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Ddx41 function in erythropoiesis
Methods
Zebrafish
Zebrafish were maintained as described12 and according to Institutional Animal Care and Use Committee-approved proto- cols in accordance with the Albert Einstein College of Medicine research guidelines. Genotyping was performed to confirm ani- mal identity. Mutants for ddx41 (ddx41sa14887) were acquired from the Zebrafish International Resource Center.13 The mutation results in a premature stop codon at tyrosine 410. For all experi- ments, sibling controls are a mix of heterozygotes and wild types. Tg(gata1:dsred)14 transgenics were used. Genotyping details are in the Online Supplementary Appendix and the Online Supplementary Table S7.
Drug treatments
All drugs were dissolved in dimethyl sulfoxide (DMSO). Dilutions were made in E3 embryo water. KU60019 (ATM inhibitor) and AZ20 (ATR inhibitor) were used with DMSO as the vehicle control.
Whole-mount in situ hybridization and o-dianisidine staining
In situ hybridization was performed as previously described.15,16 After in situ, embryos were scored manually, imaged and genotyped. The βe3-globin,17 cmyb,18 and gata119 probes were used, and in situ levels were quantified using FIJI.20 O-dianisidine staining was performed as previously described.21
Flow cytometry
Mutant and sibling embryos were binned based on morpho- logical differences. For generation of single-cell suspensions, 10- 20 embryos were processed as previously described15 (also see the Online Supplementary Appendix). Quantification for the absolute number of cells was performed by acquiring all events in a tube on the flow cytometer to determine the total number of target cells. This number was then divided by the total num- ber of embryos analyzed to calculate the number of target cells per embryo.
Cell cycle and apoptosis analyses
For 5-ethynyl-2′-deoxyuridine (EdU) incorporation experi- ments, embryos were incubated with 20 mM EdU for 2 hours. Single-cell suspensions of embryos were generated. Click-IT EdU Flow Cytometry Assay Kit was used according to the manufactur- er’s instructions. Flow cytometry analysis for active caspase-3 was performed as previously described.22 Samples were analyzed with a LSRII flow cytometer (BD Biosciences) and FlowJo software.
RNA sequencing and splicing analysis
Erythroid progenitors from ddx41 mutants and siblings were isolated by fluorescently-activated cell sorting (FACS). RNA from these cells was subsequently isolated, DNAse-digested and library prepared for sequencing. Details on library preparation, sequencing and bioinformatic analyses can be found in the Online Supplementary Appendix. All data are deposited under GEO accession number GSE160979.
Reverse transcription quantitative polymerase chain reaction
In order to validate the RNA sequencing (RNA-seq) data, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA was isolated from 40 hpf embryos. Details are listed in the Online Supplementary Appendix and Online Supplementary Table S7.
Single-cell immunofluorescence of zebrafish embryonic cells
Single-cell suspensions were prepared, and cell staining was per- formed as described in Sorrells & Nik et al.22 and detailed in the Online Supplementary Appendix. Fluorescence intensity measure- ments of γH2AX were performed using FIJI.
May-Grunwald Giemsa staining of primitive erythroid cells
May-Grunwald Giemsa staining was performed as previously described15 and as detailed in the Online Supplementary Appendix.
Statistics
Experiments were performed with a minimum of three repli- cates. Statistical analyses were performed as indicated in each fig- ure using unpaired Student’s t-test or a one-way ANOVA with Tukey’s multiple testing correction as appropriate; error bars indi- cate the standard deviation of mean, unless otherwise indicated.
Results
Ddx41 regulates erythroid development
In order to explore a function for Ddx41 in hematopoiesis, we examined erythrocyte formation and differentiation in zebrafish ddx41 homozygous loss-of- function mutants (ddx41sa14887). Maternally-deposited Ddx41 (data not shown) helps the mutants develop and survive until 3 days post fertilization (dpf) (Online Supplementary Figure S1A and B). Based on this, we consider the mutants to be functionally hypomorphic with greatly diminished but not completely absent Ddx41 levels. DDX41 is highly con- served between humans and zebrafish suggesting that les- sons learned about the in vivo role of zebrafish Ddx41 func- tion in hematopoiesis will be relevant to human DDX41. In zebrafish, primitive hematopoiesis begins ~12-24 hours post fertilization (hpf), producing embryonic erythrocytes and myeloid cells that constitute the hematopoietic system early on in development.23,24 Cells of the erythrocytic lineage first arise from the intermediate cell mass (ICM) within the posterior lateral mesoderm (PLM) (Figure 1A). These ery- throcytes express factors such as the progenitor transcrip- tion factor c-myb and the erythroid-specific transcription factor gata1 starting during somitogenesis.19,25 Using in situ hybridization, we determined that these erythroid progen- itor markers were expressed similarly in ddx41 mutants compared to siblings (mix of ddx41 heterozygotes + wild types) at 22 hpf, indicating initial erythroid specification is unaffected (Figures 1B to E). Oxygenated hemoglobinized erythrocytes are detectable beginning around 36 hpf using o-dianisidine staining.26 In ddx41 mutants, we observed lit- tle o-dianisidine-positive erythroid cells at 40 hpf (Figure 1F). We sorted gata1:dsRed+ erythrocytes at 40 hpf and found that the ddx41 mutant cells were larger than those from sibling controls (Figure 1G). This size difference could be indicative of delayed erythroid differentiation. As mutants display some developmental delay that becomes more severe as the embryos get older, it is possible that the erythroid delay is a side effect of the general developmental delay. In order to distinguish between these possibilities, we examined erythrocytes in ddx41 mutants and siblings at 48 hpf. Oxygenated hemoglobin levels remained low in mutants at 48 hpf (Figures 1H to I). In order to assess mat- uration, we also bled ddx41 mutants and sibling control embryos at 48 hpf and analyzed the morphology of isolated
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