1. Macrophage-specific gene functions in Spi1-directed innate immunity
by Anna Zakrzewska, Chao Cui, Oliver W. Stockhammer, Erica L. Benard, Herman P. Spaink, and Annemarie H. Meijer
Blood
Volume 116(3):e1-e11
July 22, 2010
©2010 by American Society of Hematology

2. Microarray experiment setup and analysis.
Microarray experiment setup and analysis. (A) The experimental setup of the microarray study. The 28-hpf time point for microarray analysis in both experiments was chosen after the onset of blood circulation in the embryo and to ensure the abundance of myeloid cells of the innate immune system as shown by lcp1 in situ staining of embryos treated with the standard control (SC) morpholino. The lack of lcp1 staining in spi1 morphants confirms the absence of myeloid cells. GFP+ myeloid cells were isolated from embryos expressing membrane-targeted EGFP under control of the spi1 promoter. The 2-step approach eliminated false positives because of leaky expression of the spi1:GFP transgene in the brain and allowed determination of the specific effect of spi1 knockdown on myeloid cells. (B) Primary analysis of microarray data. For initial comparative analysis we chose an arbitrary cut-off of absolute fold change larger than or equal to 1.2 and a P value ≤ .01. We found 3551 sequences to be down-regulated and 3684 sequences to be up-regulated in the spi1 knockdown embryos. In the GFP+ myeloid cell fraction, 6327 sequences were enriched and 6335 sequences were reduced compared with the GFP fraction. The overlap between the sequences found down-regulated in spi1 morphants and enriched in the GFP+ myeloid cell fraction was estimated and filtered against the sequence set found down-regulated in control morpholino-injected embryos. (C) GO term annotation analysis of the 249 genes found in the overlap. Category 1 indicates zebrafish genes with at least one GO term annotation; category 2, genes without any annotation in zebrafish but having a homolog with known GO term annotation in humans; and category 3, genes without any GO term annotation either in zebrafish or in humans.
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

3. Quantitative RT-PCR validation of novel genes downstream of Spi1 identified in microarray data.
Quantitative RT-PCR validation of novel genes downstream of Spi1 identified in microarray data. (A) Relative expression levels in microarray. Values are mean ± SEM of all oligos for each gene present on the array. (B) Relative expression levels in quantitative RT-PCR. For normalization, peptidylprolyl isomerase A-like (ppial), which showed no changes over the mRNA samples used, was taken as reference. Results were analyzed using the ΔΔCt method. Gene expression values for spi1 MO (morpholino)-injected group were calculated relative to the phenol red-injected control group, whereas values for GFP+ cells were calculated relative to values of the GFP− cell fraction. Values of quantitative RT-PCR data are the mean ± SEM of 2 or 3 biologic replicates.
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

4. Expression of mfap4, mpeg1, ptpn6, and cxcr3
Expression of mfap4, mpeg1, ptpn6, and cxcr3.2 is dependent on Spi1 transcription factor.
Expression of mfap4, mpeg1, ptpn6, and cxcr3.2 is dependent on Spi1 transcription factor. (A-L) Whole-mount in situ hybridization. Wild-type expression of genes mfap4 (A,C), mpeg1 (E,G), and ptpn6 (I,K) as shown in whole embryos and enlarged tail regions shows a pattern typical for myeloid cells. Expression of genes mfap4 (B,D), mpeg1 (F,H), and ptpn6 (J,L) is absent in spi1 knockdown embryos. Images were taken with a Leica M165C stereomicroscope equipped with DFC420 camera. Composite images of different focal planes were generated using Adobe Photoshop. (M-P) Single fluorescent in situ hybridization using TSA Cy3 Plus detection. Wild-type expression of gene cxcr3.2, as shown in the tail region (M) and in the magnified region of the blood island (O), shows a pattern typical for myeloid cells. Expression of gene cxcr3.2 (N,P) is absent in spi1 knockdown embryos. Images were taken with Leica TCS SPE confocal microscope, using the 532-nm laser line for scanning of signal in the red channel. The HC PL Fluotar 10.0×/0.30 dry objective was used. The images were processed in ImageJ Version 1.43 software (National Institute of Mental Health). Bars represent 100 μm (M-N) and 60 μm (O-P).
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

5. Expression of genes mfap4, cxcr3
Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 colocalizes with expression of macrophage-specific marker csf1r.
Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 colocalizes with expression of macrophage-specific marker csf1r. Double fluorescent in situ with the csf1r gene. Probes for mRNA are used as indicated in the bottom left corner of each image. Expression of genes, mfap4 (A-C), cxcr3.2 (D-F), mpeg1 (G-I), and ptpn6 (J-L) overlaps with expression of macrophage-specific marker csf1r. Images show summed Z-stacks of consecutive confocal images through the posterior blood island of 28-hpf embryos (lateral view). A Zeiss axioplan microscope with Bio-Rad MRC1024ES scanhead was used for confocal imaging. Signals in the green and red channels were scanned sequentially using, respectively, the 488-nm laser line with 522 DF32 filter for detection of emitted light, and the 568-nm laser line with 605 DF32 filter for detection of emitted light: 10×/0.30 NA and 20×/0.50 NA Plan-Neofluar objectives were used. Images were processed in ImageJ Version 1.43 software and show (from left to right) the signal in the green channel, the signal in the red channel, and the merged signals. (A-F) Bars represent 20 μm. (G-L) Bars represent 60 μm.
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

