Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/1/jth14391.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/2/jth14391-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/3/jth14391_am.pd

Loss of DDB1 Leads to Transcriptional p53 Pathway Activation in Proliferating Cells, Cell Cycle Deregulation, and Apoptosis in Zebrafish Embryos

<div><p>DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant <i>ddb1</i> allele (<i>ddb1^m863</i>) identified in zebrafish (<i>Danio rerio</i>), and analyze its effects on development. Zebrafish <i>ddb1</i> is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (<i>ddb1^m863</i> mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. <i>ddb1^m863</i> zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic <i>ddb1^m863</i> mutant embryos, which may be due to maternal rescue. In <i>ddb1^m863</i> mutant embryos, <i>pcna</i>-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor <i>p53 (tp53)</i> and the cell cycle inhibitor <i>cdkn1a (p21a/b^CIP1/WAF1</i>) in proliferating tissues. In addition, transcription of cyclin genes <i>ccna2</i> and <i>ccnd1</i> was deregulated in <i>ddb1^m863</i> mutants. Reduction of <i>p53</i> activity by anti-sense morpholinos alleviated the apoptotic phenotype in <i>ddb1^m863</i> mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.</p></div

Enhanced apoptosis in proliferation regions of the <i>ddb1</i>^<i>m863</i> mutant CNS.

<p>(A-F) The <i>pcna</i> expression pattern in <i>ddb1</i>^<i>m863</i> mutants and wild type siblings at 30 hpf (A-B) and 72 hpf (C-F). (G-J) TUNEL assay for apoptosis in <i>ddb1</i>^<i>m863</i> mutants (H-H', J) and wild type siblings (G-G', I) at 72 hpf. Embryos or larvae in lateral (A-B, E-H) and ventral views (C-D). Cross sections (20 μm; I-J) from larvae by TUNEL assay. Arrows represent affected <i>pcna</i>-expressing cells or enhanced apoptotic cells in the retina (D, J; red), pretectal region (F, J; blue), and cerebellum (F; green). Abbreviations used: ba, branchial arches; CMZ, ciliary marginal zone; Cce, cerebellum; GCL, ganglion cell layer; hb, hindbrain; H, hypothalamus; INL, inner nuclear layer; int, intestine; P, pallial proliferation zone; PO, preoptic area; tpz, tectal proliferation zones; Anterior towards the left. Scale bars in H' for G' and H', in J for all of others: 100 μm.</p

Green Synthesis of Composite Graphene Aerogels with Robust Magnetism for Effective Water Remediation

Graphene-based three-dimensional (3D) magnetic assemblies have attracted great research attention owing to their multiple natures inherited from 3D graphene assemblies and magnetic materials. However, at present, the practical applications of graphene-based magnetic materials are limited by the relative complex synthesis procedure and harsh operation conditions. Hence, a facile and green synthesis strategy is highly desired. Herein, a magnetic graphene aerogel with magnetite nanoparticles in-situ synthesized on the surface of its frameworks was fabricated through a green and facile strategy. The synthesis process was performed in a gentle condition with low energy consumption. The obtained graphene aerogels exhibited superior magnetism with a saturation magnetization of 55.7 emu&middot;g&minus;1. With the merits of well-developed pore structures, high surface area, and robust magnetic property, the obtained composite aerogels exhibited intriguing adsorption and photo-Fenton catalytic degradation performances for the organic dyes in water. Moreover, the utilized graphene aerogels could be recycled from the water due to their effective magnetic separation performance, indicating a promising capability for practical applications in the area of water remediation. We anticipate this synthesis strategy could provide some guidance for the design and development of 3D magnetic assemblies

Increase of <i>p53</i> transcript levels in <i>ddb1</i>^<i>m863</i> mutants.

<p>(A-D) Lateral (A-D) and dorsal views (inserts in A, C) of <i>p53</i> expression pattern in embryos at 36 hpf (wt, n = 30; mut, n = 8) and 48 hpf (wt, n = 25; mut, n = 11). The transcription of <i>p53</i> was prominently enhanced in <i>ddb1</i>^<i>m863</i> mutant embryos. Anterior towards the left. Abbreviations used: ba, branchial arches; Cce, cerebellum; end, endoderm, H, hypothalamus, hb, hindbrain; j, jaw; pfb, pectoral fin bud; tel, telencephalon; TeO, tectum opticum. Scale bar: 100 μm</p

Expression of <i>ddb1</i> in proliferation regions in wild type larvae at 72 hpf.

