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In JoVE (1)

Other Publications (37)

Articles by Jarema Malicki in JoVE

 JoVE Biology

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

1The Second Teaching Hospital of Jilin University, 2Department of Ophthalmology, Harvard Medical School


JoVE 1258

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Other articles by Jarema Malicki on PubMed

Genetic Analysis of Photoreceptor Cell Development in the Zebrafish Retina

To gain insight into the genetic mechanisms of photoreceptor development, we analyzed a collection of zebrafish mutations characterized by early photoreceptor cell loss. The mutant defects impair outer segment formation and are accompanied by an abnormal distribution of visual pigments. Rods and different cone types display defects of similar severity suggesting that genetic pathways common to all photoreceptors are affected. To investigate whether these phenotypes involve cell-cell interaction defects, we analyzed genetically mosaic animals. Interaction of niezerka photoreceptors with wild-type tissues improves the survival of mutant cells and restores their elongated morphology. In contrast, cells carrying mutations in the loci brudas, elipsa, fleer, and oval retain their defective phenotypes in a wild-type environment indicating cell-autonomy. These experiments identify distinct phenotypic categories of photoreceptor mutants and indicate that zebrafish photoreceptor defects involve both cell-autonomous and cell-nonautonomous mechanisms.

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.

Nagie Oko, Encoding a MAGUK-family Protein, is Essential for Cellular Patterning of the Retina

A layered organization of cells is a common architectural feature of many neuronal formations. Mutations of the zebrafish gene nagie oko (nok) produce a severe disruption of retinal architecture, indicating a key role for this locus in neuronal patterning. We show that nok encodes a membrane-associated guanylate kinase-family scaffolding protein. Nok localizes to the vicinity of junctional complexes in retinal neuroepithelium and in the photoreceptor cell layer. Mosaic analysis indicates that the nok retinal patterning phenotype is not cell-autonomous. We propose that nok function in patterning of postmitotic neurons is mediated through neuroepithelial cells and is necessary for guiding neurons to their proper destinations in retinal laminae.

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.

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.

Analysis of Gene Function in the Zebrafish Retina

Mutagenesis screens in zebrafish have uncovered several hundred mutant alleles affecting the development of the retina and established the zebrafish as one of the leading models of vertebrate eye development. In addition to forward genetic mutagenesis approaches, gene function in the zebrafish embryo is being studied using several reverse genetic techniques. Some of these rely on the overexpression of a gene product, others take advantage of antisense oligonucleotides to block function of selected loci. Here we describe these methods in the context of the developing eye.

Zebrafish N-cadherin, Encoded by the Glass Onion Locus, Plays an Essential Role in Retinal Patterning

Genetic screens in zebrafish identified several loci that play essential roles in the patterning of retinal architecture. Here, we show that one of them, glass onion, encodes the N-cadherin gene. The glo(m117) mutant allele contains a substitution of the Trp2 residue known for its essential role in the adhesive properties of classic cadherins. Both the glo(m117) and pac(tm101b) mutant N-cadherin alleles affect the polarity of the retinal neuroepithelial sheet and, unexpectedly, both result in cell-nonautonomous phenotypes in retinal patterning. The late onset of mutant N-cadherin phenotypes may be due to the ability of classic cadherins to substitute each other's function.

Cell Fate Decisions and Patterning in the Vertebrate Retina: the Importance of Timing, Asymmetry, Polarity and Waves

The differentiation of distinct cell populations in the retina is a multi-step process that involves cell cycle exit, migration, and dramatic changes of cell morphology. All these steps are tightly controlled by multiple regulatory pathways, which involve both cell-autonomous networks of transcription factors and cell-cell signaling events. Additional regulatory inputs into cell fate decisions have been recently suggested: accumulating evidence shows that the timing of cell cycle exit, the orientation of the mitotic spindle during the last cell division, and the polarity of neuronal progenitor cells could play important roles in cell fate determination.

