Characterization of HSCs in candidate gene depleted embryos.

<p>To assess the stage at which hematopoiesis of each MO injected embryo was defective, we performed whole mount <i>in situ</i> hybridization using a <i>c-myb</i> probe at 3 dpf. Although more than half of MOs had no effect on the number of HSCs, depletion of <i>rcor1</i> resulted in increased numbers of HSCs and depletion of <i>kalrn1</i>, <i>kalrn2</i>, <i>mfn2</i>, <i>pdia5</i>, <i>psmd13</i> and <i>wasplb</i> resulted in decreased numbers of HSCs in CHT at 3 dpf. Representative images of CHT region are shown. All embryos are oriented with anterior to the left and dorsal to the top.</p

<i>In vivo</i> morpholino screen in zebrafish identifies 15 new regulators of thrombopoiesis.

<p>MOs were injected into one-cell stage transgenic <i>Tg(cd41:EGFP)</i> zebrafish embryos and assayed for their effect on the number of thrombocytes (<i>cd41^high</i>) at 3 dpf. Representative confocal images were taken of the CHT. For <i>akap10</i>, <i>brd3a</i>, <i>brf1b</i>, <i>kalrn 1</i>, <i>kalrn 2</i>, <i>kif1b</i>, <i>mfn2</i>, <i>pdia5</i>, <i>psmd13</i> and <i>satb1</i> a severe decrease in the number of <i>cd41^high</i> positive cells was observed. <i>brf1a</i>, <i>rcor1</i>, <i>waspla</i>, <i>wasplb</i> and <i>wdr66</i> depletion resulted in a mild phenotype, and <i>fen1</i>, <i>grtp1b</i> and <i>tmcc2</i> MO injected embryos showed no phenotype. All embryos are oriented with anterior to the left and dorsal to the top. White arrow – thrombocytes; white arrowhead – HSCs.</p

Heat map summarising hematopoietic phenotypes of knock-down of 19 candidate genes.

<p>Data obtained from the initial knock-down were used to generate a heat map of phenotype profiles. Each colored cell in the heat map shows the severity of the observed phenotype relative to control. The most severe decrease in the number of cells is displayed in red, a moderate reduction is displayed in orange and green denotes a cell number comparable to control. A moderate increase in the cell number is displayed in blue and grey indicates that the test was not performed.</p

<i>brd3a</i> is an important regulator of thrombopoiesis.

<p>(A) Live confocal imaging of zebrafish embryos injected with h<i>BRD3-</i>GFP mRNA revealed the nuclear localization of h<i>BRD3</i> and that it binds to mitotic chromosomes (white arrows). Expression of h<i>BRD3</i> in <i>brd3a</i> MO injected embryos resulted in a partial rescue of the number of thrombocytes as shown in (B). (C) A graph to illustrate the number of thrombocytes in control, splice <i>brd3a</i> MO and splice <i>brd3a</i> MO plus h<i>BRD3</i> mRNA injected embryos. Each dot represents the number of thrombocytes in the individual MO-injected embryos with respect to control. A blue horizontal line represents the mean value of the number of thrombocytes for each group of embryos. Student t test, * p = 0.016; n = 17. All embryos are oriented with anterior to the left and dorsal to the top.</p

Supplemental gene list to accompany Figure 1j, Figure 2d. DeTCT 3' end sequencing carried out on various neural crest mutants at different time points

3' end sequencing of multiple neural crest mutants; tfap2a, tfap2c, sox10, mitfa and yap1 at differing time points. Lists represent relevant pairwise comparisons

Supplemental gene lists to accompany Figure 1i, Figure 2e. FACS of neural crest cells at 22 hpf

FACS followed by RNA-Seq of sox10:mg cells in zebrafish at 22 hpf

Supplemental gene list to accompany Figure 2f. 15 individual subset gene lists and full ZFA enrichment.

15 gene lists which are derived from the 15 different subsets found in Figure 2f. An UpSet comparison of tfap2a;tfap2c knockouts at 4 somites, 15 somites, 24 hpf and a neural crest gene enriched set from 22 hpf FACSed and RNA-Seq sequenced cells. Full ZFA enrichment shown in the magenta box

Supplemental gene lists to accompany Figure 5b-g. MCL Clustering of tfap2a;tfap2c knockout associated genes.

Gene list to accompany Figure 5b-g. MCL clustering of tfap2a and tfap2c knockout associated genes. the log2 fold changes and adj p-value are derived from tfpa2a/c knockouts compared to wild-type siblings

Supplemental gene lists to accompany Figure 4c & h. 14 subset gene lists derived from Figure 4c.

14 Subset gene lists to accompany Figure 4c

Raw data of sex ratios

Sex ratio (as percentage Male) of zebrafish lines compared to the age in days of the mother and father