6. Expression of genes mfap4, cxcr3
Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 does not colocalize with expression of neutrophil-specific marker mpx.
Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 does not colocalize with expression of neutrophil-specific marker mpx. Double fluorescent in situ with the mpx gene. Probes for mRNA are used as indicated in the bottom left corner of each image. Expression of genes, mfap4 (A-C), cxcr3.2 (D-F), mpeg1 (G-I), and ptpn6 (J-L) does not colocalize with expression of the mpx gene. Images show summed Z-stacks of consecutive confocal images through the posterior blood island of 28-hpf embryos (lateral view). In addition, no colocalization was observed in the head and yolk regions or in embryos at 48 hpf when mpx expression marks differentiated neutrophils (data not shown). Imaging conditions are as in Figure 4. Bars represent 20 μm.
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

7. Hematopoiesis pathway regulation on spi1 knockdown and in myeloid cells of a developing embryo at 28 hpf.
Hematopoiesis pathway regulation on spi1 knockdown and in myeloid cells of a developing embryo at 28 hpf. Expression profiles of the spi1 knockdown embryos and GFP+ myeloid fractions at 28 hpf are simultaneously mapped on a schematic representation of hematopoiesis in zebrafish embryos based on the current knowledge of this process19,42,43 and including the novel macrophage-specific markers identified in this study. The primitive wave of hematopoiesis in zebrafish initiates at 2 distinct sites. At the anterior lateral plate mesoderm (ALPM), hemangioblasts (H) differentiate into myeloid cells (M) or endothelial cells (EC). At the second site, the posterior lateral plate mesoderm (PLPM), which later converts into the intermediate cell mass (ICM), hemangioblasts give rise to erythroid cells (E) and also ECs. In a transient wave of hematopoiesis occurring in posterior blood island (PBI), the EMPs, which are the first multipotent hematopoietic progenitor cells, differentiate into either myeloid or erythroid cells. The definitive wave of hematopoiesis starts in the aorta-gonad-mesonephros (AGM), where hemogenic endothelium (HE) gives rise to hematopoietic stem cells (HSC) and ECs. Gene boxes are color-coded with the microarray data from spi1 knockdown on the left and GFP+ myeloid data on the right side. Expression of gene csf1r was determined by quantitative RT-PCR as the microarray contained only one probe for that gene, and it did not return a significant result. Up-regulation is indicated in yellow, down-regulation in blue, and no significant change in white. Genes marked by asterisks are the genes that were both down-regulated in spi1 knockdown embryos and enriched in the spi1-GFP myeloid cell fraction, and are the focus of this study. It should be noted that it is currently unknown to what extent the microarray expression data from 28-hpf embryos can be extrapolated to later waves of hematopoiesis. Macrophage specificity of cxcr3.2 in embryos is based on colocalization data at 28 hpf, and macrophage specificity of mpeg1, mfap4, and ptpn6 is based on colocalization studies at 28 and 48 hpf.
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology

8. Knockdown analysis of cxcr3
Knockdown analysis of cxcr3.2 using splice donor and acceptor morpholinos revealed a significantly reduced accumulation of macrophages at sites of bacterial infection.
Knockdown analysis of cxcr3.2 using splice donor and acceptor morpholinos revealed a significantly reduced accumulation of macrophages at sites of bacterial infection. (A) Schematic of the cxcr3.2 gene and MO targeting sites. Red boxes represent the exons in the cxcr3.2 gene. The intron (∼ 4 kb) is not drawn to scale. Red and blue bars represent the splice donor (D) and acceptor (A) MOs, which bind to the pre-mRNA on the intron/exon boundaries, inducing an insertion of the intron into the final transcripts. (B) RT-PCR detection of cxcr3.2 transcripts on MO knockdown. Altered splicing induced by D or A MOs resulted in an effective knockdown of the original cxcr3.2 mRNA at 24 hpf, whereas the combination of D + A MOs extended the knockdown effect until 48 hpf. The altered splice product with insertion of the approximately 4 kb intron is not amplified by RT-PCR. (C) Schematic of the experimental setup and representative images showing the attraction of macrophages and neutrophils to a local infection site. S typhimurium bacteria were injected into the embryo tail muscle at 28 hpf followed by a 3-hour incubation. Leukocytes were visualized at 3 hpi after double fluorescent in situ hybridization: (i) mfap4+ macrophages; (ii) mpx+ myeloid cells; (iii) merged; and (iv) bright-field image; bar represents 200 μm. (D) Migration of innate immune cells after MO knockdown. Accumulation of macrophages marked by mfap4 (i-iii) and accumulation of the nonoverlapping mpx+ myeloid cell population (iv) were quantified by determining the percentages of cells accumulated at the infection site with respect to the total number of cells in the tail. Data were collected from 3 independent experimental setups, in which the cxcr3.2 D MO (i), A MO (ii), or D + A MOs (iii-iv) were tested versus the Standard Control (SC) MO. Each data point represents an individual embryo, and each graph represents the combined results of 2 replicate experiments with the indicated MO combination. Lines indicate the mean, and P values indicate the level of statistical significance by Student t test. The knockdown of cxcr3.2 did not have a significant effect on migration of either macrophages or mpx+ myeloid cells in response to sterile injury (data not shown).
Anna Zakrzewska et al. Blood 2010;116:e1-e11
©2010 by American Society of Hematology