Fig. 1. Expression of pax9 during zebrafish development. (A) UMAP visualization of the hematopoietic lineage at 48hpf. (B) Distribution of cells expressing individual Pax family members. Pax9 expression is localized within a single cluster of the hematopoietic lineage. (C) Expression of pax9 during embryonic development. In situ hybridization results show dynamic changes of expression patterns at indicated stages. (D) pax9 is strongly expressed within the axial vessels and CHT (right side, arrow heads) at 48hpf. (E) Schematic illustration on the location of Pax9+ domains within the paraxial structures. pax9 expression appears to be enriched within the ventromedial region of the somites prior to the its expression within the axial vessels.

Fig. 2. Lack of pax9 selectively abrogates neutrophils. (A) Structure of pax9 locus in zebrafish. Second exon was targeted by gRNA to generate pax9 knockout mutants. (B) Nucleotide sequence of pax9 locus was affected by two independent mutations. (C) The presence of mutation was validated by PCR-gel electrophoresis combination (top) and heteroduplex analyses using T7 endonuclease 1 (bottom). (D) In silico protein structure prediction of two mutants showing signaling peptide (Red), paired-box domain (Blue), and trans-activating domain (Green). (E) Gross morphology of 96hpf pax9 mutants. (F) The number of neutrophils was assessed by Sudan black staining. (G) Quantification of (F). Blue and Red dashes represent dynamic changes of neutrophil numbers in WT and mutant, respectively. dpf; day post fertilization (H) The expression of neutrophil specific markers was significantly reduced in 1.5dpf pax9 mutants compared to WT while other hematopoietic markers show no change except cmyb. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Pax9 is required for emergency granulopoiesis upon bacterial infection. (A) List of bacteria used to infect 3.5dpf zebrafish larvae. (B) Schematic illustration of infection regimen. Bacteria stock was introduced to zebrafish larvae at 1.5dpf, and the number of neutrophils was assessed at 3.5dpf. (C–D) The number of neutrophils in 3.5dpf pax9 morpholino-injected and WT embryos was assessed by Sudan black staining upon exposure to Gram-negative (C) or positive (D) bacteria. Right, bar graphs show percentage of embryos depending on their number neutrophils: low (white), average (light grey), and high (dark grey). (E) Survival curve of pax9 morpholino-injected and WT embryos upon bacterial exposure. The mortality of pax9 morpholino-injected and WT embryos upon bacterial infection was assessed and plotted at observed stages. The mortality of pax9 morpholino-injected embryos was substantially increased compared to WT.

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