JNK mediates differentiation, cell polarity and apoptosis during amphioxus development by regulating actin cytoskeleton dynamics and ERK signalling

c-Jun terminal kinase (JNK) is a multi-functional protein involved in a diverse array of context-dependent processes, including apoptosis, cell cycle regulation, adhesion and differentiation. It is integral to a number of signalling cascades, notably downstream of non-canonical Wnt and MAPK signalling pathways. As such, it is a key regulator of cellular behaviour and patterning during embryonic development across the animal kingdom. The cephalochordate amphioxus is an invertebrate chordate model system straddling the invertebrate to vertebrate transition and is thus ideally suited for comparative studies of morphogenesis. However, next to nothing is known about JNK signalling or cellular processes in this lineage. Pharmacological inhibition of JNK signalling using SP600125 during embryonic development arrests gastrula invagination and causes convergence extension-like defects in axial elongation, particularly of the notochord. Pharynx formation and anterior oral mesoderm derivatives like the preoral pit are also affected. This is accompanied by tissue-specific transcriptional changes, including reduced expression of six3/6 and wnt2 in the notochord, and ectopic wnt11 in neurulating embryos treated at late gastrula stages. Cellular delamination results in accumulation of cells in the gut cavity and a dorsal fin-like protrusion, followed by secondary Caspase3-mediated apoptosis of polarity-deficient cells, a phenotype only partly rescued by co-culture with the pan-caspase inhibitor Z-VAD-fmk. Ectopic activation of ERK signalling in the neighbours of extruded notochord and neural cells, possibly due to altered adhesive and tensile properties

The evolution of ependymin-related proteins

This research was funded by Australian Research Council grants to BMD and SFC (DP130102543). IMLS gratefully acknowledges start-up funding for her lab from MASTS (Marine Alliance for Science and Technology Scotland) and seedcorn funding through the Wellcome Trust ISSF3 grant number 204821/Z/16/Z.Background: Ependymins were originally defined as fish-specific secreted glycoproteins involved in central nervous system plasticity and memory formation. Subsequent research revealed that these proteins represent a fish-specific lineage of a larger ependymin-related protein family (EPDRs). EPDRs have now been identified in a number of bilaterian animals and have been implicated in diverse non-neural functions. The recent discoveries of putative EPDRs in unicellular holozoans and an expanded EPDR family with potential roles in conspecific communication in crown-of-thorns starfish suggest that the distribution and diversity of EPDRs is significantly broader than currently understood. Results :We undertook a systematic survey to determine the distribution and evolution of EPDRs in eukaryotes. In addition to Bilateria, EPDR genes were identified in Cnidaria, Placozoa, Porifera, Choanoflagellatea, Filasterea, Apusozoa, Amoebozoa, Charophyta and Percolozoa, and tentatively in Cercozoa and the orphan group Malawimonadidae. EPDRs appear to be absent from prokaryotes and many eukaryote groups including ecdysozoans, fungi, stramenopiles, alveolates, haptistans and cryptistans. The EPDR family can be divided into two major clades and has undergone lineage-specific expansions in a number of metazoan lineages, including in poriferans, molluscs and cephalochordates. Variation in a core set of conserved residues in EPDRs reveals the presence of three distinct protein types; however, 3D modelling predicts overall protein structures to be similar. Conclusions:  Our results reveal an early eukaryotic origin of the EPDR gene family and a dynamic pattern of gene duplication and gene loss in animals. This research provides a phylogenetic framework for the analysis of the functional evolution of this gene family.Publisher PDFPeer reviewe

Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

Background: Armadillo, the Drosophila orthologue of vertebrate beta-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex'' is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. Methodology/Principal Findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional'' and "adhesive'' pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, Delta NArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-Delta NArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. Conclusions/Significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-CadherinPublisher PDFPeer reviewe

Epidermal changes during tail regeneration in the Bahamas lancelet, <i>Asymmetron lucayanum</i> (Cephalochordata)

The epidermis of a cephalochordate is described by scanning electron microscopy before tail amputation and at the following intervals thereafter: 1 day, 3 days, 6 days, 10 days and 14 days. Before amputation, the epidermis covering the entire body, including the tail, was a monolayer of non-ciliated cells in a hexagonal array. In one-day amputees, epidermal cells from the wound edge migrated, evidently by means of contractile lobopodia, forming a loosely associated monolayer on the cut surface. In the three-day amputee, cells covering the regenerate had resumed their tight-packed hexagonal array; however, in the six- and ten-day samples, the cells covering the regenerating tail were loosely associated again and smaller than before. Surprisingly, in the same six- and ten-day samples, the epidermal cells covering all body regions anterior to the regenerate had changed conspicuously - their apical cell membranes had shrunk, thereby opening up an intercellular gap, although the cells maintained their hexagonal shape and appeared to have the same neighbours as before. This gapped stage (of unknown significance) lasted about a week, after which the gaps closed up, and all the epidermal cells, including those on the regenerating tail, resumed their close association with their neighbours in a hexagonal grid

Epidermal changes during tail regeneration in the Bahamas lancelet, <i>Asymmetron lucayanum</i> (Cephalochordata)

