<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

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

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

<i>nol9</i>^{<i>sa1022/sa1022</i>} embryos display tissue-specific upregulation of <i>tp53</i>.

<p>Representative images of embryos stained by whole-mount <i>in situ</i> hybridization against <i>tp53</i>. (A) At 48 hpf, similar levels of <i>tp53</i> signal (arrow) was detected in the CHT of <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos and their wild-type siblings. (B) At 72 hpf, <i>nol9</i>^{<i>sa1022/sa1022</i>} mutant embryos were characterized by more <i>tp53</i> signal in the CHT than their wt siblings. Mann-Whitney U test, p<0.05. (A-B) All embryos are oriented with anterior to the left and dorsal to the top. (C) Schematic representation of digestive organs in a wild-type 72 hpf zebrafish larva. I–intestine, L- liver, P- pancreas. (D) At 72 hpf, <i>nol9</i>^{<i>sa1022/sa1022</i>} mutant embryos display strong <i>tp53</i> signal in the liver (arrowhead) and intestine (arrow), compared to weak signal in the intestine of wild-type siblings. (C-D) Dorsal view anterior up.</p

The number of HSPCs is rescued in a <i>tp53</i> mutant background.

<p>(A) The CHT of 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross stained by WISH against <i>c-myb</i>. Signal extracted from the corresponding WISH photograph is shown. Numbers represent larvae with the displayed phenotype out of the total number of larvae examined. (B) Quantification of <i>c-myb in situ</i> signal for 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross, depending on their genotype. Data are represented as the mean number of pixels +/- SEM. <i>nol9</i>^<i>+/+</i><i>;tp53</i>^<i>+/+</i> n = 9; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/+</i> n = 11; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^{<i>+/zdf1</i>} n = 19; <i>nol9</i>^<i>-/-</i><i>; tp53</i>^{<i>zdf1/zdf1</i>} n = 10. Two-tailed Student’s <i>t</i>-Test, *, p<0.05; **, p<0.01. (C) Representative pictures of the CHT of 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross stained by WISH against <i>hbae1</i>. Signal extracted from the corresponding WISH photograph is shown. (D) Quantification of <i>hbae1</i> WISH signal for larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross, depending on their genotype. Data are represented as the mean number of pixels +/- SEM. <i>nol9</i>^<i>+/+</i><i>;tp53</i>^<i>+/+</i> n = 16; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/+</i> n = 14; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/-</i> n = 23; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>-/-</i> n = 16. Two-tailed Student’s <i>t</i>-Test, *, p<0.05; **, p<0.01. Within the figure, <i>nol9</i>^{<i>sa1022</i>} allele has been denoted as <i>nol9</i>^<i>-</i> and <i>tp53</i>^<i>zdf1</i> as <i>tp53</i>^<i>-</i>.</p

Loss-of-function <i>nol9</i> mutation leads to a defect in ITS2 pre-rRNA processing.

<p>(A) Expression pattern of <i>nol9</i> by WISH at sphere stage (4 hpf), 12 hpf, 48 hpf, 72 hpf, 96 hpf and 120 hpf. Black arrows indicate branchial arches, white arrow indicates pancreas and arrowhead indicates <i>nol9</i>-expressing cells in the CHT. (B) Representative Northern blot analysis of RNA isolated from 5 dpf <i>nol9</i>^{<i>sa102/sa10222</i>} mutants and control (<i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>+/sa1022</i>}) siblings using 5’ETS, ITS1 and ITS2 probes to detect rRNA processing intermediates. Corresponding rRNA intermediates (a, b, c, d) are indicated in B) and C). (C) Schematic representation of the rRNA intermediates detected in the Northern blot analysis. The sites of hybridization of the 5’ETS, ITS1 and ITS2 probes are indicated in red. (D) Methylene blue staining of the membrane was used to control for equal loading of RNA. wt–<i>nol9</i>^<i>+/+</i>/<i>nol9</i>^{<i>+/sa1022</i>}, mut–<i>nol9</i>^{<i>sa1022/sa1022</i>}.</p

Ultrastructural studies of caudal hematopoietic tissue (CHT) in <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae.

