in the Kupffer's vesicle, a conserved embryonic organ that establishes laterality. Together, such findings show the utility of a combinatorial zebrafish and Chlamydomonas approach to dissecting the mechanistic relationship between the cilium and signaling molecules such as TOR. Here, we describe tools, reagents, and methodologies for the manipulation of the TOR pathway and the analysis of cilia morphology and function in zebrafish and Chlamydomonas.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function. © 2013 The American Society of Human Genetics.

The cilium is a cell-surface antenna that is well conserved from single-cell eukaryotes to humans. In recent years, the cilium has garnered immense attention due to revelations of the indispensible role that the organelle plays in orchestrating vertebrate development and homeostasis. This tiny organelle senses extracellular signals and couples them with a plethora of downstream functions, such as growth, division and motility.1 As such, ciliary dysfunction is intimately associated with numerous disease etiologies, including polycystic kidney disease, obesity and cancer.2 Despite this, the mechanisms regulating the morphology and function of the cilium, as well as the relationship between ciliary morphology and function, are not well understood. Emerging work in cell culture, zebrafish and mice suggest that broad and diverse signaling pathways may play a crucial role in regulating ciliary size.3-7 Some of these pathways seem to execute cilia size changes through shared downstream targets, which suggests the existence of a common mechanism that integrates signaling from numerous inputs. For example, knockdown of fibroblast growth factor receptor 1 (Fgfr1), a component of the fibroblast growth factor (FGF) signaling cascade, results in shortened cilia and left-right body patterning defects in zebrafish4 (Fig. 1A). FGF abrogation results in decreased expression of transcription factors foxj1 and rfx2, which have been previously demonstrated to be essential for motile ciliogenesis.4,8,9 Similarly, zebrafish that lack DeltaD, a ligand of the Notch pathway, display shortened cilia, left-right body defects and decreased expression of foxj1 (Fig. 1A). However, how ciliogenic transcription factors such as foxj1 and rfx2 mediate cilia length changes and the identity of their downstream effectors remains largely unknown. Figure 1. Diverse mechanisms of cilia length control in vertebrates. (A) FGF and Notch signaling influence cilia length, motility and cilia-directed fluid flow via the ciliogenic factor fox1j. (B) cAMP signaling and intracellular calcium levels ... Alternatively, the delivery rate of cargo necessary for ciliogenesis by intraflagellar transport (IFT) has been suggested to be a mechanism for cilia length control.10 A small-molecule screen performed in tissue culture revealed that decreased intracellular calcium or increased cyclic AMP (cAMP) signaling results in abnormally elongated primary cilia.5 These elongated cilia display elevated anterograde IFT particle velocities, which suggests that alterations in IFT kinetics may influence ciliary length. (Fig. 1B). In addition, a small-molecule screen performed in C. reinhardtii identified a compound that induced flagellar shortening and decreased anterograde IFT speed and frequency.11 Interestingly, a genome-wide RNAi screen performed in mammalian cultured cells revealed a role for actin dynamics and endocytic vesicular trafficking in regulating cilia length and ciliogenesis.6 Silencing of actin-related protein 3 (ARP3), a component of the ARP2/3 complex that initiates actin filament polymerization, resulted in elongated primary cilia and increased ciliogenesis (Fig. 1C). In addition, silencing of phospholipase A2, group III (PLA2G3), a secreted phospholipase that has been implicated in vesicular trafficking by inducing membrane curvature, also elongated primary cilia and increased ciliogenesis (Fig. 1D). We have recently demonstrated a novel role for target-of-rapamycin (TOR) signal transduction in modulating cilia size and function through translational regulation.12 Inspired by the observation of abnormally long cilia in the renal epithelium of tuberous sclerosis complex (TSC) models, we investigated TOR signaling as a downstream mechanism for TSC-mediated cilia elongation.3 In zebrafish, activation of TOR complex-1 (TORC1) signaling increases ciliary length through S6 kinase 1 (S6K1)-mediated protein synthesis of cilia precursors (Fig. 1E). Conversely, rapamycin inhibition of TORC1 decreases ciliary length by repressing S6K1-mediated translation. Surprisingly, we observed that this mechanism is seemingly independent of IFT speed, as inhibition of TORC1-mediated translation did not alter anterograde particle velocity (unpublished data). Rather, TORC1 inhibition diminishes the synthesis of ciliary precursors in the cytosol, thus representing a unique and novel mechanism for cilia length control. However, the identity of such precursors remains to be elucidated. We further demonstrated that ciliary length is critical for ciliary function. In the zebrafish Kupffer’s vesicle, motile cilia that are too long or short display slow beating frequencies and fail to generate the proper fluid flow that is required for establishing embryonic left-right body asymmetry. Moreover, ciliary length is also tightly correlated with ciliary motility in a wild-type population of Chlamydomonas cells with a natural distribution of flagellar lengths. Together, these studies begin to address an overlooked question in cell biology: since cellular size is carefully defined and regulated by robust mechanisms, are subcellular organelles also regulated in this manner? Strikingly, our findings suggest that this is indeed the case, as cellular and subcellular size may be regulated in part by common and shared mechanisms, with TOR signaling being a prominent example. As a major nutrient sensor and metabolic regulator, it is not surprising that TOR may play an integral role in size control at numerous levels for the cell. However, as diverse signaling cascades have also been implicated in ciliary size control, TOR likely represents merely one instrument within an orchestra: numerous parallel and complementary organelle size control mechanisms are likely to coexist. Elucidation of this potential size control network will have profound implications for cellular and subcellular function and, consequently, core biological processes and disease etiologies.