Retinal Pattern and the Genetic Basis of Its Formation in Zebrafish

The vertebrate nervous system contains an immense diversity of distinct cellular components that are organized into precise spatial patterns. The importance of accurate neuronal architecture is particularly obvious in the retina, where it is necessary for the formation of visual images. The retina is structured in a distinct layered pattern that is remarkably conserved in evolution, including phyla as diverse as primates and teleost fish. Genetic analysis in zebrafish reveals mechanisms that are essential for the formation of this architecture.

Intraflagellar Transport Genes Are Essential for Differentiation and Survival of Vertebrate Sensory Neurons

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.

Genetics of Photoreceptor Development and Function in Zebrafish

The vertebrate photoreceptor is a cell of unique morphology and function. It is an exquisite light detector, both sensitive and adaptable. Several unusual morphological features facilitate photoreceptor function. Signal detection is accomplished by a specialized apical structure, the outer segment. There, the capture of light produces fluctuations in cell membrane potential, which are then transmitted to the downstream circuitry of the retina via a rare type of synaptic junction, the ribbon synapse. The development, maintenance and function of the vertebrate photoreceptor cell have been studied mainly in four model organisms, ranging from an amphibian to man. A teleost fish, the zebrafish, is an important recent addition to this group. Genetic screens in zebrafish have identified an impressive collection of photoreceptor cell mutants, including the absence or malformation of specific morphological features as well as functional abnormalities. These mutant strains are currently studied using both molecular and embryological tools and provide important insights into photoreceptor biology.

Approaches to Study Neurogenesis in the Zebrafish Retina

Similar to other vertebrate species, the zebrafish retina is simpler than other regions of the central nervous system (CNS). Relative simplicity, rapid development, and accessibility to genetic analysis make the zebrafish retina an excellent model system for the studies of neurogenesis in the vertebrate CNS. Numerous genetic screens have led to isolation of an impressive collection of mutations affecting the retina and the retinotectal projection in zebrafish. Mutant phenotypes are being studied using a rich variety of markers: antibodies, RNA probes, retrograde and anterograde tracers, as well as transgenic lines. Particularly impressive progress has been made in the characterization of the zebrafish genome. Consequently, positional and candidate cloning of mutant genes are now fairly easy to accomplish in zebrafish. Many mutant genes have, in fact, already been cloned and their analysis has provided important insights into the gene circuitry that regulates retinal neurogenesis. Genetic screens for visual system defects will continue in the future and progressively more sophisticated screening approaches will make it possible to detect a variety of subtle mutant phenotypes in retinal development. The remarkable evolutionary conservation of the vertebrate eye provides the basis for the use of the zebrafish retina as a model of human disorders. Some of the genetic defects of the zebrafish retina indeed resemble human retinopathies. As new techniques are being introduced and improved at a rapid pace, the zebrafish will continue to be an important organism for the studies of the vertebrate visual system.

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.

Genomic Organization of Zebrafish Cone-rod Homeobox Gene and Exclusion As a Candidate Gene for Retinal Degeneration in Niezerka and Mikre Oko

To determine the genomic organization of the zebrafish crx gene and to evaluate if mutations in crx are responsible for the retinal degeneration phenotype in the zebrafish (Danio rerio) mutants niezerka (nie(m743)) and mikre oko (mok(m632)).

Reverse Genetic Analysis of Neurogenesis in the Zebrafish Retina

To gain an understanding of molecular events that underlie pattern formation in the retina, we evaluated the expression profiles of over 8000 transcripts randomly selected from an embryonic zebrafish library. Detailed analysis of cDNAs that display restricted expression patterns revealed factors that are specifically expressed in single cell classes and are potential regulators of neurogenesis. These cDNAs belong to numerous molecular categories and include cell surface receptors, cytoplasmic enzymes, and transcription factors. To test whether expression patterns that we have uncovered using this approach are indicative of function in neurogenesis, we used morpholino-mediated knockdown approach. The knockdown of soxp, a transcript expressed in the vicinity of the inner plexiform layer, revealed its role in cell type composition of amacrine and ganglion cell layers. Blocking the function of cxcr4b, a chemokine receptor specifically expressed in ganglion cells, suggests a role in ganglion cell survival. These experiments demonstrate that in situ hybridization-based reverse genetic screens can be applied to isolate genetic regulators of neurogenesis. This approach very well complements forward genetic mutagenesis studies previously used to study retinal neurogenesis in zebrafish.