<p>(A) Schematic representation of a transversal section in the CHT region of a 120 hpf zebrafish, dorsal up. NT–neural tube, NC–notochord, M—myotome, CA–caudal artery, CHT–caudal hematopoietic tissue, CV–caudal vein. (B-C) A low-magnification TEM image of the CHT from a <i>nol9</i>^<i>+/+</i> (B, n = 2) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (C, n = 2) larvae. Dashed line denotes the width of the CHT. Arrowheads denote mitochondrial profiles visible within myotomes (M). Asterisks denote extracellular matrix (ECM). (D-E) High magnification TEM image of cells present in the CHT of <i>nol9</i>^<i>+/+</i> (D) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (E) larvae. (F-G) High magnification TEM image of endothelial cells in the caudal vein of <i>nol9</i>^<i>+/+</i> (F) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (G) larvae. Arrow denotes vesicles visible in the cytoplasm of the endothelial cell, reminiscent of lipofuscin. E–endothelium. Scale bars: 2 μm.</p

<i>nol9</i>^{<i>sa1022/sa1022</i>} mutants show a decrease in the number of lymphocytes and HSPCs.

<p>A) Whole-mount <i>in situ</i> hybridization with lymphocyte-specific <i>rag1</i> probe (arrowhead) revealed that <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae displayed weak to medium <i>rag1</i> expression at 96 hpf, compared to medium to strong signal in <i>nol9</i>^<i>+/+</i> or <i>nol9</i>^{<i>+/sa1022</i>} siblings. Larvae are oriented anterior to the top and ventral up. (B) The number of larvae displaying different degrees of <i>rag1</i> expression as assessed by WISH. Data are represented as the number of larvae belonging to each phenotypic group. Fisher’s exact test, ** p<0.01. (C) Whole-mount <i>in situ</i> hybridization against the thymic epithelium marker <i>foxn1</i> at 96 hpf revealed a similar level of signal (arrow) in <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} siblings. (D) Whole-mount <i>in situ</i> hybridization using a <i>c-myb</i> riboprobe was used to assess the number of HSCs emerging in the AGM region at 36 hpf (arrow) and the number of HSPCs in the CHT (arrow) at 72 hpf. Representative pictures of the AGM region (36 hpf) and the CHT (72 hpf) are shown. Mutant <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos displayed normal <i>c-myb</i> signal at 36 hpf and decreased <i>c-myb</i> signal at 72 hpf compared to wt siblings. Fisher’s exact test, **, p<0.01. All embryos are oriented with anterior to the left and dorsal to the top. Numbers represent embryos with the displayed phenotype out of the total number of embryos examined.</p

<i>nol9</i> mutation affects the development of the exocrine but not the endocrine pancreas.

<p>(A) Representative single channel confocal images of the pancreas of 96 hpf <i>Tg(ptf1a</i>:<i>EGFP;ins</i>:<i>mCherry)</i> larvae. <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae were characterized by smaller <i>ptf1a</i>-positive area (green) and similar <i>ins</i>-positive area (red) compared to <i>nol9</i>^<i>+/+</i> siblings. (B) TEM pictures of the exocrine pancreas of <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae at 120 hpf. The white arrow denotes endoplasmic reticulum in the <i>nol9</i>^<i>+/+</i> cell. Asterisks indicate zymogen granules. Arrowheads indicate mitochondria. Scale bar: 2 μm. (C-D) Confocal images of the pancreas of <i>Tg(ptf1a</i>:<i>EGFP)</i> larvae subjected to immunohistochemistry against α-Carboxypeptidase-a (α-Cpa) (C) and α-Cytokeratin (D) at 120 hpf. (C) The exocrine pancreas differentiation marker α-Cpa (red) was detected in <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} siblings. (D) The pancreatic ducts expressing α-Cytokeratin (red) are not apparent in <i>nol9</i>^{<i>sa1022/sa1022</i>} mutants, in contrast to the ductal network clearly visible in <i>nol9</i>^<i>+/+</i> siblings.</p