Temporal control of Wnt signaling is required for habenular neuron diversity and brain asymmetry.

Precise temporal coordination of signaling processes is pivotal for cellular differentiation during embryonic development. A vast number of secreted molecules are produced and released by cells and tissues, and travel in the extracellular space. Whether they induce a signaling pathway and instruct cell fate, however, depends on a complex network of regulatory mechanisms, which are often not well understood. The conserved bilateral left-right asymmetrically formed habenulae of the zebrafish are an excellent model for investigating how signaling control facilitates the generation of defined neuronal populations. Wnt signaling is required for habenular neuron type specification, asymmetry and axonal connectivity. The temporal regulation of this pathway and the players involved have, however, have remained unclear. We find that tightly regulated temporal restriction of Wnt signaling activity in habenular precursor cells is crucial for the diversity and asymmetry of habenular neuron populations. We suggest a feedback mechanism whereby the tumor suppressor Wnt inhibitory factor Wif1 controls the Wnt dynamics in the environment of habenular precursor cells. This mechanism might be common to other cell types, including tumor cells.

Left/right asymmetric collective migration of parapineal cells is mediated by focal FGF signaling activity in leading cells.

The ability of cells to collectively interpret surrounding environmental signals underpins their capacity to coordinate their migration in various contexts, including embryonic development and cancer metastasis. One tractable model for studying collective migration is the parapineal, a left-sided group of neurons that arises from bilaterally positioned precursors that undergo a collective migration to the left side of the brain. In zebrafish, the migration of these cells requires Fgf8 and, in this study, we resolve how FGF signaling correlates with-and impacts the migratory dynamics of-the parapineal cell collective. The temporal and spatial dynamics of an FGF reporter transgene reveal that FGF signaling is activated in only few parapineal cells usually located at the leading edge of the parapineal during its migration. Overexpressing a constitutively active Fgf receptor compromises parapineal migration in wild-type embryos, while it partially restores both parapineal migration and mosaic expression of the FGF reporter transgene in fgf8-/- mutant embryos. Focal activation of FGF signaling in few parapineal cells is sufficient to promote the migration of the whole parapineal collective. Finally, we show that asymmetric Nodal signaling contributes to the restriction and leftwards bias of FGF pathway activation. Our data indicate that the first overt morphological asymmetry in the zebrafish brain is promoted by FGF pathway activation in cells that lead the collective migration of the parapineal to the left. This study shows that cell-state differences in FGF signaling in front versus rear cells is required to promote migration in a model of FGF-dependent collective migration.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses.

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

Biallelic IARS Mutations Cause Growth Retardation with Prenatal Onset, Intellectual Disability, Muscular Hypotonia, and Infantile Hepatopathy.

tRNA synthetase deficiencies are a growing group of genetic diseases associated with tissue-specific, mostly neurological, phenotypes. In cattle, cytosolic isoleucyl-tRNA synthetase (IARS) missense mutations cause hereditary weak calf syndrome. Exome sequencing in three unrelated individuals with severe prenatal-onset growth retardation, intellectual disability, and muscular hypotonia revealed biallelic mutations in IARS. Studies in yeast confirmed the pathogenicity of identified mutations. Two of the individuals had infantile hepatopathy with fibrosis and steatosis, leading in one to liver failure in the course of infections. Zinc deficiency was present in all affected individuals and supplementation with zinc showed a beneficial effect on growth in one.

Cutis laxa, exocrine pancreatic insufficiency and altered cellular metabolomics as additional symptoms in a new patient with ATP6AP1-CDG.

Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. >100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T>G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H+-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the ß-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.

The Risk Genes for Neuropsychiatric Disorders negr1 and opcml Are Expressed throughout Zebrafish Brain Development.

