Loss of Deacetylation Enzymes Hdac6 and Sirt2 Promotes Acetylation of Cytoplasmic Tubulin, but Suppresses Axonemal Acetylation in Zebrafish Cilia.

Cilia are evolutionarily highly conserved organelles with important functions in many organs. The extracellular component of the cilium protruding from the plasma membrane comprises an axoneme composed of microtubule doublets, arranged in a 9 + 0 conformation in primary cilia or 9 + 2 in motile cilia. These microtubules facilitate transport of intraflagellar cargoes along the axoneme. They also provide structural stability to the cilium, which may play an important role in sensory cilia, where signals are received from the movement of extracellular fluid. Post-translational modification of microtubules in cilia is a well-studied phenomenon, and acetylation on lysine 40 (K40) of alpha tubulin is prominent in cilia. It is believed that this modification contributes to the stabilization of cilia. Two classes of enzymes, histone acetyltransferases and histone deacetylases, mediate regulation of tubulin acetylation. Here we use a genetic approach, immunocytochemistry and behavioral tests to investigate the function of tubulin deacetylases in cilia in a zebrafish model. By mutating three histone deacetylase genes (Sirt2, Hdac6, and Hdac10), we identify an unforeseen role for Hdac6 and Sirt2 in cilia. As expected, mutation of these genes leads to increased acetylation of cytoplasmic tubulin, however, surprisingly it caused decreased tubulin acetylation in cilia in the developing eye, ear, brain and kidney. Cilia in the ear and eye showed elevated levels of mono-glycylated tubulin suggesting a compensatory mechanism. These changes did not affect the length or morphology of cilia, however, functional defects in balance was observed, suggesting that the level of tubulin acetylation may affect function of the cilium.

Ciliopathy genes are required for apical secretion of Cochlin, an otolith crystallization factor.

Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.

STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena.

The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet-Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

Identification of additional outer segment targeting signals in zebrafish rod opsin.

In vertebrate photoreceptors, opsins are highly concentrated in a morphologically distinct ciliary compartment known as the outer segment (OS). Opsin is synthesized in the cell body and transported to the OS at a remarkable rate of 100 to 1000 molecules per second. Opsin transport defects contribute to photoreceptor loss and blindness in human ciliopathies. Previous studies revealed that the rhodopsin C-terminal tail, of 44 amino acids, is sufficient to mediate OS targeting in Xenopus photoreceptors. Here, we show that, although the Xenopus C-terminus retains this function in zebrafish, the homologous zebrafish sequence is not sufficient to target opsin to the OS. This functional difference is largely caused by a change of a single amino acid present in Xenopus but not in other vertebrates examined. Furthermore, we find that sequences in the third intracellular cytoplasmic loop (IC3) and adjacent regions of transmembrane helices 6 and 7 are also necessary for opsin transport in zebrafish. Combined with the cytoplasmic tail, these sequences are sufficient to target opsin to the ciliary compartment.

The role of endothelial cilia in postembryonic vascular development.

BACKGROUND: Cilia are essential for morphogenesis and maintenance of many tissues. Loss-of-function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development. We therefore used a conditional rescue approach to induce endothelial cilia ablation after early embryonic development and examined the effect on vascular development and mural cell development in postembryonic, juvenile, and adult Zebrafish. RESULTS: ift54(elipsa)-mutant Zebrafish are unable to form cilia. We rescued cilia formation and ameliorated the phenotype of ift54 mutants using a novel Tg(ubi:loxP-ift54-loxP-myr-mcherry,myl7:EGFP)sh488 transgene expressing wild-type ift54 flanked by recombinase sites, then used a Tg(kdrl:cre)s898 transgene to induce endothelial-specific inactivation of ift54 at postembryonic ages. Fish without endothelial ift54 function could survive to adulthood and exhibited no vascular defects. Endothelial inactivation of ift54 did not affect development of tagln-positive vascular mural cells around either the aorta or the caudal fin vessels, or formation of vessels after tail fin resection in adult animals. CONCLUSIONS: Endothelial cilia are not essential for development and remodeling of the vasculature in juvenile and adult Zebrafish when inactivated after embryogenesis.

The Nuclear Arsenal of Cilia.

Several recent studies have revealed that nuclei and cilia share molecular components implicated in DNA damage response, splicing, gene expression, and sub-compartmentalization of the cell. We review evidence that exchange of components between the nucleus and cilia is facilitated by the centrosome, which contributes both to the mitotic apparatus of the nucleus and to the cilia structure. Moreover, the centrosome and the pericentriolar material form condensates that share components with stress granules and P-bodies, membrane-less organelles enriched in RNA and RNA-processing proteins. These features may largely explain the origin of similar molecular mechanisms in nuclei and cilia.