Oko Meduzy and Related Crumbs Genes Are Determinants of Apical Cell Features in the Vertebrate Embryo

Polarity is an essential attribute of most eukaryotic cells. One of the most prominent features of cell polarity in many tissues is the subdivision of cell membrane into apical and basolateral compartments by a belt of cell junctions. The proper formation of this subdivision is of key importance. In sensory cells, for example, the apical membrane compartment differentiates specialized structures responsible for the detection of visual, auditory, and olfactory stimuli. In other tissues, apical specializations are responsible for the propagation of fluid flow. Despite its importance, the role of genetic determinants of apico-basal polarity in vertebrate embryogenesis remains poorly investigated.

The Zebrafish Cornea: Structure and Development

To evaluate the zebrafish as a model for the studies of corneal development and disease.

A Screen for Genetic Defects of the Zebrafish Ear

To advance the understanding of genetic mechanisms involved in the patterning and the differentiation of the vertebrate auditory system, we screened for mutations affecting ear development in the zebrafish larva. Fifteen recessive mutant alleles have been isolated and analyzed. The phenotypes of these mutants involve abnormalities in ear morphology, otolith formation, or both processes in parallel. Among morphological defects, we found mutations affecting early patterning of the otic vesicle, the morphogenesis of semicircular canals, and the expansion of the ear lumen. The two most severe mutant phenotypes involve the absence of anterior and posterior cristae, as well as a severely misshapen morphology of the ear. In the category of otolith mutants, we found defects in otolith formation, growth, and shape. As it proved to be the case in past screening efforts of this type, these mutant lines represent an asset in the studies of molecular mechanisms that regulate vertebrate ear development.

Genetic Defects of Pronephric Cilia in Zebrafish

Cilia play key roles in many aspects of embryogenesis and adult physiology in vertebrates. Past genetic screens in zebrafish identified numerous defects of ciliogenesis, including several mutations in the components of the intraflagellar transport machinery. In contrast to previous studies, here we describe a collection of mutants that affect subpopulations of cilia. Mutant embryos are characterized by a shortening and an abnormal movement of kidney cilia, and in one case also a reduction of cilia length in the Kupffer's vesicle. In contrast to that, the cilia of sensory neurons, including photoreceptor cells, hair cells, and olfactory sensory cells, appear grossly intact. Motility defects of pronephric cilia vary in mutant strains from complete paralysis to an increased frequency of movement, and are associated with left-right asymmetry defects. While ciliary ultrastructure is normal in most mutants, one of the mutant loci is essential for the formation of proper microtubule architecture in the axoneme of pronephric cilia. Mutants characterized in this study reveal intriguing genetic differences between subpopulations of embryonic cilia, and provide an opportunity to study several aspects of cilia structure and function.

Detection of Blob Objects in Microscopic Zebrafish Images Based on Gradient Vector Diffusion

The zebrafish has become an important vertebrate animal model for the study of developmental biology, functional genomics, and disease mechanisms. It is also being used for drug discovery. Computerized detection of blob objects has been one of the important tasks in quantitative phenotyping of zebrafish. We present a new automated method that is able to detect blob objects, such as nuclei or cells in microscopic zebrafish images. This method is composed of three key steps. The first step is to produce a diffused gradient vector field by a physical elastic deformable model. In the second step, the flux image is computed on the diffused gradient vector field. The third step performs thresholding and nonmaximum suppression based on the flux image. We report the validation and experimental results of this method using zebrafish image datasets from three independent research labs. Both sensitivity and specificity of this method are over 90%. This method is able to differentiate closely juxtaposed or connected blob objects, with high sensitivity and specificity in different situations. It is characterized by a good, consistent performance in blob object detection.

