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First published online 6 October 2004
doi: 10.1242/dev.01409

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Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish

Jiann-Ruey Hong^1,*, Gen-Hwa Lin², Cliff Ji-Fan Lin^2,3, Wan-ping Wang², Chien-Chung Lee², Tai-Lang Lin² and Jen-Leih Wu^2,*,

¹ Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, National Cheng-Kung University, Tainan 701, Taiwan
² Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Nankang, Taipei 115, Taiwan
³ Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 117, Taiwan

View larger version (123K): [in a new window]   Fig. 1. Electron micrographs of apoptotic cell death within the zebrafish embryo at shield stage. (A) A vertical view of a zebrafish embryo at shield (6 hpf) stage, revealing an apparently normal developmental pattern that includes the enveloping layer (EVL; black arrow), an apoptotic cell (short arrow) and a phagocytotic cell (long arrow). These features are located at the deep cell multilayer, 4-5 cell layers from the blastoderm margin (BM). (B) A horizontal view of a zebrafish embryo at the shield (6hpf) stage. The apoptotic cell corpses include a highly condensed chromatin cell (short arrow). A substantial quantity of enclosed membrane material derived from multi-micronuclei (long arrow) suggests that the apoptotic cell is entering the late apoptotic cell stage. (C) An apoptotic cell engulfed by neighboring cells, revealing a normal nucleus (N) and a substantial quantity of multi-micronuclei (arrows). Scale bars: 10 µM in A,B; 1 µM in C.

View larger version (55K): [in a new window]   Fig. 2. Sequence alignment of zebrafish psr and its expression profile. (A) A sequence alignment of the PSR protein with the positions of the functional domains shown and indicated by a series of boxes labeled A-D. Box A reveals a potential tyrosine phosphorylation site (from 100-108 aa; KCGEDNDGY), which lies within the predicted intracellular domain (SMART-TMHMM2). The jmjC, A domain (residues 143-206) depicted in box B is part of the cupin metalloenzyme superfamily. In box C (residues 257-287) is an estimate of the secondary structure based upon an assessment of topology (SMART-TMHMM2) and possible hydrophobicity domains. The membrane-associated domain (340-359) contained within box D predicts (SMART-TMHMM2) an extracellular domain that contains the rich supply of serine that potentially may be glycosylated site in this domain. In the block are included all species that are show the complete identity sequences and with the partial identity sequences in all species being shadowed. PSR-H: human PSR; PSR-M: mouse PSR; PSR-F: zebrafish PSR; PSR-D: Drosophila PSR; PSR-C: C. elegans PSR. (B) Expression pattern of PSR from early to late developmental stages probed by in situ antisense RNA hybridization. PSR staining was visualized with a blue color. Topic view of embryos shown in panels a-b, lateral view shown in panels d-f, anterior is to the right side. (a) 512-cell stage, psr is expressed in all cells examined. (b) 30% epiboly, psr is expressed only near the margin of yolk syncytial layer. (c) 2-3 somites, psr are expressed in the whole embryo including the ectoderm, mesoderm and endoderm especially, with the major location for psr being within the brain region (indicated by arrow) and the posterior of the embryo. (d) 24 hpf, psr is expressed principally in the whole notochord (indicated by arrow) from the anterior to the posterior regions, and secondarily distributed in the trunk, brain and the eyes. (e) 36 hpf, psr is expressed similarly to that for 24 hpf but here, psr can be seen to be expressed in the hatching gland and posterior of somite (indicated by arrow). (f) 3 dpf, the psr can be seen to be expressed in some organs such as the heart and the kidney (indicated by arrow). Scale bars denote 100 µm.

View larger version (68K): [in a new window]   Fig. 3. Morpholino-induced knockdown of psr results in the loss of the cell corpse-engulfment process and cell migration. (A,B) Control-MO-injected embryos. Lateral (A) and top (B) views of the normal somite (open square and arrow) reveal the clear furrow of somite at the 6-somites stage (12 hpf). (C) Lateral view of a PSR-MO-injected embryo reveals abnormal development of the somite. There is no evidence of the clear furrow and the somite is thinner. (D) In PSR-MO embryos, a substantial number of cell corpses accumulate in the somite region (arrows), and an apparent `hole' (long arrow) is formed on the surface of the somite. Scale bars: 100 µM. (E) In the control-MO group, complete epiboly and a normal brain (arrow) are observed; the clear bar of the eye can be seen. (F) In the PSR-MO group, incomplete epiboly (long arrow) and an abnormal brain (short arrow) are seen; the eye bar is not clearly evident. Scale bars: 250 µM. (G-J) The sequential pattern of cell corpse accumulation in whole embryos from 12-36 hpf. Embryonic cells were injected with control-MO and PSR-MO (40 ng per embryo) at the one-cell stage to block translation of the psr mRNA. (G,I) Control-MO groups at 12 hpf (G) and 36 hpf (I); embryos appear normal and no accumulation of cell corpses is evident. (H,J) PSR-MO groups at 12 hpf (H) and 36 hpf (J); embryos are abnormal and a gradual accumulation of corpse cells can be seen (arrows). Scale bars: 250 µM.