The immunoglobulin LAMP/OBCAM/NTM (IgLON) family of cell adhesion molecules comprises five members known for their involvement in establishing neural circuit connectivity, fine-tuning, and maintenance. Mutations in IgLON genes result in alterations in these processes and can lead to neuropsychiatric disorders. The two IgLON family members NEGR1 and OPCML share common links with several of them, such as schizophrenia, autism, and major depressive disorder. However, the onset and the underlying molecular mechanisms have remained largely unresolved, hampering progress in developing therapies. NEGR1 and OPCML are evolutionarily conserved in teleosts like the zebrafish (Danio rerio), which is excellently suited for disease modelling and large-scale screening for disease-ameliorating compounds. To explore the potential applicability of zebrafish for extending our knowledge on NEGR1- and OPCML-linked disorders and to develop new therapeutic strategies, we investigated the spatio-temporal expression of the two genes during early stages of development. negr1 and opcml are expressed maternally and subsequently in partially distinct domains of conserved brain regions. Other areas of expression in zebrafish have not been reported in mammals to date. Our results indicate that NEGR1 and OPCML may play roles in neural circuit development and function at stages earlier than previously anticipated. A detailed functional analysis of the two genes based on our findings could contribute to understanding the mechanistic basis of related psychiatric disorders.

"Vacuum-assisted staining": a simple and efficient method for screening in Drosophila.

The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

Conserved and diverged asymmetric gene expression in the brain of teleosts.

Morphological left-right brain asymmetries are universal phenomena in animals. These features have been studied for decades, but the functional relevance is often unclear. Studies from the zebrafish dorsal diencephalon on the genetics underlying the establishment and function of brain asymmetries have uncovered genes associated with the development of functional brain asymmetries. To gain further insights, comparative studies help to investigate the emergence of asymmetries and underlying genetics in connection to functional adaptation. Evolutionarily distant isogenic medaka inbred lines, that show divergence of complex traits such as morphology, physiology and behavior, are a valuable resource to investigate intra-species variations in a given trait of interest. For a detailed study of asymmetry in the medaka diencephalon we generated molecular probes of ten medaka genes that are expressed asymmetrically in the zebrafish habenulae and pineal complex. We find expression of eight genes in the corresponding brain areas of medaka with differences in the extent of left-right asymmetry compared to zebrafish. Our marker gene analysis of the diverged medaka inbred strains revealed marked inter-strain size differences of the respective expression domains in the parapineal and the habenulae, which we hypothesize may result from strain-specific gene loss. Thus, our analysis reveals both inter-species differences but also intra-species plasticity of gene expression in the teleost dorsal diencephalon. These findings are a starting point showing the potential to identify the genetics underlying the emergence and modulations of asymmetries. They are also the prerequisite to examine whether variance in habenular gene expression may cause variation of behavioral traits.

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries.

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/beta-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.

Zebrafish Tools for Deciphering Habenular Network-Linked Mental Disorders.

The prevalence of patients suffering from mental disorders is substantially increasing in recent years and represents a major burden to society. The underlying causes and neuronal circuits affected are complex and difficult to unravel. Frequent disorders such as depression, schizophrenia, autism, and bipolar disorder share links to the habenular neural circuit. This conserved neurotransmitter system relays cognitive information between different brain areas steering behaviors ranging from fear and anxiety to reward, sleep, and social behaviors. Advances in the field using the zebrafish model organism have uncovered major genetic mechanisms underlying the formation of the habenular neural circuit. Some of the identified genes involved in regulating Wnt/beta-catenin signaling have previously been suggested as risk genes of human mental disorders. Hence, these studies on habenular genetics contribute to a better understanding of brain diseases. We are here summarizing how the gained knowledge on the mechanisms underlying habenular neural circuit development can be used to introduce defined manipulations into the system to study the functional behavioral consequences. We further give an overview of existing behavior assays to address phenotypes related to mental disorders and critically discuss the power but also the limits of the zebrafish model for identifying suitable targets to develop therapies.

Expression of a Barhl1a reporter in subsets of retinal ganglion cells and commissural neurons of the developing zebrafish brain.

