Hemoglobin staining of embryos injected with rps24 MO using o-dianisidine and effectiveness of rps24 MO. (A-F) O-staining of rps24 MO embryos showed a drastic reduction in the number of hemoglobin-stained blood cells when rps24 is knockdown (A and D are controls, B and E are rps24 knockdown) and partially rescued phenotype by co-injection of p53 MO (C and F). (G-H) The sequence of rps24 MO is a compliment of 1-24 bp of rps24 cDNA. Embryos co-injected with 25 ng rps24: egfp DNA and 5 ng control MO produced green fluorescent protein (G), and the expression of green fluorescent fusion protein was inhibited by co-injection with 2 ng rps24 Mo (H). A, B, C are ventral view; D, E, F, G and H are lateral view.

RPS24 is required for both primitive and definitive hematopoiesis zebrafish partly mediated by p53 pathway. (A-C) The expression of gata1 (black arrow) was significantly decreased in rps24 MO and can be partly rescued in rps24 and p53 double MO at 48 hpf. (D-F) The expression of scl was significantly decreased in rps24 MO and can be partly rescued in rps24 and p53 double MO at 48 hpf. (G-L) The expression of cmyb and runx1 was significantly decreased in rps24 MO and can be partly rescued in rps24 and p53 double MO at 48 hpf.

Differentially expressed hematological genes in RPS24 MO. (A) A total of 25 differentially expressed hematological genes (by fold-change >1.3 and p-value <0.05) were detected in RPS24-deficient zebrafish embryos in 48hpf. The expression intensity of these genes were normalized to (-1, 1) and represented as heat map by R script. (B) Representative experimental validation of the regulated genes by Real-time PCR analysis. Gene expression was represented as the mean ± SD and One-way ANOVA was performed for comparison between Control and RPS24-deficient zebrafish embryos (**P <0.001, *P <0.01, n = 3). Gene expression in Control samples was normalized to 1.

Differentially regulated known miRNAs. (A) Specific expressed miRNAs in RPS24 and Control are represented in the form of Venn diagrams. A subset of miRNAs that are differentially regulated by fold-change >2.0 and p-value <0.05 is in the overlapped area and their normalized expression levels can be seen in the heat map. (B) Representative experimental validation of the regulated miRNAs by Real-time PCR analysis. Gene expression was represented as the mean ± SD and One-way ANOVA was performed for comparison between Control and RPS24-deficient zebrafish embryos (**P <0.001, *P <0.01, n = 3). Gene expression in Control samples was normalized to 1.