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Ddx41 function in erythropoiesis
erythrocytes using May-Grunwald-Giemsa staining (Figure 1J). The ddx41 mutant erythrocytes displayed a megaloblas- toid-like phenotype, suggesting some abnormalities in ery- throcyte maturation.
In order to acquire enough erythrocytes to perform the morphological assessment, we needed to bleed four times as many ddx41 mutant embryos as compared to sibling control embryos, suggesting mutants had fewer erythro- cytes than siblings. In order to test this hypothesis, we assessed the number of gata1:dsred+ erythroid progenitors in ddx41 mutants and siblings using flow cytometry quan- tification. We determined that the absolute number of gata1:dsred+ erythrocytes per embryo was significantly reduced in ddx41 mutants compared to siblings at both 28 and 40 hpf (Figures 2A to D). These data indicate that decreased erythrocyte number contributes to the develop- ment of anemia in ddx41 mutants.
Erythroid progenitors arising from both primitive and definitive erythroid-myeloid progenitor (EMP)-derived waves are present during the developmental time points analyzed. The gene programs for the specification and dif- ferentiation of primitive and EMP-derived erythropoiesis are highly similar, but the developmental timings are dis- tinct (Figure 2E). EMP specification begins around 26 hpf.27 In order to determine whether there were defects in EMP- derived erythropoiesis, we performed in situ hybridization for the progenitor marker c-myb at 26 hpf and 36 hpf and gata1 at 26 hpf in siblings and ddx41 mutants (Figures 2F and G; Online Supplementary Figure S1C to F). Expression of both c-myb and gata1 within the posterior blood island (PBI) region where EMP form were not decreased and in fact were increased in ddx41 mutants as compared to siblings. As the gene programs are highly similar between these two waves of erythroid development, these data indicate that the reduction in erythrocytes in ddx41 mutants is occurring at an erythroid progenitor stage after c-myb and gata1 are both expressed, which is shortly after erythroid lineage specification.
In order to further characterize the maturation state of the erythrocytes at 40 hpf in ddx41 mutants and siblings, we performed RT-qPCR for embryonic and larval globins. Expression of the embryonic globins αε1, αε3, βε1, and βε3 begins during somitogenesis with expression of all of these globins except βε3 persisting in primitive and EMP-derived erythrocytes throughout larval development.28 In contrast, levels of βε3 globin diminish dramatically from 24-48 hpf, somewhat concomitant with the increasing expression of the larval βε2 globin. The other larval globin αε5 is not expressed significantly until 14 dpf. In ddx41 mutants, we determined that while the levels of the embryonic/larval globins αε1 and βε1 were diminished, the levels of the embryonic-restricted βε3 globin remained high, consistent with a maturational defect in primitive erythrocytes. Additionally, expression of the larval βε2 globin was lower in mutants compared to sibling controls. Although ddx41 mutants die before there are expansive numbers of matur- ing erythrocytes derived from EMP, these data indicate that mutants have fewer definitive erythrocytes compared to siblings. This finding suggests that similar to primitive ery- throid progenitors, EMP are specified normally, but there is a later stage defect, although the underlying cause (e.g., diminished expansion, maturation or differentiation) can- not be deciphered. Together, our findings establish that Ddx41 is critical for erythrocyte expansion and maturation.
Cell cycle genes are mis-expressed and alternatively spliced in ddx41 mutant erythroid progenitors
In order to mechanistically assess the underlying cause of the erythrocytic defect in ddx41 mutants, we conducted RNA-seq on gata1:dsred+ erythrocytes isolated from ddx41 mutants and siblings at 40 hpf. Over 1,800 genes were downregulated and more than 1,900 were upregulated in ddx41 mutants compared to siblings (Figure 3A; Online Supplementary Table S1, log2 fold-change ≥1, adjusted P- value <0.05). In order to understand if particular pathways were enriched in the differentially expressed genes, we per- formed gene set level analysis on the upregulated and downregulated gene lists by comparing each to the Molecular Signature Database (MSigDb), a platform that computes overlaps between classes of genes that are over- or underrepresented in lists of genes in known pathways.29,30 In the downregulated gene list, mRNA splicing was the top gene set with DNA replication, cell cycle, and DNA repair also enriched (Figure 3B; Online Supplementary Table S2). In the upregulated gene list, genes associated with adaptive immunity, posttranslational modifications, innate immune system, and cell cycle were enriched (Figure 3C; Online Supplementary Table S3). We validated the expression changes in several cell cycle and DNA-damage-associated genes using RT-qPCR (Figure 3D).
Ddx41 interacts with components of the spliceosome.1 Additionally, the top downregulated pathway in our gene set was pre-mRNA splicing, thus we examined how ddx41 loss affected mRNA splicing in erythrocytes. When com- paring splicing between ddx41 mutants and siblings, a total of 370 alternative splicing events were observed (Figures 3E; Online Supplementary Table S4). The specific splicing defects detected included exon skipping (SE), which was the most frequently altered splicing event, intron retention (RI), alter- native 5’-splice site usage, alternative 3’-splice site usage, and changes in mutually exclusive exon usage. Alternative splicing within protein coding regions of a transcript can result in the introduction of premature termination codon (PTC) or generation of a novel peptide. For all SE and RI events (comprising nearly 85% of all splicing changes), we determined how the alternative splicing event might alter the protein sequence (Figure 3F; Online Supplementary Table S5). More than 50% of SE events altered the protein sequence and are predicted to generate novel peptides. Approximately 43% of SE and 90% of RI events are pre- dicted to target the alternatively spliced transcript for non- sense-mediated decay (NMD) due to the introduction of a PTC. For example, the retained intron variant for homolo- gous repair-associated factor structural maintenance of chromosome 5 (smc5) identified in ddx41 mutants is predict- ed to result in NMD that could result in elevated DNA dam- age (Figure 3G). Another example of an NMD isoform expressed in ddx41 mutant is the exon 3 skipped isoform of signal transducer and activator of transcription 1a (stat1a) that would diminish signaling by numerous cytokine path- ways. Pathway analysis of these alternatively spliced fac- tors revealed that those resulting in novel peptide sequences are enriched in mRNA metabolism, morphogen- esis, and cell cycle, and those predicted to result in NMD are enriched for mRNA processing, DNA replication, and gene expression (Figure 3H; Online Supplementary Table S6). These results depict that Ddx41 influences the expression and splicing of cell cycle, DNA repair, and mRNA process- ing genes in erythrocytes.
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