The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed “ciliopathies.” Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear. Here, we show that the target-of-rapamycin (TOR) growth pathway modulates cilia size and function during zebrafish development. Knockdown of tuberous sclerosis complex 1a ( tsc1a ), which encodes an upstream inhibitor of TOR complex 1 (Torc1), increases cilia length. In contrast, treatment of embryos with rapamycin, an inhibitor of Torc1, shortens cilia length. Overexpression of ribosomal protein S6 kinase 1 ( S6k1 ), which encodes a downstream substrate of Torc1, lengthens cilia. Furthermore, we provide evidence that TOR-mediated cilia assembly is evolutionarily conserved and that protein synthesis is essential for this regulation. Finally, we demonstrate that TOR signaling and cilia length are pivotal for a variety of downstream ciliary functions, such as cilia motility, fluid flow generation, and the establishment of left-right body asymmetry. Our findings reveal a unique role for the TOR pathway in regulating cilia size through protein synthesis and suggest that appropriate and defined lengths are necessary for proper function of the cilium.

Defects in the cilium, a once thought vestigial organelle, have recently been implicated in many human diseases, including a number of cystic kidney diseases such as polycystic kidney disease (PKD), Bardet Bieldl Syndrome, and Meckel-Gruber Syndrome. In a forward genetic screen, qilin was identified as a novel gene important in the pathogenesis of kidney cysts in zebrafish. In this paper we characterized qilin(hi3959A) mutant's phenotypes in detail, investigated cilia formation in this mutant and performed structural and functional analysis of the Qilin protein. Results reveal Qilin's essential role in cilia assembly and maintenance in multiple organs, including the kidney, the lateral line organ, and the outer segment of the photoreceptor cell. In addition, rescue experiments suggest that defective pronephric cilia correlate with the formation of kidney cysts in qilin(hi3959A) mutants. Further, genetic analysis suggests that qilin interacts with multiple intraflagellar transport (IFT) complex B genes, which is supported by the striking phenotypic similarities between qilin(hi3959A) and IFT complex B mutants. Finally, through deletion analysis we provide evidence that the well-conserved N-terminus and the coiled-coil domain of Qilin are both essential and sufficient for its function. Taken all the observations together, we propose that Qilin acts in a similar role as IFT complex B proteins in cilia assembly, maintenance and kidney development in zebrafish.

The cilium, a previously little studied cell surface protrusion, has emerged as an important organelle in vertebrate cells. This tiny structure is essential for normal embryonic development, including the formation of left-right asymmetry, limb morphogenesis, and the differentiation of sensory cells. In the adult, cilia also function in a variety of processes, such as the survival of photoreceptor cells, and the homeostasis in several tissues, including the epithelia of nephric ducts. Human ciliary malfunction is associated with situs inversus, kidney cysts, polydactyly, blindness, mental retardation, obesity, and many other abnormalities. The genetic accessibility and optical transparency of the zebrafish make it an excellent vertebrate model system to study cilia biology. In this chapter, we describe the morphology and distribution of cilia in zebrafish embryonic and larval organs. We also provide essential protocols to analyze cilia formation and function.

arl13b was initially cloned as the novel cystic kidney gene scorpion (sco) in zebrafish and was shown to be required for cilia formation in the kidney duct. In mouse, a null mutant of Arl13b shows abnormal ultrastructure of the cilium and defective sonic hedgehog (Shh) signaling. Importantly, a recent study linked mutations in ARL13B to a classical form of Joubert syndrome (JS), an autosomal recessive disorder characterized by a distinctive cerebellar malformation. In this study, we analyzed the zebrafish arl13b (sco) mutant and gene products in detail. We first demonstrate that Arl13b is a protein that is highly enriched in the cilium and is required for cilia formation in multiple organs in zebrafish, and that knockdown of arl13b leads to multiple cilia-associated phenotypes. We additionally show that multiple regions of Arl13b are required for its localization to the cilium. By means of rescuing experiments with a series of deletion and point mutants, we further demonstrate that the ciliary localization is crucial for the in vivo function of Arl13b. Together, these results strongly support the hypothesis that JS-related disease (JSRD) is a ciliopathy, or a disease caused by ciliary defects, and that Arl13b functions mainly through the cilium.

Summary Background Bilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades is unknown. Results Here, we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically-encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink demonstrates that they are essential for LR development. Conclusions These findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling., Graphical Abstract