MOESM2 of Plasmodium falciparum genetic variation of var2csa in the Democratic Republic of the Congo

Additional file 2. Neighbour-joining tree of samples from DRC, Benin and Senegal, produced in Figtree ( http://tree.bio.ed.ac.uk/software/figtree/ ). Edges are coloured from red to blue according to their bootstrap percentage (black edges are terminal and so have no bootstrap value). Dotted lines leading away from the tree are coloured to indicate the origin of the sample

Comparison of miR-126 expression levels in <i>klf2a</i>^sh317 mutants with wildtypes at 72hpf.

<p>Expression levels of miR-126 do not differ between <i>klf2a</i>^sh317 and wildtypes at the observed time point. Expression of miR-126 was normalized to U6 for relative quantification. Individual points represent relative quantification of miR-126 in a pool of embryos relative to the mean expression in the wild-type group. Summary data is displayed as mean ± SEM.</p

<i>klf2a</i> expression patterns in developing zebrafish embryos.

<p><b>(A)</b><i>klf2a</i> expression patterns were examined using whole-mount in situ hybridisation. Grey arrowheads indicate cloaca, black arrows indicate cells lateral to the most posterior notochord, white arrows indicate pectoral fin, red arrows indicate trunk vasculature, black arrowheads indicate the cardiac outflow tract, white arrowheads indicate neuromasts and green arrows indicate subintestinal veins (3dpf) or hepatic portal vein (5dpf). Numbers in the top left corners indicate number of embryos with similar staining patterns out of total number of embryos examined. Scale bar = 500μm. <b>(B)</b> Cross sections of a 48hpf wildtype embryo showing <i>klf2a</i> expression in dorsal aorta (red arrow), parachordal vessel (black arrow), intersegmental vessel (ISV) (black arrowhead) and dorsal longitudinal anastomotic vessel (DLAV) (dotted black arrow). Anatomical positions of sections are indicated by the red lines and numbers on the top panel figure with corresponding cross sections images in the bottom panel. Scale bar = 500μm (top panel) and 100μm (bottom panel).</p

Comparison of the morphology of wildtype and <i>klf2a</i>^sh317 mutant embryos.

<p>There are no obvious morphological differences between wildtype and <i>klf2a</i>^sh317 mutant embryos up to 5dpf and also beyond up to adulthood (not shown). Scale bar = 500μm.</p

Expression of HSC markers <i>runx1</i> and <i>cmyb</i> is unaffected in homozygous <i>klf2a</i>^sh317 mutants at 36hpf.

<p>Expression patterns of HSC markers <i>runx1</i> (top panel) and <i>cmyb</i> (bottom panel) do not differ between the wildtype and <i>klf2a</i>^sh317 mutants at 36hpf. Black arrows indicate the aorta-gonad-mesonephros (AGM) region and red arrows indicate caudal haematopoietic tissue (CHT). Numbers in the top left corners indicate number of embryos with similar staining patterns out of total number of embryos examined. Scale bar = 500μm.</p

<i>klf2b</i> knockdown does not alter heart rate or AA5x formation in <i>klf2a</i>^sh317 mutants.

<p><b>(A)</b><i>klf2b</i> morpholino injection did not affect the heart rate of <i>klf2a</i>^sh317 mutants at either 48 or 72hpf. <b>(B)</b> AA5x vessel formation (white arrowheads) is intact in <i>klf2b</i> morphants in either a homozygous or heterozygous <i>klf2a</i>^sh317 mutant background. White arrows indicate lateral dorsal aortae. DA indicates dorsal aorta. Scale bar = 200μm.</p

<i>klf2b</i> expression patterns do not differ between wildtype and <i>klf2a</i>^sh317 mutants.

<p><i>klf2b</i> mRNA is detectable in the developing pectoral fin bud (black arrows). Most of the embryos examined exhibit <i>klf2b</i> mRNA presence on the surface of the embryos representing epidermal cells as described before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141611#pone.0141611.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141611#pone.0141611.ref048" target="_blank">48</a>] (red arrowheads). A small proportion of both wildtype and <i>klf2a</i>^sh317 mutant embryos show vascular staining in the ISVs (red arrows) and/or in the subintestinal veins (green arrows). There were no differences in staining patterns and especially in the level of vascular staining observed between wildtype and <i>klf2a</i>^sh317 mutant embryos. Figures in bottom left corner of each image indicate the number of embryos with similar staining patterns out of total number of embryos examined. Scale bar = 500μm.</p

<i>klf4a and biklf/klf4b/klf17</i> expression patterns do not differ between wildtype and <i>klf2a</i>^sh317 mutants at 48hpf.

<p><i>klf4a</i> expression was detected in epidermis (black arrows) and pectoral fins (red arrowheads) and <i>biklf/klf4b/klf17</i> expression was detected in neuromasts of lateral line organ (red arrows) and in hatching glands (black arrowheads) of wildtype embryos and <i>klf2a</i>^sh317 mutants in keeping with previously published data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141611#pone.0141611.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141611#pone.0141611.ref047" target="_blank">47</a>] No vascular expression of <i>klf4a</i> or <i>biklf/klf4b/klf17</i> could be detected in any of the wildtype embryos or <i>klf2a</i>^sh317 mutants examined. Figures in bottom left corner of each image indicate the number of embryos with similar staining patterns out of total number of embryos examined. Scale bar = 500μm.</p

Vascular development in <i>vhl</i>^{<i>hu2117</i>} mutants is unaffected by the <i>klf2a</i>^sh317 mutation.

<p>Wildtype embryos exhibit angiogenesis typical for this developmental stage– 3dpf (left figure, white arrow). Homozygous <i>vhl</i>^hu2117 embryos show enlargement of vessels (ISVs and DLAV) with increased tortuosity and looping of the DLAV (middle figure, right arrow). The same vascular phenotype was be observed in <i>vhl</i>^hu2117 embryos in a homozygous <i>klf2a</i>^sh317 background (right figure, red arrow). Confocal images of <i>Tg</i>(<i>fli1</i>:<i>eGFP)</i> zebrafish embryos at 3dpf.. Scale bar = 70μm.</p

Comparison of heart rates and blood flow velocities of wildtype and <i>klf2a</i>^sh317 mutant embryos at 48hpf (A) No significant differences in heart rates were observed. (B) Instantaneous blood flow velocities throughout a single cardiac cycle in wildtype and <i>klf2a</i>^sh317 mutants. (C) Average instantaneous blood flow velocities per cardiac cycle (n = 24, unpaired t-test).

<p>Comparison of heart rates and blood flow velocities of wildtype and <i>klf2a</i>^sh317 mutant embryos at 48hpf (A) No significant differences in heart rates were observed. (B) Instantaneous blood flow velocities throughout a single cardiac cycle in wildtype and <i>klf2a</i>^sh317 mutants. (C) Average instantaneous blood flow velocities per cardiac cycle (n = 24, unpaired t-test).</p