ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.

Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development.

Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.

Myofibrillar myopathy hallmarks associated with ZAK deficiency.

The ZAK gene encodes two functionally distinct kinases, ZAKα and ZAKß. Homozygous loss of function mutations affecting both isoforms causes a congenital muscle disease. ZAKß is the only isoform expressed in skeletal muscle and is activated by muscle contraction and cellular compression. The ZAKß substrates in skeletal muscle or the mechanism whereby ZAKß senses mechanical stress remains to be determined. To gain insights into the pathogenic mechanism, we exploited ZAK-deficient cell lines, zebrafish, mice and a human biopsy. ZAK-deficient mice and zebrafish show a mild phenotype. In mice, comparative histopathology data from regeneration, overloading, ageing and sex conditions indicate that while age and activity are drivers of the pathology, ZAKß appears to have a marginal role in myoblast fusion in vitro or muscle regeneration in vivo. The presence of SYNPO2, BAG3 and Filamin C (FLNC) in a phosphoproteomics assay and extended analyses suggested a role for ZAKß in the turnover of FLNC. Immunofluorescence analysis of muscle sections from mice and a human biopsy showed evidence of FLNC and BAG3 accumulations as well as other myofibrillar myopathy markers. Moreover, endogenous overloading of skeletal muscle exacerbated the presence of fibres with FLNC accumulations in mice, indicating that ZAKß signalling is necessary for an adaptive turnover of FLNC that allows for the normal physiological response to sustained mechanical stress. We suggest that accumulation of mislocalized FLNC and BAG3 in highly immunoreactive fibres contributes to the pathogenic mechanism of ZAK deficiency.

Machine learning discriminates a movement disorder in a zebrafish model of Parkinson's disease.

Animal models of human disease provide an in vivo system that can reveal molecular mechanisms by which mutations cause pathology, and, moreover, have the potential to provide a valuable tool for drug development. Here, we have developed a zebrafish model of Parkinson's disease (PD) together with a novel method to screen for movement disorders in adult fish, pioneering a more efficient drug-testing route. Mutation of the PARK7 gene (which encodes DJ-1) is known to cause monogenic autosomal recessive PD in humans, and, using CRISPR/Cas9 gene editing, we generated a Dj-1 loss-of-function zebrafish with molecular hallmarks of PD. To establish whether there is a human-relevant parkinsonian phenotype in our model, we adapted proven tools used to diagnose PD in clinics and developed a novel and unbiased computational method to classify movement disorders in adult zebrafish. Using high-resolution video capture and machine learning, we extracted novel features of movement from continuous data streams and used an evolutionary algorithm to classify parkinsonian fish. This method will be widely applicable for assessing zebrafish models of human motor diseases and provide a valuable asset for the therapeutics pipeline. In addition, interrogation of RNA-seq data indicate metabolic reprogramming of brains in the absence of Dj-1, adding to growing evidence that disruption of bioenergetics is a key feature of neurodegeneration.This article has an associated First Person interview with the first author of the paper.

An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos.

Myogenic regulatory factors (MRFs) are known to have essential roles in both the establishment and differentiation of the skeletal muscle cell lineage. MyoD is expressed early in the Xenopus mesoderm where it is present and active several hours before the activation of muscle differentiation genes. Previous studies in cultured cells and in Xenopus laevis have identified sets of genes that require MyoD prior to differentiation of skeletal muscle. Here we report results from experiments using CRISPR/Cas9 to target the MyoD gene in the diploid frog Xenopus tropicalis, that are analysed by RNA-seq at gastrula stages. We further investigate our data using cluster analysis to compare developmental expression profiles with that of MyoD and α-cardiac actin, reference genes for skeletal muscle determination and differentiation. Our findings provide an assessment of using founder (F0) Xenopus embryos from CRISPR/Cas9 protocols for transcriptomic analyses and we conclude that although targeted F0 embryos are genetically mosaic for MyoD, there is significant disruption in the expression of a specific set of genes. We discuss candidate target genes in context of their role in the sub-programs of MyoD regulated transcription.