Promoting the regeneration or survival of retinal ganglion cells (RGCs) is one focus of regenerative medicine. Homeobox Barhl transcription factors might be instrumental in these processes. In mammals, only barhl2 is expressed in the retina and is required for both subtype identity acquisition of amacrine cells and for the survival of RGCs downstream of Atoh7, a transcription factor necessary for RGC genesis. The underlying mechanisms of this dual role of Barhl2 in mammals have remained elusive. Whole genome duplication in the teleost lineage generated the barhl1a and barhl2 paralogues. In the Zebrafish retina, Barhl2 functions as a determinant of subsets of amacrine cells lineally related to RGCs independently of Atoh7. In contrast, barhl1a expression depends on Atoh7 but its expression dynamics and function have not been studied. Here we describe for the first time a Barhl1a reporter line in vivo showing that barhl1a turns on exclusively in subsets of RGCs and their post-mitotic precursors. We also show transient expression of barhl1a:GFP in diencephalic neurons extending their axonal projections as part of the post-optic commissure, at the time of optic chiasm formation. This work sets the ground for future studies on RGC subtype identity, axonal projections and genetic specification of Barhl1a-positive RGCs and commissural neurons.

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos.

BACKGROUND: The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms. RESULTS: We describe zebrafish (Danio rerio) embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs), with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase) interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested. CONCLUSION: Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

Bioenergetic dysfunction in a zebrafish model of acute hyperammonemic decompensation.

Acute hyperammonemic encephalopathy is a life-threatening manifestation of individuals with urea cycle disorders, which is associated with high mortality rates and severe neurological sequelae in survivors. Cerebral bioenergetic failure has been proposed as one of the key mechanisms underlying hyperammonemia-induced brain damage, but data supporting this hypothesis remain inconclusive and partially contradictory. Using a previously established zebrafish model of acute hyperammonemic decompensation, we unraveled that acute hyperammonemia leads to a transamination-dependent withdrawal of 2-oxoglutarate (alpha-ketoglutarate) from the tricarboxylic acid (TCA) cycle with consecutive TCA cycle dysfunction, ultimately causing impaired oxidative phosphorylation with ATP shortage, decreased ATP/ADP-ratio and elevated lactate concentrations. Thus, our study supports and extends the hypothesis that cerebral bioenergetic dysfunction is an important pathophysiological hallmark of hyperammonemia-induced neurotoxicity.

QDPR homologues in Danio rerio regulate melanin synthesis, early gliogenesis, and glutamine homeostasis.

Dihydropteridine reductase (QDPR) catalyzes the recycling of tetrahydrobiopterin (BH4), a cofactor in dopamine, serotonin, and phenylalanine metabolism. QDPR-deficient patients develop neurological symptoms including hypokinesia, truncal hypotonia, intellectual disability and seizures. The underlying pathomechanisms are poorly understood. We established a zebrafish model for QDPR deficiency and analyzed the expression as well as function of all zebrafish QDPR homologues during embryonic development. The homologues qdpra is essential for pigmentation and phenylalanine metabolism. Qdprb1 is expressed in the proliferative zones of the optic tectum and eye. Knockdown of qdprb1 leads to up-regulation of pro-proliferative genes and increased number of phospho-histone3 positive mitotic cells. Expression of neuronal and astroglial marker genes is concomitantly decreased. Qdprb1 hypomorphic embryos develop microcephaly and reduced eye size indicating a role for qdprb1 in the transition from cell proliferation to differentiation. Glutamine accumulation biochemically accompanies the developmental changes. Our findings provide novel insights into the neuropathogenesis of QDPR deficiency.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division. Endoplasmic reticulum (ER)-localized ADP-dependent glucokinase (ADPGK) has been shown to play a critical role in T cell receptor activation-induced remodeling of energy metabolism, however the underlying mechanisms remain unclear. Therefore, we established and characterized in vitro and in vivo models for ADPGK-deficiency using Jurkat T cells and zebrafish. Upon activation, ADPGK knockout Jurkat T cells displayed increased cell death and ER stress. The increase in cell death resulted from a metabolic catastrophe and knockout cells displayed severely disturbed energy metabolism hindering induction of Warburg phenotype. ADPGK knockdown in zebrafish embryos led to short, dorsalized body axis induced by elevated apoptosis. ADPGK hypomorphic zebrafish further displayed dysfunctional glucose metabolism. In both model systems loss of ADPGK function led to defective N- and O-glycosylation. Overall, our data illustrate that ADPGK is part of a glucose sensing system in the ER modulating metabolism via regulation of N- and O-glycosylation.