Mechanism of Positioning the Cell Nucleus in Vertebrate Photoreceptors

Organelles are frequently distributed in a nonrandom manner in a cell's cytoplasm. A particular distribution pattern often facilitates a specific function of a cell, whereas its aberrations can lead to cell death. We show that a mutation in the zebrafish mikre oko (mok) locus, which encodes dynactin 1 subunit of the dynactin complex, produces a severe displacement of the photoreceptor cell nucleus toward the synaptic terminus. Interference with the function of other dynein complex constituents, including p50/dynamitin, the Lis1 polypeptide, and the disruption of a nuclear envelope component of the syne gene family in vertebrate photoreceptors also result in the mispositioning of nuclei. Although the overall photoreceptor polarity is not affected, this phenotype is accompanied by a misdistribution of the Bardet-Biedl syndrome 4 polypeptide and a decreased photoreceptor survival. These findings reveal an important mechanism that regulates nuclear position in vertebrate neurons.

An Automated Method for Cell Detection in Zebrafish

Quantification of cells is a critical step towards the assessment of cell fate in neurological disease or developmental models. Here, we present a novel cell detection method for the automatic quantification of zebrafish neuronal cells, including primary motor neurons, Rohon-Beard neurons, and retinal cells. Our method consists of four steps. First, a diffused gradient vector field is produced. Subsequently, the orientations and magnitude information of diffused gradients are accumulated, and a response image is computed. In the third step, we perform non-maximum suppression on the response image and identify the detection candidates. In the fourth and final step the detected objects are grouped into clusters based on their color information. Using five different datasets depicting zebrafish cells, we show that our method consistently displays high sensitivity and specificity of over 95%. Our results demonstrate the general applicability of this method to different data samples, including nuclear staining, immunohistochemistry, and cell death detection.

Elipsa is an Early Determinant of Ciliogenesis That Links the IFT Particle to Membrane-associated Small GTPase Rab8

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.

Drosophila Asterless and Vertebrate Cep152 Are Orthologs Essential for Centriole Duplication

The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl was previously reported to be a centrosomal protein essential for centrosome function. Here we identify mecD, a severe loss-of-function allele of the asl gene, and demonstrate that it is required for centriole and cilia formation. Similarly, Cep152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, we found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication.

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.

CRB1 Gene Mutations Are Associated with Keratoconus in Patients with Leber Congenital Amaurosis

To present an association of mutations in the CRB1 gene with keratoconus in patients with Leber congenital amaurosis (LCA).

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.

What Drives Cell Morphogenesis: a Look Inside the Vertebrate Photoreceptor

Vision mediating photoreceptor cells are specialized light-sensitive neurons in the outer layer of the vertebrate retina. The human retina contains approximately 130 million of such photoreceptors, which enable images of the external environment to be captured at high resolution and high sensitivity. Rod and cone photoreceptor subtypes are further specialized for sensing light in low and high illumination, respectively. To enable visual function, these photoreceptors have developed elaborate morphological domains for the detection of light (outer segments), for changing cell shape (inner segments), and for communication with neighboring retinal neurons (synaptic terminals). Furthermore, rod and cone subtypes feature unique morphological variations of these specialized characteristics. Here, we review the major aspects of vertebrate photoreceptor morphology and key genetic mechanisms that drive their formation. These mechanisms are necessary for cell differentiation as well as function. Their defects lead to cell death.

Zebrafish Ale Oko, an Essential Determinant of Sensory Neuron Survival and the Polarity of Retinal Radial Glia, Encodes the P50 Subunit of Dynactin

Although microtubule-dependent motors are known to play many essential functions in eukaryotic cells, their role in the context of the developing vertebrate embryo is less well understood. Here we show that the zebrafish ale oko (ako) locus encodes the p50 component of the dynactin complex. Loss of ako function results in a degeneration of photoreceptors and mechanosensory hair cells. Additionally, mutant Müller cells lose apical processes and their perikarya translocate rapidly towards the vitreal surface of the retina. This is accompanied by the accumulation of the apical determinants Nok and Has/aPKC in their cell bodies. ako is required cell-autonomously for the maintenance of the apical process but not for cell body positioning in Müller glia. At later stages, the retinotectal projection also degenerates in ako mutants. These results indicate that the p50 component of the dynactin complex is essential for the survival of sensory neurons and the maintenance of ganglion cell axons, and functions as a major determinant of apicobasal polarity in retinal radial glia.