Apico-basal Polarity Determinants Encoded by crumbs Genes Affect Ciliary Shaft Protein Composition, IFT Movement Dynamics, and Cilia Length.

One of the most obvious manifestations of polarity in epithelia is the subdivision of the cell surface by cell junctions into apical and basolateral domains. crumbs genes are among key regulators of this form of polarity. Loss of crumbs function disrupts the apical cell junction belt and crumbs overexpression expands the apical membrane size. Crumbs proteins contain a single transmembrane domain and localize to cell junction area at the apical surface of epithelia. In some tissues, they are also found in cilia. To test their role in ciliogenesis, we investigated mutant phenotypes of zebrafish crumbs genes. In zebrafish, mutations of three crumbs genes, oko meduzy/crb2a, crb3a, and crb2b, affect cilia length in a subset of tissues. In oko meduzy (ome), this is accompanied by accumulation of other Crumbs proteins in the ciliary compartment. Moreover, intraflagellar transport (IFT) particle components accumulate in the ciliary shaft of ome;crb3a double mutants. Consistent with the above, Crb3 knockdown in mammalian cells affects the dynamics of IFT particle movement. These findings reveal crumbs-dependent mechanisms that regulate the localization of ciliary proteins, including Crumbs proteins themselves, and show that crumbs genes modulate intraflagellar transport and cilia elongation.

The Cilium: Cellular Antenna and Central Processing Unit.

Cilia mediate an astonishing diversity of processes. Recent advances provide unexpected insights into the regulatory mechanisms of cilium formation, and reveal diverse regulatory inputs that are related to the cell cycle, cytoskeleton, proteostasis, and cilia-mediated signaling itself. Ciliogenesis and cilia maintenance are regulated by reciprocal antagonistic or synergistic influences, often acting in parallel to each other. By receiving parallel inputs, cilia appear to integrate multiple signals into specific outputs and may have functions similar to logic gates of digital systems. Some combinations of input signals appear to impose higher hierarchical control related to the cell cycle. An integrated view of these regulatory inputs will be necessary to understand ciliogenesis and its wider relevance to human biology.

Unexpected Roles for Ciliary Kinesins and Intraflagellar Transport Proteins.

Transport of proteins in the ciliary shaft is driven by microtubule-dependent motors, kinesins. Prior studies suggested that the heterotrimeric ciliary kinesin may be dispensable for certain aspects of transport in specialized cilia of vertebrate photoreceptor cells. To test this possibility further, we analyzed the mutant phenotype of the zebrafish kif3a gene, which encodes the common motor subunit of heterotrimeric ciliary kinesins. Cilia are absent in all organs examined, leading to the conclusion that kif3a is indispensable for ciliogenesis in all cells, including photoreceptors. Unexpectedly, kif3a function precedes ciliogenesis as ciliary basal bodies are mispositioned in mutant photoreceptors. This phenotype is much less pronounced in intraflagellar transport (IFT) mutants and reveals that kif3a has a much broader role than previously assumed. Despite the severity of their basal body phenotype, kif3a mutant photoreceptors survive longer compared to those in IFT mutants, which display much weaker basal body mispositioning. This effect is absent in kif3a;IFT double mutants, indicating that IFT proteins have ciliary transport-independent roles, which add to the severity of their photoreceptor phenotype. kif3a is dispensable for basal body docking in otic vesicle sensory epithelia and, surprisingly, short cilia form in mechanosensory cristae even in the absence of kif3a In contrast to Kif3a, the functions of the Kif3c-related protein, encoded by the kif3c-like (kif3cl) gene, and the homodimeric ciliary kinesin, kif17, are dispensable for photoreceptor morphogenesis. These studies demonstrate unexpected new roles for both ciliary heterotrimeric kinesins and IFT particle genes and clarify the function of kif17, the homodimeric ciliary kinesin gene.

The role of crumbs genes in the vertebrate cornea.