Computational approaches for understanding the diagnosis and treatment of Parkinson's disease.

This study describes how the application of evolutionary algorithms (EAs) can be used to study motor function in humans with Parkinson's disease (PD) and in animal models of PD. Human data is obtained using commercially available sensors via a range of non-invasive procedures that follow conventional clinical practice. EAs can then be used to classify human data for a range of uses, including diagnosis and disease monitoring. New results are presented that demonstrate how EAs can also be used to classify fruit flies with and without genetic mutations that cause Parkinson's by using measurements of the proboscis extension reflex. The case is made for a computational approach that can be applied across human and animal studies of PD and lays the way for evaluation of existing and new drug therapies in a truly objective way.

Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling.

Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b.

Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.

Genetic studies have established that heparan sulphate proteoglycans (HSPGs) are required for signalling by key developmental regulators, including Hedgehog, Wnt/Wg, FGF, and BMP/Dpp. Post-synthetic remodelling of heparan sulphate (HS) by Sulf1 has been shown to modulate these same signalling pathways. Sulf1 codes for an N-acetylglucosamine 6-O-endosulfatase, an enzyme that specifically removes the 6-O sulphate group from glucosamine in highly sulfated regions of HS chains. One striking aspect of Sulf1 expression in all vertebrates is its co-localisation with that of Sonic hedgehog in the floor plate of the neural tube. We show here that Sulf1 is required for normal specification of neural progenitors in the ventral neural tube, a process known to require a gradient of Shh activity. We use single-cell injection of mRNA coding for GFP-tagged Shh in early Xenopus embryos and find that Sulf1 restricts ligand diffusion. Moreover, we find that the endogenous distribution of Shh protein in Sulf1 knockdown embryos is altered, where a less steep ventral to dorsal gradient forms in the absence of Sulf1, resulting in more a diffuse distribution of Shh. These data point to an important role for Sulf1 in the ventral neural tube, and suggests a mechanism whereby Sulf1 activity shapes the Shh morphogen gradient by promoting ventral accumulation of high levels of Shh protein.

Early transcriptional targets of MyoD link myogenesis and somitogenesis.

In order to identify early transcriptional targets of MyoD prior to skeletal muscle differentiation, we have undertaken a transcriptomic analysis on gastrula stage Xenopus embryos in which MyoD has been knocked-down. Our validated list of genes transcriptionally regulated by MyoD includes Esr1 and Esr2, which are known targets of Notch signalling, and Tbx6, mesogenin, and FoxC1; these genes are all are known to be essential for normal somitogenesis but are expressed surprisingly early in the mesoderm. In addition we found that MyoD is required for the expression of myf5 in the early mesoderm, in contrast to the reverse relationship of these two regulators in amniote somites. These data highlight a role for MyoD in the early mesoderm in regulating a set of genes that are essential for both myogenesis and somitogenesis.

Neural crest migration requires the activity of the extracellular sulphatases XtSulf1 and XtSulf2.

In vertebrates, there are two related genes, Sulf1 and Sulf2 that code for extracellular heparan sulphate 6-0-endosulphatases. These enzymes act to post-synthetically remodel heparan sulphate chains, generating structural diversity of cell surface HSPGs; this activity provides an important mechanism to modulate developmental cell signalling. Here we describe the expression and activity of Xenopus tropicalis Sulf2 (XtSulf2), which like XtSulf1, can act extracellularly to inhibit BMP4 and FGF4 signalling. Consistent with its discrete expression in regions of the anterior developing nervous system, we found that overexpression of XtSulf2 disrupts the expression of a set of neural markers and inhibits the migration of the neural crest. Using a combination of grafting experiments and antisense morpholino based knockdown studies in Xenopus embryos, we demonstrate that endogenous XtSulf1 and XtSulf2 play an important role during cranial neural crest cell migration in vivo.

