Jennifer O. Liang

Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling.

Zebrafish cyclops (cyc) mutations cause deficiencies in the dorsal mesendoderm, and ventral neural tube,, leading to neural defects and cyclopia,. Here we report that cyc encodes a transforming growth factor-β (TGF-β)-related intercellular signalling molecule that is similar to mouse nodal. cyc is expressed in dorsal mesendoderm at gastrulation and in the prechordal plate until early somitogenesis. Expression reappears transiently in the left lateral-plate mesoderm, and in an unprecedented asymmetric pattern in the left forebrain. Injection of cyc RNA non-autonomously restores sonic hedgehog -expressing cells of the ventral brain and floorplate that are absent in cyc mutants, whereas inducing activities are abolished by cycm294, a mutation of a conserved cysteine in the mature ligand. Our results indicate that cyc provides an essential non-cell-autonomous signal at gastrulation, leading to induction of the floorplate and ventral brain.

Otx5 regulates genes that show circadian expression in the zebrafish pineal complex

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus—the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.

Leaning to the left: laterality in the zebrafish forebrain

How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left-right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left-right biases are established and propagated in the developing vertebrate brain.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System

Background: Klotho is an age suppressor protein whose brain function is unknown. Results: Klotho protects hippocampal neurons from glutamate and amyloid β-induced oxidative damage through the induction of the thioredoxin/peroxiredoxin system. Conclusion: Klotho is neuroprotective via the regulation of the redox system. Significance: Understanding the mechanism underlying Klotho-induced neuroprotection may lead to the development of novel therapeutic approaches against neurodegeneration. Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.

Comparative Activity of ADP-ribosylation Factor Family Members in the Early Steps of Coated Vesicle Formation on Rat Liver Golgi Membranes

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPγS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 μM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling

In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium-containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor-related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.

Amyloid precursor protein is required for convergent-extension movements during Zebrafish development.

Amyloid precursor protein (APP) has been a focus of intense investigation because of its role in Alzheimers disease (AD), however, its biological function remains uncertain. Loss of APP and APP-like proteins results in postnatal lethality in mice, suggesting a role during embryogenesis. Here we show that in a zebrafish model system, knock down of APP results in the generation of fish with dramatically reduced body length and a short, curly tail. In situ examination of gene expression suggests that the APP morphant embryos have defective convergent-extension movements. We also show that wild-type human APP rescues the morphant phenotype, but the Swedish mutant APP, which causes familial AD (fAD), does not rescue the developmental defects. Collectively, this work demonstrates that the zebrafish model is a powerful system to define the role of APP during embryonic development and to evaluate the functional activity of fAD mutant APP.

Novel functions for Period 3 and Exo-rhodopsin in rhythmic transcription and melatonin biosynthesis within the zebrafish pineal organ

Entrainment of circadian clocks to environmental cues such as photoperiod ensures that daily biological rhythms stay in synchronization with the Earths rotation. The vertebrate pineal organ has a conserved role in circadian regulation as the primary source of the nocturnal hormone melatonin. In lower vertebrates, the pineal has an endogenous circadian clock as well as photoreceptive cells that regulate this clock. The zebrafish opsin protein Exo-rhodopsin (Exorh) is expressed in pineal photoreceptors and is a candidate to mediate the effects of environmental light on pineal rhythms and melatonin synthesis. We demonstrate that Exorh has an important role in regulating gene transcription within the pineal. In developing embryos that lack Exorh, expression of the exorh gene itself and of the melatonin synthesis gene serotonin N-acetyl transferase 2 (aanat2) are significantly reduced. This suggests that the Exorh protein at the cell membrane is part of a signaling pathway that positively regulates transcription of these genes, and ultimately melatonin production, in the pineal. Like many other opsin genes, exorh is expressed with a daily rhythm: mRNA levels are higher at night than during the day. We found that the transcription factor Orthodenticle homeobox 5 (Otx5) activates exorh transcription, while the putative circadian clock component Period 3 (Per3) represses expression during the day, thereby contributing to the rhythm of transcription. This work identifies novel roles for Exorh and Per3, and gives insight into potential interactions between the sensory and circadian systems within the pineal.

Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain

BackgroundThe mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc) mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression.ResultsWe find that pineal rhythms are present in cyc mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin) is a peptide hormone expressed in and around the SCN. We find avpl mRNA is absent in cyc mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, cryptochrome 1b (cry1b) and cryptochrome 3 (cry3), in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, exo-rhodopsin (exorh) and serotonin-N-acetyltransferase (aanat2), involved in photoreception and melatonin synthesis, respectively, is also similar between cyc embryos and their wildtype (WT) siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in cyc embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in cyc mutants kept in constant conditions decreased more quickly than in their WT siblings.ConclusionOur data suggests that circadian rhythms can be initiated and maintained in the absence of SCN and other tissues in the ventral brain. However, the SCN may have a role in regulating the amplitude of rhythms when environmental cues are absent. This provides some of the first evidence that the SCN of teleosts is not essential for establishing circadian rhythms during development. Several SCN-independent circadian rhythms have also been found in mammalian species. Thus, zebrafish may serve as a model system for understanding how vertebrate embryos coordinate rhythms that are controlled by different circadian clocks.

Identification of cleavage sites leading to the shed form of the anti-aging protein klotho.

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50–60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70–80%. Deletion of amino acids 954–962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.