The epidermis of a cephalochordate is described by scanning electron microscopy before tail amputation and at the following intervals thereafter: 1 day, 3 days, 6 days, 10 days and 14 days. Before amputation, the epidermis covering the entire body, including the tail, was a monolayer of non-ciliated cells in a hexagonal array. In one-day amputees, epidermal cells from the wound edge migrated, evidently by means of contractile lobopodia, forming a loosely associated monolayer on the cut surface. In the three-day amputee, cells covering the regenerate had resumed their tight-packed hexagonal array; however, in the six- and ten-day samples, the cells covering the regenerating tail were loosely associated again and smaller than before. Surprisingly, in the same six- and ten-day samples, the epidermal cells covering all body regions anterior to the regenerate had changed conspicuously - their apical cell membranes had shrunk, thereby opening up an intercellular gap, although the cells maintained their hexagonal shape and appeared to have the same neighbours as before. This gapped stage (of unknown significance) lasted about a week, after which the gaps closed up, and all the epidermal cells, including those on the regenerating tail, resumed their close association with their neighbours in a hexagonal grid

Asymmetric distribution of <i>pl10</i> and <i>bruno2</i>, new members of a conserved core of early germline determinants in cephalochordates

Molecular fingerprinting of conserved germline and somatic ¨stemness¨ markers in different taxa have been key in defining the mechanism of germline specification ("preformation" or "epigenesis"), as well as expression domains of somatic progenitors. The distribution of molecular markers for primordial germ cells (PGCs), including vasa, nanos and piwil1, as well as Vasa antibody staining, support a determinative mechanism of germline specification in the cephalochordate Branchiostoma lanceolatum, similarly to other amphioxus species. pl10 and bruno2, but not bruno4/6, are also expressed in a pattern consistent with these other germline genes, adding to our repertoire of PGC markers in lancelets. Expression of nanos, vasa and the remaining markers (musashi, pufA, pufB, pumilio and piwil2) may define populations of putative somatic progenitors in the tailbud, the amphioxus posterior growth zone, or zones of proliferative activity. Finally, we also identify a novel expression domain for musashi, a classic neural stem cell marker, during notochord development in amphioxus. These results are discussed in the context of germline determination in other taxa, stem cell regulation and regenerative capacity in adult amphioxus

Distribution of temperature tolerance quantitative trait loci in Arctic charr (Salvelinus alpinus) and inferred homologies in rainbow trout (Oncorhynchus mykiss).

We searched for quantitative trait loci (QTL) affecting upper temperature tolerance (UTT) in crosses between the Nauyuk Lake and Fraser River strains of Arctic charr (Salvelinus alpinus) using survival analysis. Two QTL were detected by using two microsatellite markers after correcting for experiment-wide error. A comparative mapping approach localized these two QTL to homologous linkage groups containing UTT QTL in rainbow trout (Oncorhynchus mykiss). Additional marginal associations were detected in several families in regions homologous to those with QTL in rainbow trout. Thus, the genes underlying UTT QTL may antedate the divergence of these two species, which occurred by approximately 16 MYA. The data also indicate that one pair of homeologs (ancestrally duplicated chromosomal segments) have contained QTL in Arctic charr since the evolution of salmonids from a tetraploid ancestor 25-100 MYA. This study represents one of the first examples of comparative QTL mapping in an animal polyploid group and illustrates the fate of QTL after genome duplication and reorganization

E-Cadherin levels change in the cytoplasm or basolateral membrane in response to changes in levels of endogenous Armadillo protein upon N-terminal deletion mutant overexpression.

<p>(A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm^1–155. (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm^1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔC^XM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.</p

Distribution of Armadillo through 3^rd instar larval wing disc development.

<p>(A) Diagrammatic representation of a third instar larval wing disc, highlighting the compartments of the wing pouch formed by intersecting Anterior/Posterior (A/P) and Dorsal/Ventral (D/V) boundaries. The dashed box outlines the confocal view at 60× magnification. (B) Diagrammatic representation of apicobasal confocal sections through a single cell of the epithelium, with AJs in red and the nucleus shown in blue. The AJ is considered to be the 0% baseline, with subapical (top 10%), midcellular (50%) and basal (100%) reference points shown. (C–F) The subcellular distribution of endogenous Armadillo changes throughout the development of the 3^rd instar larval wing disc as assessed by Armadillo-GFP under the Armadillo promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002893#pone.0002893-Marygold1" target="_blank">[52]</a>. Panels C to F show subsequently older wing discs at 20× magnification. Panels C′ to F^IV show changes in subcellular localisation of Armadillo in discs of similar age to C–F at 60× magnification. (C′–F′) Cells at the level of the AJ (0%). Note the distribution of cells along the A/P boundary (red arrowheads), forming a “line” of cells. A single row of cells in early 3^rd instar (C′) becomes a series of aligned cells along the D/V boundary by the late 3^rd instar in response to Wg and Notch signalling (F′ black arrowheads). (C″–F″) Within the top 10% of the cell, Armadillo has a punctate distribution within the domain of Wg signalling (C″) that resolves into a tramtrack pattern around the D/V domain of expression (F″, white arrows). (C′″–F′″) In addition, at approximately 50% of cell height Armadillo puncta are also stabilised in two vertical stripes along either side of the Hh signalling domain (F′″ yellow arrowheads), most visibly dorsally. These patterns are evident at the basalmost point in the cell as well (C^IV–F^IV) The antibody staining with N27A1 recapitulates that of the Armadillo-GFP (not shown).</p

Example illustrating the quantification method developed to compare changes in Armadillo protein levels at the level of the AJ across experiments.

<p>(A, A′, A″) UAS Arm^S10 is overexpressed in the <i>dppGAL4</i> domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Arm^S10, respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of Arm^S10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).</p