The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.The study was supported by Cancer Research UK (grant number C45041/A14953 to AC and LF), Wellcome Trust (grants number 084183/Z/07/Z to EBM and number 098051 to DLS and LLH), Specialist Programme from Bloodwise [12048], the Medical Research Council [MC_U105161083] and Ted’s Gang (to AJW), a Wellcome Trust strategic award to the Cambridge Institute for Medal Research [100140] and the Cambridge NIHR Biomedical Research Centre (to AJW and AC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It was first available from PLOS via http://dx.doi.org/10.1371/journal.pgen.100567

A Loss of Function Screen of Identified Genome-Wide Association Study Loci Reveals New Genes Controlling Hematopoiesis

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits

<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

<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

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

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

<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

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

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