Characterisation of the fibroblast growth factor dependent transcriptome in early development.

BACKGROUND: FGF signaling has multiple roles in regulating processes in animal development, including the specification and patterning of the mesoderm. In addition, FGF signaling supports self renewal of human embryonic stem cells and is required for differentiation of murine embryonic stem cells into a number of lineages. METHODOLOGY/PRINCIPAL FINDINGS: Given the importance of FGF signaling in regulating development and stem cell behaviour, we aimed to identify the transcriptional targets of FGF signalling during early development in the vertebrate model Xenopus laevis. We analysed the effects on gene expression in embryos in which FGF signaling was inhibited by dominant negative FGF receptors. 67 genes positively regulated by FGF signaling and 16 genes negatively regulated by FGF signaling were identified. FGF target genes are expressed in distinct waves during the late blastula to early gastrula phase. Many of these genes are expressed in the early mesoderm and dorsal ectoderm. A widespread requirement for FGF in regulating genes expressed in the Spemann organizer is revealed. The FGF targets MKP1 and DUSP5 are shown to be negative regulators of FGF signaling in early Xenopus tissues. FoxD3 and Lin28, which are involved in regulating pluripotency in ES cells are shown to be down regulated when FGF signaling is blocked. CONCLUSIONS: We have undertaken a detailed analysis of FGF target genes which has generated a robust, well validated data set. We have found a widespread role for FGF signaling in regulating the expression of genes mediating the function of the Spemann organizer. In addition, we have found that the FGF targets MKP1 and DUSP5 are likely to contribute to the complex feedback loops involved in modulating responses to FGF signaling. We also find a link between FGF signaling and the expression of known regulators of pluripotency.

A consensus Oct1 binding site is required for the activity of the Xenopus Cdx4 promoter.

Cdx homeodomain transcription factors have multiple roles in early vertebrate development. Furthermore, mis-regulation of Cdx expression has been demonstrated in metaplasias and cancers of the gut epithelium. Given the importance of Cdx genes in development and disease, the mechanisms underlying their expression are of considerable interest. We report an analysis of the upstream regulatory regions from the amphibian Xenopus laevis Cdx4 gene. We show that a GFP reporter containing 2.8 kb upstream of the transcription start site is expressed in the posterior of transgenic embryos. Deletion analysis of the upstream sequence reveals that a 247-bp proximal promoter fragment will drive posterior expression in transgenic embryos. We show that 63 bp of upstream sequence, that includes a consensus site for POU-domain octamer-binding proteins, retains significant promoter activity. Co-expression of the octamer-binding protein Oct1 induces expression from a Cdx4 reporter and mutation of the octamer site abolishes activity of the same reporter. We show that the octamer site is highly conserved in the promoters of the human, mouse, chicken, and zebrafish Cdx4 genes and within the promoters of amphibian Cdx1 and Cdx2. These data suggest a conserved function for octamer-binding proteins in the regulation of Cdx family members.

Xenopus tropicalis peroxidasin gene is expressed within the developing neural tube and pronephric kidney.

Peroxidasin, originally identified in Drosophila, is a member of the myeloperoxidase family with a novel domain structure. It is proposed that peroxidasin is secreted and has functions associated with stabilization of the extracellular matrix. We report the identification of the Xenopus tropicalis orthologue of the peroxidasin gene. We show that the predicted protein sequence of Xenopus peroxidasin shows high sequence identity with the human orthologue and that the exon structure is highly conserved between the two species. We describe the first detailed developmental expression pattern for peroxidasin in a vertebrate species. Maternal expression of Xtpxn is localized to the animal hemisphere where it persists through early cleavage stages. Initial zygotic Xtpxn expression is detected in the developing neural tube and becomes localized to the hindbrain and midbrain. Xtpxn is expressed in the primordium of the pronephric kidney and expression persists in the pronephric tubules and duct throughout development. Potential roles for peroxidasin during early vertebrate development are discussed.

Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos.

Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.