Supplemental Table from Conserved but flexible modularity in the zebrafish skull: implications for craniofacial evolvability

Morphological variation is the outward manifestation of development and provides fodder for adaptive evolution. Because of this contingency, evolution is often thought to be biased by developmental processes and functional interactions among structures, which are statistically detectable through forms of covariance among traits. This can take the form of substructures of integrated traits, termed modules, which together comprise patterns of variational modularity. While modularity is essential to an understanding of evolutionary potential, biologists currently have little understanding of its genetic basis and its temporal dynamics over generations. To address these open questions, we compared patterns of craniofacial modularity among laboratory strains, defined mutant lines and a wild population of zebrafish (<i>Danio rerio</i>). Our findings suggest that relatively simple genetic changes can have profound effects on covariance, without greatly affecting craniofacial shape. Moreover, we show that instead of completely deconstructing the covariance structure among sets of traits, mutations cause shifts among seemingly latent patterns of modularity suggesting that the skull may be predisposed towards a limited number of phenotypes. This new insight may serve to greatly increase the evolvability of a population by providing a range of ‘preset’ patterns of modularity that can appear readily and allow for rapid evolution

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

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

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