<p>(A-H) Characterization of <i>pcna</i> (A-D) and <i>ddb1</i> (E-H) expression in wild type larvae at 72 hpf. Lateral views (A, E) and sections (20 μm; B-D, F-H). (A, E) <i>ddb1</i> was broadly expressed in the brain while <i>pcna</i> was restricted to proliferation zones. (B-D, F-H) In the medial (m), lateral (l) and basal (b) proliferation zones of the tectum opticum (TeO) and the hypothalamic proliferation zone (H), elevated expression of <i>ddb1</i> was detected compared to other areas of the brain. <i>ddb1</i> was also highly expressed in the ciliary marginal zone (CMZ) and in proliferation regions of the pharyngeal endoderm (p) (black arrow head), jaw (j) and branchial arches (ba). The expression of <i>ddb1</i> was also found in <i>pcna</i>-negative regions such as the ganglion cell layer (GCL) and inner nuclear layer (INL) of the retina. Transcripts of <i>ddb1</i> were detected throughout the medullar oblongata (MO) where <i>pcna</i> was only expressed in the dorsal part, the rhombic lip (rl). Anterior towards the left. Stars mark the otic vesicle. Scale bar: 100 μm.</p

Genetic mapping and identification of the <i>m863</i> gene locus.

<p>(A) Schematic diagram of zebrafish linkage group (LG) 18 and the <i>m863</i> genomic region (Ensembl Zv8). Numbers in brackets represent recombination event per number of meiosis analyzed. The <i>m863</i> mutation was mapped to the genomic interval defined by the microsatellite markers z13220 and z59637. The SSLP marker18s56 generated in this study showed one recombinant, while no recombinants were recovered for the SSLP markers 18s18a and z1417. The gene <i>zgc</i>:<i>63840</i> mapped to the critical interval of the <i>m863</i> mutation. (B) Chromatogram of partial cDNA sequences from wild type and homozygous mutant <i>m863</i> embryos. The insertion of 4 bp occurred in <i>m863</i> embryos at position 2402–2405 (exon 19) of the <i>ddb1</i> ORF. (C) Chromatogram of genomic sequences of 3'-end of exon 19 and 5'-splice site from wild type, heterozygous embryos, and homozygous <i>m863</i> mutants. The mononucleotide substitution (T→C) in homozygous <i>m863</i> mutants affected the second core base of the splice donor consensus sequence, which is highly conserved in eukaryotic splice donor sites. (D) The frame shift by partial intron insertion introduced a stop codon after a stretch of novel 55 variant amino acids in the C-terminal part of the mutant protein. (E) Schematic diagram of the domain structure of Ddb1 protein based on human DDB1 structure prediction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134299#pone.0134299.ref010" target="_blank">10</a>]. The partial BPC propeller is truncated and C-terminal helical domain absent in the truncated <i>m863</i> Ddb1 protein. Arrows represent the point mutation (C), the last wild type amino acid (D), and premature stop (E) in mutant <i>m863</i>.</p

Knockdown of <i>p53</i> by anti-sense morpholino compensates the apoptotic phenotype of <i>ddb1</i>^<i>m863</i> mutants.

<p>The <i>ddb1</i>^<i>m863</i> mutants at one-cell stage were injected with <i>p53</i> morpholino as indicated (amount of morpholino per embryo provided in ng), and analyzed by TUNEL staining at 72 hpf. Based on the TUNEL staining, genotyped mutant larvae were categorized in the following classes: Class I—severe increase in apoptosis; Class IV weak phenotype with only few apoptotic cells similar to wild type siblings. The apoptosis levels in class II and III were between class I and IV (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134299#pone.0134299.g006" target="_blank">Fig 6</a>).</p><p>Knockdown of <i>p53</i> by anti-sense morpholino compensates the apoptotic phenotype of <i>ddb1</i>^<i>m863</i> mutants.</p

Increased <i>p21b</i>^{<i>CIP1/WAF1</i>} expression in homozygous <i>ddb1</i>^<i>m863</i> mutants.

<p>(A-B) Enhanced expression of <i>p21b</i>^{<i>CIP1/WAF1</i>} (<i>cdkn1a</i>) in <i>ddb1</i>^<i>m863</i> homozygous embryos in the proliferation regions of the optic tectum, compared to wild type siblings at 36 hpf. (C-D) At 48 hpf, <i>p21b</i>^{<i>CIP1/WAF1</i>} expression was strongly elevated in proliferation regions of the optic tectum, cerebellum, telencephalon, retina, branchial arches and pectoral fin bud in <i>ddb1</i>^<i>m863</i> mutants compared to wild type siblings. In contrast, expression of <i>p21b</i>^{<i>CIP1/WAF1</i>} in ventral hindbrain nuclei (arrow heads in B, D) was unaltered in <i>ddb1</i>^<i>m863</i> mutants. All views are lateral except for inserts (dorsal views). Abbreviations used: Cce, cerebellum; H, hypothalamus; L, lens; pfb, pectal fin bud; ret, retina; tel, telencephalon; TeO, tectum opticum. Anterior towards the left. Scale bar: 100 μm.</p