Zebrafish mutants provide insights into Apolipoprotein B functions during embryonic development and pathological conditions
Hanoch Templehof, Noga Moshe, Inbal Avraham-Davidi, Karina Yaniv
Hanoch Templehof, Noga Moshe, Inbal Avraham-Davidi, Karina Yaniv
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Research Article Development Vascular biology

Zebrafish mutants provide insights into Apolipoprotein B functions during embryonic development and pathological conditions

Abstract

Apolipoprotein B (ApoB) is the primary protein of chylomicrons, VLDLs, and LDLs and is essential for their production. Defects in ApoB synthesis and secretion result in several human diseases, including abetalipoproteinemia and familial hypobetalipoproteinemia (FHBL1). In addition, ApoB-related dyslipidemia is linked to nonalcoholic fatty liver disease (NAFLD), a silent pandemic affecting billions globally. Due to the crucial role of APOB in supplying nutrients to the developing embryo, ApoB deletion in mammals is embryonic lethal. Thus, a clear understanding of the roles of this protein during development is lacking. Here, we established zebrafish mutants for 2 apoB genes: apoBa and apoBb.1. Double-mutant embryos displayed hepatic steatosis, a common hallmark of FHBL1 and NAFLD, as well as abnormal liver laterality, decreased numbers of goblet cells in the gut, and impaired angiogenesis. We further used these mutants to identify the domains within ApoB responsible for its functions. By assessing the ability of different truncated forms of human APOB to rescue the mutant phenotypes, we demonstrate the benefits of this model for prospective therapeutic screens. Overall, these zebrafish models uncover what are likely previously undescribed functions of ApoB in organ development and morphogenesis and shed light on the mechanisms underlying hypolipidemia-related diseases.

Authors

Hanoch Templehof, Noga Moshe, Inbal Avraham-Davidi, Karina Yaniv

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Figure 4

apoB mutants display hyperangiogenic phenotypes.

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apoB mutants display hyperangiogenic phenotypes. (A) Schematic represen...
(A) Schematic representation of the zebrafish embryonic vasculature, with red square marking the subintestinal vessels (SIVs). (B–G) Confocal images at 3 dpf showing ectopic sprouts (arrowheads) arising in the SIVs of apoba–/– apoBb.1–/– double mutants (E), and apoba–/– apoBb.1MO (F) and stalactite (stl) mutants (G), but not in WT (B), apoBa–/– (C), or apoBb.1–/– (D) animals. (H and I) Quantification of the number (H) and length (I) of ectopic sprouts at 3 dpf. n = 3, nWT = 45, napoBa–/– = 55, napoBb.1–/– = 23, napoBa–/– apoBb.1–/– = 31, napoBa–/– apoBb.1MO = 40, napoBb.1–/– apoBaMO = 21. The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey’s multiple-comparison test. ****P < 0.0001, **P < 0.01. (J and K) Confocal images at 5 dpf of apoBa–/– and apoba–/– apoBb.1MO embryos in the Tg(12xNRE:Egfp) reporter background. White arrowheads in apoBa–/– apoBb.1MO embryos point to endothelial cells (ECs) with active Notch signaling, in ectopic sprouts that failed to retract. n = 2, napoBa–/– = 10, napoBa–/– apoBb.1MO = 12. (L and M) Alkaline phosphatase (AP) staining of the SIVs at 3 dpf, showing inhibition of ectopic sprouting following intravascular injection of DiI-LDL into apoBa–/– apoBb.1MO. (N) Quantification of number and length of ectopic sprouts following intravascular injection of DiI-LDL (n = 3, napoBa–/– = 57, napoBa–/–+LDL = 43, napoBa–/– apoBb.1MO = 67, napoBa–/– apoBb.1MO+LDL = 57). The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey’s multiple-comparison test. ****P < 0.0001, ***P < 0.001. (O) AP staining of the SIVs of 3 dpf apoBa–/– apoBb.1MO embryos treated with OA, as quantified (P) (n = 3, napoBa–/– apoBb.1MO = 20, napoBa–/– apoBb.1MO+6μg OA = 19, napoBa–/– apoBb.1MO+10μg OA = 21, napoBa–/– apoBb.1MO+20μg OA = 22). The data are shown as the mean ± SEM, calculated using ANOVA followed by Tukey’s multiple-comparison test. Scale bar: (B–G) 50 μm, (J–O) 100 μm. P < 0.05 (considered significant versus control group).