Generation and characterization of the zebrafish transgenic strain expressing SBDS^R126T.

(A) Constructs to create the transgenic line expressing zebrafish sbds and human SBDS^R126T were injected into fertilized eggs and, subsequently, strains were inbred. (B) Western blotting showed Sbds/SBDS^R126T expression in zebrafish fins on 1-yr-old fish. (C) The number of transgene copies was determined in the zebrafish SBDS^R126T line. (D) mRNA levels of human SBDS in 2 dpf larvae with one and two copies of the transgene Tg(SBDS^R126T). (E) Immunoblot showing protein levels in fish with one and two copies of the transgene comparing with WT sbds. ubi-ubiquitin promoter, cmlc2 green heart marker to aid screening of transgenesis.

Phenotype and biochemical analysis of adult zebrafish lines expressing the human SBDS^R126T.

(A) Zebrafish cross between sbds heterozygous and transgenic line. (B) Standard length in 1-yr-old fish. (C) H&E staining of internal organs of 1-yr-old male fish. (D) Flow cytometric analysis of whole kidney marrows showing the different blood cell types in the presence or absence of the transgene SBDS^R126T, in the different zebrafish sbds backgrounds. (E) Polysome profile of the liver. (F) Western blotting demonstrated Eif6 accumulation in zebrafish sbds KO with the transgene. (G, H) Protein quantification (H) qRT-PCR analysis of liver, brain, and GI showing up-regulation of cdkn1a in all tissues. AUFS, absorbance units full scale.

Phenotype of 10 dpf larvae expressing the human SBDS^R126T.

(A) Western blot showed Eif6 without RPL5, and Rpl11 accumulation in the sbds KO fish, but there was no Eif6 accumulation in the SBDS^R126T strain. (B) Protein quantification of Western blots as performed with NIH ImageJ. (C) After Sudan black staining, neutrophils were counted and found to be significant lower in the sbds KO background with or without the transgene. (D) qRT-PCR analysis of critical genes involved in ribosomal, metabolic, and tp53-associated pathways.

Level of SBDS^R126T protein expression affected embryonic development.

(A) Incross between WT sbds fish with two copies of the transgenic line produced healthy embryos. (B) Incross between sbds KO fish with two copies of the transgenic line produced healthy embryos and deformed embryos. Black star shows healthy embryo, green star shows developmentally delayed, and red star shows deformed embryo. (C) qRT-PCR analysis of critical genes involved in ribosomal, metabolic, and tp53-associated pathways. (D) Western blot showing Eif6 accumulation in zebrafish-deformed embryos. (E) Protein quantification of Western blots as performed with NIH ImageJ. (F) Sudan black staining of 10 dpf larvae to count neutrophils. (G) Standard length and neutrophil number were reduced in sbds KO larvae expressing SBDS^R126T.

Incrosses of sbds KO with one copy of the transgene have severe malformations in early stages of development.

(A) Crosses between WT and sbds KO fish. (B) Embryos obtained from crosses at 24 and 48 hpf. (C) Percentage of deformed fish in the first 72 hpf. (D) qRT-PCR analysis of critical genes involved in ribosomes, metabolism, and tp53 pathways.

Two copies of the SBDS^R126T transgene rescues neutropenia at 5 dpf.

(A) Crosses between sbds^+/− zebrafish with two copies of the transgene. (B) Neutrophil count in 5 dpf larvae.

Tp53^M214K does not rescue neutropenia or survival in sbds mutants.

(A) Scheme of an incross of sbds^+/−; p53^M214K/M214K. (B) Sudan black staining and neutrophil count in 10 dpf larvae. (C) qRT-PCR analysis of sbds, tp53, and cdkn1a in the p53^M214K/M214K background comparing sbds KO and WT siblings. (D) Incross between WT sbds fish with 1x copy of the transgenic line in the tp53^M214K/M214K background. (E) Embryos obtained from crosses at 24 and 48 hpf. (F) qRT-PCR analysis of critical genes involved in ribosomes, metabolism, and tp53 pathways.

Summary of variable SBDS protein levels on the sbds-null zebrafish.

Sbds dose affects survival during development. Note the difference in zebrafish WT sbds rescue Tg(ubi:sbds:pA) and the transgenic line expressing the human SBDS^R126TTg(ubi:SBDS^R126T:pA). Closed arrows show mortality and dash arrows shows partial mortality. MZ, maternal zygotic.