Tol2 gene trap integrations in the zebrafish amyloid precursor protein genes appa and aplp2 reveal accumulation of secreted APP at the embryonic veins

Abstract

Background: The single spanning transmembrane amyloid precursor protein (APP) and its proteolytic product, amyloid-beta (Ab) peptide, have been intensely studied due to their role in the pathogenesis of Alzheimer's disease. However, the biological role of the secreted ectodomain of APP, which is also generated by proteolytic cleavage, is less well understood. Here, we report Tol2 red fluorescent protein (RFP) transposon gene trap integrations in the zebrafish amyloid precursor protein a (appa) and amyloid precursor-like protein 2 (aplp2) genes. The transposon integrations are predicted to disrupt the appa and aplp2 genes to primarily produce secreted ectodomains of the corresponding proteins that are fused to RFP.

Results: Our results indicate the Appa-RFP and Aplp2 fusion proteins are likely secreted from the central nervous system and accumulate in the embryonic veins independent of blood flow.

Conclusions: The zebrafish appa and aplp2 transposon insertion alleles will be useful for investigating the biological role of the secreted form of APP.

Fig. 1

Genomic organization of zebrafish appa and aplp2 genes showing the location of Tol2 gene trap integration sites and predicted polypeptides. A: Genomic structure of appa and aplp2. Blue boxes indicate exons. Location of Tol2 gene trap insertion site is shown. Black arrowheads indicate transposon arms. Purple, red and orange boxes represent the splice acceptor (SA), red fluorescent protein cDNA (RFP), and transcriptional termination/poly adenylation sequences (poly A), respectively. B: Schematic of domain organization of the App proteins and their proteolytic processing. The predicted Appa-RFP and Aplp2-RFP proteins contain nearly the entire ectodomain. Grey circles with attached lines represent the lipid bilayer positioned at the transmembrane domain. HBD1/GFLD: heparin-binding domain/growth factor like domain; Cu/Zn BD: copper- ad zinc- binding domain; DE: acidic-rich domain; KPI: Kunitz protease inhibitor domain; α, β and γ: α-, β- and γ-secretase cleavage sites; p3, α-secretase proteolytic product; Aβ: β-secretase proteolytic product, amyloid β peptide; AICD, APP intracellular domain; YENPTY, conserved C-terminal interaction motif.

Fig. 2

Phylogenetic and expression analysis of zebrafish appa and aplp2. A: Phylogenetic tree of amyloid precursor proteins. Zebrafish Appa and Aplp2 are shown in bold. The horizontal bars represent the percentage of amino acid substitutions required to generate the corresponding tree. Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis. B: Relative expression levels of appa and aplp2 throughout zebrafish development and in adult tissues as evaluated by reverse transcriptase-polymerase chain reaction. Expression of the ribosomal protein S6 kinase b, polypeptide 1b (rps6kb1) gene was used as a control.

Fig. 3

Expression of appa and aplp2 in developing central and peripheral nervous system of 36 hpf zebrafish embryos. A: Lateral view, appa expression in the eye lens, optic tectum, midbrain tegmentum, hindbrain, and posterior lateral line ganglion. B: Dorsal view, appa expression in the cerebellar plate and ganglia. C: Lateral view, appa expression in the neural tube and lateral line neuromasts (arrowheads). Line labeled “F” refers to position of cross-section shown in panel F. D: Camera lucida drawing of 35 hours post fertilization (hpf) zebrafish embryo (Kimmel et al., 1995). Line labeled “E, J” refers to position of cross-sections in panels E and J. E: appa expression in the posterior lateral line ganglia and lateral regions of medulla oblongata (arrowheads). F: appa expression in the neural tube, neuromasts of the lateral line, and pronephric ducts. G: Lateral view of aplp2 expression in the epiphysis, olfactory placode, cerebellum, ganglia, and neural tube. Line labeled “K” refers to position of cross-section shown in panel K. H: Dorsal view, aplp2 expression in the epiphysis, trigeminal ganglia, and ganglia in the hindbrain. I: Lateral view, aplp2 expression in the trigeminal ganglia, anterodorsal and posterior lateral line ganglia. J: aplp2 expression in posterior lateral line ganglion and lateral regions of medulla oblongata (arrowheads). K: aplp2 expression in lateral regions of the neural tube, floor plate, and pronephric duct. Inset shows higher magnification view of boxed region of neural tube. Arrow points to expression of aplp2 in the floor plate region. ADLG, anterodorsal lateral line ganglia; Cb, cerebellum; CeP, cerebellar plate; EL, eye lens; Ep, epiphysis; FP, floor plate; Ga, ganglion; Hb, Hindbrain; MO, medulla oblongata; MT, midbrain tegmentum; NL, neuromasts of lateral line; NT, neural tube; Op, olfactory placode; OT, optic tectum; PD, pronephric duct; PLLG, posterior lateral line ganglion; TG, trigeminal ganglia. Scale bars = 50 μm.

Fig. 4

Red fluorescent protein (RFP) expression in the Appa^is22Gt gene trap line. A–G,I: Fluorescence images of live Appa^is22Gt; Tg(flk1:moesin-gfp)^is1 embryos. A: Appa-RFP was detected throughout the veins and weakly in CNS and neural tube in 34 hours post fertilization (hpf) embryos. B: Enlargement of boxed area in (A) shows RFP fluorescence in the caudal vein and intersegmental vessels. C–F: Appa-RFP localization at 32 hpf embryos in the trunk (C,D) and the head (E,F). Appa-RFP fluorescence overlaps with GFP expression in endothelial cells in the veins (C,E), but not the arterial vessels (MTA, PPrA, LDA marked with yellow arrows in E). H: Immunolocalization with anti-RFP antibody shows Appa-RFP is localized to the caudal vein but absent from the dorsal aorta. G,I: Confocal images of cross-section through the trunk of a 36 hpf embryo shows Appa-RFP accumulation in the posterior caudal vein. CCV, common cardinal vein; CV, caudal vein; LDA, lateral dorsal aorta; MCeV, midcerebral vein; MtA, metencephalic artery; NT, neural tube; PHBC, primordial hindbrain channel; PPrA, primitive prosencephalic artery. Scale bars = 20 μm in C,D, 50 μm in E–I.

Fig. 5

Red fluorescent protein (RFP) expression in the aplp2^is23Gt gene trap line. Fluorescence images of live aplp2^is23Gt; Tg(flk1:moesin-gfp)^is1 embryos. A: Aplp2-RFP fluorescence in the caudal vein and intersegmental vessels in a 34 hours post fertilization (hpf) embryo. B: Enlargement of boxed area in (A). C–F: RFP fluorescence at 32 hpf was observed in the trunk (C,D) and the head (E,F). C,E: Aplp2-RFP fluorescence overlaps with green fluorescent protein (GFP) expression in endothelial cells in the veins, but not the arterial vessels (MTA, PPrA, LDA marked with yellow arrows in E). Weak expression is detected in the dorsal aorta. H: Immunolocalization with anti-RFP antibody shows Appa-RFP was in the caudal vein but absent from the dorsal aorta. G,I: Confocal images of cross-sections through the trunk of 36 hpf embryo shows Aplp2-RFP accumulation at the posterior caudal vein. CV, caudal vein; LDA, lateral dorsal aorta; MCeV, midcerebral vein; MtA, metencephalic artery; PHBC, primordial hindbrain channel; PMBC, primordial midbrain channel; PPrA, primitive prosencephalic artery. Scale bars = 20 μm in C,D, 50 μm in E–I.