Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-β, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.


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April 2023

Data Policy information about availability of data
All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Single-cell RNA sequencing data was deposited in the Gene Expression Omnibus (GEO) and can be accessed under GSE252788.The two samples generated are accessible under: GSM8007849 MF317_A1_GEX_zebrafish_multiseq GSM8007850 MF317_A2_GEX_zebrafish_multiseq We made use of the following publicly available datasets in our study: • Single-cell RNA-seq: E-GEOD-100911, E-MTAB-5530, GSE137539, GSE165276, GSE149938, GSE142754, Myeloid cells from the fetal immune atlas, Tabula Sapiens -Immune, syn16816566 • Bulk RNA-seq: GSE79044, GSE109467, GSE172184 (neuroblastoma data provided by the authors) , GSE153263 , GSE151682 , GSE175880 • Others: neutrotime model, AnimalTFDB3.0-zebrfish,xCell signatures Additional file 5, additional metadata of GSE109467 was kindly provided by the authors.
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Reporting on sex and gender
Bone marrow samples from patients (male and female children) with metastatic and localized neuroblastoma and ganglioneuroma have been included .There was no selection done based on sex.Neuroblastoma is slightly more abundant in male than in female children (1:1.2),but no differences in clinical outcome has been reported.Sex was considered as covariate in the bioinformatics analysis, but was found insignificant.Sex was not considered as a co-variable in the statistical analysis of imaging data, since the number of samples did not allow for a statistically meaningful interpretation.Gender was not assessed, since the patient cohort is comprised of children aged 0-18 and the median age at diagnosis is 2 years.

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In this study bone marrow aspirates from children and adolescents (age at diagnosis 0-18 years) with neuroblastoma or ganglioneuroma were analysed.The cohort used for imaging contained 21 patient samples.

Recruitment
Patients with high-risk metastatic (stage M) neuroblastoma were enrolled in the SIOPEN/HR-NBL-1 trial (NCT01704716) according to the trial's inclusion and exclusion criteria.Samples were available as left over material from diagnostic procedures and selected for this study based on their availability in the CCRI Biobank.

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The number of replicates for each experiment is given in the figure legends.All attempts of reproduction were successful.Experiments showed variability due to natural differences between zebrafish clutches.

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April 2023 Randomization We did not perform randomization of study participants as it was not relevant for our study.Samples were available from diagnostic procedures.There were no different treatments of patients assessed.

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Fertilized eggs for standard or Morpholino injection were randomly picked from the same clutches.

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To avoid bias when assessing neutrophil morphology in Neuroblastoma patient bone marrow samples, diagnostic information (localized control or metastatic tumor) was single-blinded.Morphology assessment was done in two lots each containing samples of both diagnoses (1st lot: controls n = 4 and metastatic n = 9; 2nd lot: controls n = 5 and metastatic n = 3) .Morphology of samples was first linked to the patient number and later unblinded for each lot.Similarly, cytospin slides for assessing zebrafish neutrophil morphology were masked (singleblinded).
For other experiments (e.g.flow cytometry of standard-morpholino or Cebpb-morpholino treated larvae or Cebpb mutant analysis) no blinding was used due to technical reasons and limited staff availability.
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