Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

AbstractThe establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channelkcnk5bas a key player in integrating calcineurin’s growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen insofand wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.</jats:p

Bioelectric Signaling Regulates Size in Zebrafish Fins

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish

Development of DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios

Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm (Amyelois transitella), peach twig borer (Anarsia lineatella), oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), raisin moth (Cadra figulilella), and Indian meal moth (Plodia interpunctella). In this approach, the dissociation (melt) temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 was determined using quantitative PCR (qPCR). Within these six species, the distribution and the number of melt peak temperatures provide an unambiguous species level identification that is reproducible when unsheared DNA can be extracted. The test is robust across a variety of sampling approaches including insects removed from sticky card traps, museum specimens, and samples that were left in the field for up to 7 days. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change

Vertebrate <i>kcnk5</i> homologs and expression in zebrafish development.

<p>(A) Due to a whole genome duplication event, teleost fish have two <i>kcnk5</i> paralogs that show early divergence. Numbers indicate bootstrap values in percentage (100 bootstrap replications). Nodes with a bootstrap value lower than 95 were collapsed. Dre, <i>Danio rerio</i>; Ola, <i>Oryzias latipes</i>; Gac, <i>Gasterosteus aculeatus</i>, Tru, <i>Takifugu rubripes</i>; Tni <i>Tetraodon nigridoviridis</i>, Gmo, <i>Gadus morhua</i>; Mmu, <i>mus musculus</i>; Gga, <i>Gallus gallus</i>; Xtr <i>Xenopus tropicalis</i>. (B) RT-PCR of <i>kcnk5a</i> and <i>kcnk5b</i> shows comparable expression between the two paralogs in multiple adult tissues, including fins.</p

Overexpression of <i>kcnk5b</i> is sufficient to cause fin overgrowth.

<p>(A) Construct used to create <i>kcnk5b</i>-expressing clones via Tol2 transgenesis. (B) Individual fish expressing <i>kcnk5b</i> (W169L) (left) or <i>kcnk5b</i> (wt) (right) in mosaic clones display localized fin and barbel overgrowth. (C–F) Overgrowth is associated with DsRed expression (in red) within mesenchymal cells. (C) Calcein staining labels bone tissue (in green) of an overgrown fin (DsRed; <i>kcnk5</i>(W169L) expressing clone). (D) Mesenchymal clones are associated with increased segment length in the fin compared to non-overgrown DsRed negative regions. (E) Fibroblast-like cells appear as DsRed positive cells within the fin rays (dotted line) that surround DsRed negative vasculature (arrows in E and F) which extend along the actinotrichia (fibrils within dotted lines in F) towards the distal end of the fin. (G) Overgrown barbels show DsRed signal within the mesenchyme (area within dotted line) but not in the vasculature (arrow). (H) Number of clones associated with overgrowth in different <i>kcnk5b</i> variants. (I) Proportion of different cell types labeled in overgrown tissues. (J) Electrophysiological recordings of the non-conductive <i>kcnk5b</i> (GFGAAA) mutant in oocytes. Squares: <i>kcnk5b</i> (wt), purple stars: <i>kcnk5b</i> (F241Y)+<i>kcnk5b</i> (wt), blue circles: <i>kcnk5b</i> (W169L)+<i>kcnk5b</i> (wt), green triangles: + <i>kcnk5b</i> (GFGAAA)+<i>kcnk5b</i> (wt). Current was normalized to the measurement of wt current at 60 mV. Inset: DsRed+ fibroblasts in fish injected with the non-conductive construct do not lead to fin overgrowth.</p

Cell proliferation is increased in <i>alf</i> mutants.

<p>(A) Sections of wild type and heterozygous <i>alf</i> fins. No significant difference in cell size is seen in the two groups. (B) Antibody staining against PCNA on paraffin sections of regenerating fins 4 days post amputation (dpa). Chart shows percentage of proliferating nuclei (PCNA) over total nuclei (Hoechst). N = 3–4 sections of 4 individual fish **: p-value<0.01.</p

Gain-of-function mutations in <i>kcnk5b</i> affect ionic conduction and lead to hyperpolarization of the cell.

<p>(A) Location of the amino acids altered in <i>kcnk5b</i> gain-of-function mutants. Kcnk5b protein was modeled on human KCNK4 (K2p4.1). GFG and GYG domains represent the selectivity pore of the channel. (B) Voltage clamp recordings from <i>Xenopus</i> oocytes injected with cRNA of wild type and mutant <i>kcnk5b</i>. The membrane potential was clamped at a reference potential of −80 mV and then stepped to a test potential from +60 mV to −100 mV for 500 ms. The current that is applied in order to clamp the voltage to a certain value corresponds to the current passing through the plasma membrane. Representative electrophysiological traces are shown. (C) The mutant channels display increased conductance over wild type channels expressed at comparable levels. Error bars represent standard deviation. (D) Kcnk5b influences membrane potential (V_m) in oocytes. The mutant variants tend to hyperpolarize the cell (each point represents one oocyte).</p