The Apical Complex Couples Cell Fate and Cell Survival to Cerebral Cortical Development

Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling.

A Male with Unilateral Microphthalmia Reveals a Role for TMX3 in Eye Development

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

The Role of Crumbs Genes in the Vertebrate Cornea

To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea.

Genetic Defects of GDF6 in the Zebrafish out of Sight Mutant and in Human Eye Developmental Anomalies

The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Analysis of the Retina in the Zebrafish Model

The zebrafish is one of the leading models for the analysis of the vertebrate visual system. A wide assortment of molecular, genetic, and cell biological approaches is available to study zebrafish visual system development and function. As new techniques become available, genetic analysis and imaging continue to be the strengths of the zebrafish model. In particular, recent developments in the use of transposons and zinc finger nucleases to produce new generations of mutant strains enhance both forward and reverse genetic analysis. Similarly, the imaging of developmental and physiological processes benefits from a wide assortment of fluorescent proteins and the ways to express them in the embryo. The zebrafish is also highly attractive for high-throughput screening of small molecules, a promising strategy to search for compounds with therapeutic potential. Here we discuss experimental approaches used in the zebrafish model to study morphogenetic transformations, cell fate decisions, and the differentiation of fine morphological features that ultimately lead to the formation of the functional vertebrate visual system.

Analysis of Cilia Structure and Function 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.

Nephrocystins and MKS Proteins Interact with IFT Particle and Facilitate Transport of Selected Ciliary Cargos

Cilia are required for the development and function of many organs. Efficient transport of protein cargo along ciliary axoneme is necessary to sustain these processes. Despite its importance, the mode of interaction between the intraflagellar ciliary transport (IFT) mechanism and its cargo proteins remains poorly understood. Our studies demonstrate that IFT particle components, and a Meckel-Gruber syndrome 1 (MKS1)-related, B9 domain protein, B9d2, bind each other and contribute to the ciliary localization of Inversin (Nephrocystin 2). B9d2, Inversin, and Nephrocystin 5 support, in turn, the transport of a cargo protein, Opsin, but not another photoreceptor ciliary transmembrane protein, Peripherin. Interestingly, the components of this mechanism also contribute to the formation of planar cell polarity in mechanosensory epithelia. These studies reveal a molecular mechanism that mediates the transport of selected ciliary cargos and is of fundamental importance for the differentiation and survival of sensory cells.

Kinesin-2 Family in Vertebrate Ciliogenesis

The differentiation of cilia is mediated by kinesin-driven transport. As the function of kinesins in vertebrate ciliogenesis is poorly characterized, we decided to determine the role of kinesin-2 family motors-heterotrimeric kinesin-II and the homodimeric Kif17 kinesin-in zebrafish cilia. We report that kif17 is largely dispensable for ciliogenesis; kif17 homozygous mutant animals are viable and display subtle morphological defects of olfactory cilia only. In contrast to that, the kif3b gene, encoding a heterotrimeric kinesin subunit, is necessary for cilia differentiation in most tissues, although exceptions exist, and include photoreceptors and a subset of hair cells. Cilia of these cell types persist even in kif3b/kif17 double mutants. Although we have not observed a functional redundancy of kif3b and kif17, kif17 is able to substitute for kif3b in some cilia. In contrast to kif3b/kif17 double mutants, simultaneous interference with kif3b and kif3c leads to the complete loss of photoreceptor and hair cell cilia, revealing redundancy of function. This is in agreement with the idea that Kif3b and Kif3c motor subunits form complexes with Kif3a, but not with each other. Interestingly, kif3b mutant photoreceptor cilia differentiate with a delay, suggesting that kif3c, although redundant with kif3b at later stages of differentiation, is not active early in photoreceptor ciliogenesis. Consistent with that, the overexpression of kif3c in kif3b mutants rescues early photoreceptor cilia defects. These data reveal unexpected diversity of functional relationships between vertebrate ciliary kinesins, and show that the repertoire of kinesin motors changes in some cilia during their differentiation.

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