An Eye on the Wnt Inhibitory Factor Wif1.

The coordinated interplay between extrinsic activating and repressing cell signaling molecules is pivotal for embryonic development and subsequent tissue homeostasis. This is well exemplified by studies on the evolutionarily conserved Wnt signaling pathways. Tight temporal and spatial regulation of Wnt signaling activity is required throughout lifetime, from maternal stages before gastrulation until and throughout adulthood. Outside cells, the action of numerous Wnt ligands is counteracted and fine-tuned by only a handful of well characterized secreted inhibitors, such as for instance Dickkopf, secreted Frizzled Related Proteins and Cerberus. Here, we give an overview of our current understanding of another secreted Wnt signaling antagonist, the Wnt inhibitory factor Wif1. Wif1 can directly interact with various Wnt ligands and inhibits their binding to membrane bound receptors. Epigenetic promoter methylation of Wif1, leading to silencing of its transcription and concomitant up-regulation of Wnt signaling, is a common feature during cancer progression. Furthermore, an increasing number of reports describe Wif1 involvement in regulating processes during embryonic development, which so far has not received as much attention. We will summarize our knowledge on Wif1 function and its mode of action with a particular focus on the zebrafish (Danio rerio). In addition, we highlight the potential of Wif1 research to understand and possibly influence mechanisms underlying eye diseases and regeneration.

Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase.

Hyperammonemia is the common biochemical hallmark of urea cycle disorders, activating neurotoxic pathways. If untreated, affected individuals have a high risk of irreversible brain damage and mortality. Here we show that acute hyperammonemia strongly enhances transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death in ammoniotelic zebrafish larvae via synergistically acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate. Intriguingly, specific and irreversible inhibition of ornithine aminotransferase (OAT) by 5-fluoromethylornithine rescues zebrafish from lethal concentrations of ammonium acetate and corrects hyperammonemia-induced biochemical alterations. Thus, OAT inhibition is a promising and effective therapeutic approach for preventing neurotoxicity and mortality in acute hyperammonemia.

Expression of marker genes during early ear development in medaka.

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.

Prediction of volume response under open-chest conditions during coronary artery bypass surgery.

INTRODUCTION: Adequate fluid loading is the first step of hemodynamic optimization in cardiac patients undergoing surgery. Neither a clinical approach alone nor conventional parameters like central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP) are thought to be sufficient for recognizing fluid deficiency or overload. The aim of this study was to evaluate the suitability of CVP, PCWP, global end-diastolic volume index (GEDVI), pulse pressure variation (PPV), and stroke volume variation (SVV) for predicting changes in the cardiac index (CI) and stroke volume index (SVI) after sternotomy. METHODS: In 40 patients, CVP, PCWP, GEDVI, PPV, SVV, and the CI were measured at two points of time. One measurement was performed after inducing anesthesia and one after sternotomy. RESULTS: A significant increase in heart rate, CI, and GEDVI was observed during the study period. CVP, SVV, and PPV decreased significantly. There were no significant correlations between CVP and PCWP and changes in CI. In contrast, GEDVI, SVV, and PPV significantly correlated with CI changes. Only relative changes of GEDVI, SVV, and PPV predicted changes in SVI. CONCLUSION: During cardiac surgery and especially after sternotomy, CVP and PCWP are not suitable for monitoring fluid status. Direct volume measurement like GEDVI and dynamic volume responsive measurements like SVV and PPV may be more suitable for monitoring the volume status of patients, particularly under open-chest conditions.

The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages.

Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers.