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

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

AbstractAmacrine 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 ratios 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 Role of crumbs Genes in the Vertebrate Cornea

crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are found in zebrafish mutants and human carriers of CRB1 gene mutations

The Role of crumbs Genes in the Vertebrate Cornea

crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are found in zebrafish mutants and human carriers of CRB1 gene mutations