PURPOSE: To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea. METHODS: The authors used histologic analysis and electron microscopy to evaluate the corneas of zebrafish mutant for a crumbs locus oko meduzy (ome) and in mutants of four other loci, nagie oko (nok), heart and soul (has), mosaic eyes (moe), and ncad (formerly glass onion), that function in the same or related genetic pathways. In parallel, they performed an evaluation of corneas in human carriers of a crumbs gene, CRB1, and mutations using topography and biomicroscopy. The expression of the CRB1 gene in the normal human cornea was examined by polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: The corneas of zebrafish mutants display severe abnormalities of the epithelial and stromal layers. The epithelial cells do not properly adhere to each other, and fluid-filled spaces form between them. In addition, the layering of the corneal stroma is poorly formed or absent. The corneas of human carriers of CRB1 mutations display shape deviations compared with what has been observed in normal individuals. A PCR product of the correct size was obtained from normal human corneal samples. Sequence analyses confirmed its identity to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea. CONCLUSIONS: crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are very severe in the zebrafish model and, in comparison, appear clinically less pronounced in the human eye.

Small molecule screen for compounds that affect vascular development in the zebrafish retina.

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.

Spatiotemporal features of neurogenesis in the retina of medaka, Oryzias latipes.

The vertebrate retina is very well conserved in evolution. Its structure and functional features are very similar in phyla as different as primates and teleost fish. Here, we describe the spatiotemporal characteristics of neurogenesis in the retina of a teleost, medaka, and compare them with other species, primarily the zebrafish. Several intriguing differences are observed between medaka and zebrafish. For example, photoreceptor differentiation in the medaka retina starts independently in two different areas, and at more advanced stages of differentiation, medaka and zebrafish retinae display obviously different patterns of the photoreceptor cell mosaic. Medaka and zebrafish evolutionary lineages are thought to have separated from each other 110 million years ago, and so the differences between these species are not unexpected, and may be exploited to gain insight into the architecture of developmental pathways. Importantly, this work highlights the benefits of using multiple teleost models in parallel to understand a developmental process.

Mutations that affect the survival of selected amacrine cell subpopulations define a new class of genetic defects in the vertebrate retina.

Amacrine neurons are among the most diverse cell classes in the vertebrate retina. To gain insight into mechanisms vital to the production and survival of amacrine cell types, we investigated a group of mutations in three zebrafish loci: kleks (kle), chiorny (chy), and bergmann (bgm). Mutants of all three genes display a severe loss of selected amacrine cell subpopulations. The numbers of GABA-expressing amacrine interneurons are sharply reduced in all three mutants, while cell loss in other amacrine cell subpopulations varies and some cells are not affected at all. To investigate how amacrine cell loss affects retinal function, we performed electroretinograms on mutant animals. While the kle mutation mostly influences the function of the inner nuclear layer, unexpectedly the chy mutant phenotype also involves a loss of photoreceptor cell activity. The precise ration and arrangement of amacrine cell subpopulations suggest that cell-cell interactions are involved in the differentiation of this cell class. To test whether defects of such interactions may be, at least in part, responsible for mutant phenotypes, we performed mosaic analysis and demonstrated that the loss of parvalbumin-positive amacrine cells in chy mutants is due to extrinsic (cell-nonautonomous) causes. The phenotype of another amacrine cell subpopulation, the GABA-positive cells, does not display a clear cell-nonautonomy in chy animals. These results indicate that environmental factors, possibly interactions among different subpopulations of amacrine neurons, are involved in the development of the amacrine cell class.

The zebrafish eye: developmental and genetic analysis.

In this review, we have attempted to cover all the major points of zebrafish eye development, and have found that, for the most part, it has much in common with other eyes, in both vertebrates and the fly. In addition to the confirmation and extension of earlier studies, however, the work on zebrafish has provided some new insights that should be assessed for their applicability to the development of other vertebrates. Among these are the modulated cellular proliferation in the optic vesicle, the complex spatiotemporal pattern of central retinal neurogenesis, the emergence of spatial order among the photoreceptors, the genetic controls of cell fates, and the genetic mechanisms underlying retinal stratification. Substantial though it is, this contribution will grow rapidly in the next few years as the advances of zebrafish genetics are accelerated by progress of genomics, especially the zebrafish genome project.

High-throughput behavioral screening method for detecting auditory response defects in zebrafish.

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles.

Forward and reverse genetic approaches to the analysis of eye development in zebrafish.

The zebrafish has been established as a mainstream research system, largely due to the immense success of genetic screens. Over 2000 mutant alleles affecting zebrafish's early development have been isolated in two large-scale morphological screens and several smaller efforts. So far, over 50 mutant strains display retinal defects and many more have been shown to affect the retinotectal projection. More recently, mutant isolation and characterization have been successfully followed by candidate and positional cloning of underlying genes. To supplement forward genetic mutational analysis, several reverse genetic techniques have also been developed. These recent advances, combined with the genome project, have established the zebrafish as one of the leading models for studies of visual system development.