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

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]

Genetic defects of GDF6 in the zebrafish out of sight mutant and in human eye developmental anomalies

Contains fulltext : 88522.pdf (publisher's version ) (Open Access)BACKGROUND: 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. RESULTS: In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed. CONCLUSIONS: This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype

Kinesin-2 family motors in the unusual photoreceptor cilium

This review focuses on recent advances in the understanding of kinesin-2 family motors in vertebrate photoreceptor development. Zebrafish photoreceptors develop by the 3rd day of embryogenesis, making it possible to study mutant phenotypes without the use of conditional alleles. Recent work using a zebrafish kif3b mutant allele validates the concept that the heterotrimeric kinesin II motor is generally required for ciliogenesis. In zebrafish photoreceptors, however, loss of kif3b function delays but does not block cilium formation. This is thought to occur because both kif3b or kif3c can dimerize with kif3a and function redundantly. The second ciliary kinesin thought to function in photoreceptor cells is kif17. Prior work has shown that either morpholino knockdown of this gene or the overexpression of its dominant negative form can reduce or delay photoreceptor cilium development without any evident impact on ciliogenesis in general. This has led to the idea that kif17 may play an important role only in some specialized cilium types, such the one in photoreceptor cells. In a recently identified kif17 mutant, however, photoreceptor outer segments are formed by 5 dpf and an obvious delay of outer segment formation is seen only at the earliest stage analyzed (3 dpf). This work suggests that kif17 plays a significant role mainly at an early stage of photoreceptor development. Taken together, these studies lead to an intriguing concept that as they differentiate photoreceptors alter their kinesin repertoire

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

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

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

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar transport

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the 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 the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling 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 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis

oko meduzy and Related crumbs Genes Are Determinants of Apical Cell Features in the Vertebrate Embryo

SummaryBackgroundPolarity 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.ResultsWe show that zebrafish oko meduzy (ome) locus encodes a crumbs gene homolog, essential for the proper apico-basal polarity of neural tube epithelia. Two ome paralogs, crb2b and crb3a, promote the formation of apical cell features: photoreceptor inner segments and cilia in renal and auditory systems. The motility of cilia is defective following the impairment of crb2b function. Apical surface defects in ome- and crb2b-deficient animals are associated with profound disorganization of neuronal architecture and with the formation of pronephric cysts, respectively. Unexpectedly, despite differences in their structure and expression patterns, crumbs genes are, at least partially, functionally interchangeable.Conclusionsome and related crumbs genes are necessary for the formation of gross morphological features in several organs, including the CNS and the renal system. On the cellular level, crumbs genes regulate the formation of both ciliary and nonciliary apical membrane compartment

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