kcnj13 regulates pigment cell shapes in zebrafish and has diverged by cis-regulatory evolution between Danio species.

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma.

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38-high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma.

Parental mutations influence wild-type offspring via transcriptional adaptation.

Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.

NRASQ61K melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.

Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish.

The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.

PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation.

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.

The gene regulatory basis of genetic compensation during neural crest induction.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.

Dicer1 is required for pigment cell and craniofacial development in zebrafish.

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1sa9205, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1sa9205 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1sa9205 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1sa9205 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10.

Kinematic and Biomechanical Response of Post-Mortem Human Subjects Under Various Pre-Impact Postures to High-Rate Vertical Loading Conditions.

Limited data exist on the injury tolerance and biomechanical response of humans to high-rate, under-body blast (UBB) loading conditions that are commonly seen in current military operations, and there are no data examining the influence of occupant posture on response. Additionally, no anthropomorphic test device (ATD) currently exists that can properly assess the response of humans to high-rate UBB loading. Therefore, the purpose of this research was to examine the response of post-mortem human surrogates (PMHS) in various seated postures to high-rate, vertical loading representative of those conditions seen in theater. In total, six PMHS tests were conducted using loading pulses applied directly to the pelvis and feet of the PMHS: three in an acute posture (foot, knee, and pelvis angles of 75°, 75°, and 36°, respectively), and three in an obtuse posture (15° reclined torso, and foot, knee, and pelvis angles of 105°, 105°, and 49.5°, respectively). Tests were conducted with a seat velocity pulse that peaked at ~4 m/s with a 30-40 ms time to peak velocity (TTP) and a floor velocity that peaked at 6.9-8.0 m/s (2-2.75 ms TTP). Posture condition had no influence on skeletal injuries sustained, but did result in altered leg kinematics, with leg entrapment under the seat occurring in the acute posture, and significant forward leg rotations occurring in the obtuse posture. These data will be used to validate a prototype ATD meant for use in high-rate UBB loading scenarios.

Similitude assessment method for comparing PMHS response data from impact loading across multiple test devices.

Biological tissue testing is inherently susceptible to the wide range of variability specimen to specimen. A primary resource for encapsulating this range of variability is the biofidelity response corridor or BRC. In the field of injury biomechanics, BRCs are often used for development and validation of both physical, such as anthropomorphic test devices, and computational models. For the purpose of generating corridors, post-mortem human surrogates were tested across a range of loading conditions relevant to under-body blast events. To sufficiently cover the wide range of input conditions, a relatively small number of tests were performed across a large spread of conditions. The high volume of required testing called for leveraging the capabilities of multiple impact test facilities, all with slight variations in test devices. A method for assessing similitude of responses between test devices was created as a metric for inclusion of a response in the resulting BRC. The goal of this method was to supply a statistically sound, objective method to assess the similitude of an individual response against a set of responses to ensure that the BRC created from the set was affected primarily by biological variability, not anomalies or differences stemming from test devices.

A high-resolution mRNA expression time course of embryonic development in zebrafish.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.

Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form.

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1.

Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.

Evaluation of WIAMan Technology Demonstrator Biofidelity Relative to Sub-Injurious PMHS Response in Simulated Under-body Blast Events.

Three laboratory simulated sub-injurious under-body blast (UBB) test conditions were conducted with whole-body Post Mortem Human Surrogates (PMHS) and the Warrior Assessment Injury Manikin (WIAMan) Technology Demonstrator (TD) to establish and assess UBB biofidelity of the WIAMan TD. Test conditions included a rigid floor and rigid seat with independently varied pulses. On the floor, peak velocities of 4 m/s and 6 m/s were applied with a 5 ms time to peak (TTP). The seat peak velocity was 4 m/s with varied TTP of 5 and 10 ms. Tests were conducted with and without personal protective equipment (PPE). PMHS response data was compiled into preliminary biofidelity response corridors (BRCs), which served as evaluation metrics for the WIAMan TD. Each WIAMan TD response was evaluated against the PMHS preliminary BRC for the loading and unloading phase of the signal time history using Correlation Analysis (CORA) software to assign a numerical score between 0 and 1. A weighted average of all responses was calculated to determine body region and whole body biofidelity scores for each test condition. The WIAMan TD received UBB biofidelity scores of 0.62 in Condition A, 0.59 in Condition B, and 0.63 in Condition C, putting it in the fair category (0.44-0.65). Body region responses with scores below a rating of good (0.65-0.84) indicate potential focus areas for the next generation of the WIAMan design.

Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BACKGROUND: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations. RESULTS: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward. CONCLUSIONS: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BACKGROUND: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. RESULTS: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. CONCLUSIONS: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.

Comparison of the efficacy and safety of low molecular weight heparins for venous thromboembolism prophylaxis in medically ill patients.

OBJECTIVE: To conduct a systematic review and mixed-treatment comparison (MTC) meta-analysis to compare the efficacy and safety of low molecular weight heparins (LMWHs) for venous thromboembolism (VTE) prophylaxis in hospitalized medically ill patients. As a secondary objective we compared all therapies within the network to each other. METHODS: We conducted a systematic literature search for randomized trials that evaluated pharmacologic VTE prophylaxis in hospitalized medically ill patients. We conducted a traditional meta-analysis for all pairwise comparisons using a random effects model, reporting relative risks (RRs) and 95% confidence intervals for each outcome. To determine the relative efficacy and safety of included therapies we conducted a MTC meta-analysis using a Bayesian framework, reporting odds ratios (OR) and 95% credible intervals. RESULTS: Twenty trials met inclusion criteria. Enoxaparin, dalteparin, nadroparin and certoparin were the LMWHs evaluated although none in direct comparative trials. Upon MTC, the relative efficacy of all LMWHs was similar in preventing mortality and VTE as well as in the odds of major and minor bleeding. Dalteparin was not included in the network to evaluate deep vein thrombosis (DVT) and pulmonary embolism (PE) due to lack of reported data and the remaining LMWHs were found to be similar in relative efficacy in preventing these outcomes. LIMITATIONS: Traditional meta-analysis was not possible for many drug comparisons made within the MTC. Heterogeneity was observed in several of the traditional meta-analyses although this may be an inherent limitation of the studied population. Overall rarity of events contributed to imprecise estimates demonstrated by the wide confidence intervals. CONCLUSIONS: Enoxaparin, dalteparin, nadroparin and certoparin are similar in relative efficacy for the prevention of mortality and VTE and in the odds of major or minor bleeding while enoxaparin, nadroparin and certoparin are similar in relative efficacy for the prevention of PE and DVT in hospitalized medical patients.