View larger version (82K): [in a new window]   Fig. 4. Apoptotic cells identified by Acridine Orange staining and TUNEL assay. Embryonic cells were injected with control-MO or PSR-MO (40 ng per embryo) at the one-cell stage to block translation of psr mRNA. (A-C,E) Acridine Orange staining of 17 hpf control-MO (A), 17 hpf PSR-MO (B), 14 hpf PSR-MO (C) and 17 hpf PSR-MO (E) embryos. (D) Phase-contrast image of the PSR-MO group at 17 hpf. (F-I) TUNEL assay at 12 hpf stage. (F,H) Phase-contrast images of control-MO (F) and PSR-MO (H) groups. (G,I) Observation of the embryos under a fluorescent microscope allows the identification of positive apoptotic cells in the PSR-MO group (I), when compared with the control-MO group (G). (D,E) Corpse cells released (see B) accumulate predominantly in the furrow between somites. AC, apoptotic cells; S, clear somites. Scale bars: 100 µM.

View larger version (46K): [in a new window]   Fig. 5. Effective delivery of morpholinos and ubiquitous psr inhibition by PSR-MO. (A) Detection of PSR protein in 24 hpf PSR-MO-injected embryos by western-blot analysis. Note the dose-dependent loss of PSR protein from these embryos (panel a, lanes 3-5). (panel b) Actin internal control. (B) One-cell stage embryos were injected with either 40 ng control-MO or 40 ng PSR-MO, then fixed at either the 36 hpf or 3 dpf stage for in situ hybridization. (a,d) Phase-contrast images of the control-MO group, showing normal embryonic development and psr expression patterns. (b,c,e,f) Phase-contrast images of the PSR-MO group, showing abnormal development, with delayed (b,arrows) or distorted (c, arrows) psr expression patterns. (g-i) Fluorescence microscopy of an Acridine Orange-stained control-MO embryo (g), a weakly defective PSR-MO embryo (h) and a severely defective PSR-MO embryo (i). Accumulated corpse cells are indicated by arrows.

View larger version (75K): [in a new window]   Fig. 6. Morpholino-induced knockdown of psr results in defective brain, heart and notochord development. Morphological analysis of embryos injected with 40 ng control (A,C,E,G,I,J) or PSR morpholinos (B,D,F,H,K,L), and examined at 36 hpf (A,B,E,F) or 3 dpf (C,D,G-I), following staining for pax2.1 (A,B) or nkx2.5 (E-H), or with Acridine Orange (AO; I-L), or in the absence of stain (C,D). (A,B) Top view, anterior to the right; stained with pax2.1. (C,D) Lateral views. The PSR-MO-injected embryo (B) shows an enlarged brain (long arrow) and an abnormal pax2.1 expression pattern (short arrows), when compared with the control-MO-injected embryo (A). The enlarged brain includes fore-, mid- and hindbrain morphologies (indicated by the open square in Fig. 6D; comparative control is shown in C). (E-L) Investigation of heart development. (E-H) At 36 hpf, the PSR-MO-injected embryos reveal an absence of normal heart formation (F, arrows and asterisks), when compared to the atria (A) and ventricles (V) observed in controls (E). By 3 dpf a tube-like heart has formed in the PSR-MO-injected embryos (H,K,L; arrows), as compared with normal controls (G,I,J). (M-U) The effect of knockdown of psr on notochord formation at 3 dpf. PSR-MO-injected embryos (N,P,R,T,U) show abnormal morphological formation such as the bending of the notochord (N, arrows), when compared with the control-MO group (M,O,Q,S). Scale bars: 100 µM.

View larger version (76K): [in a new window]   Fig. 7. The PSR morpholino-induced defect morphant can be rescued by injection of psr mRNA. PSR-MO at 20 ng and psr mRNA at 20 pg were injected at the one- to two-cell stage, embryonic development was traced at 12 (A-D), 48 (E-H) and 72 (I-L) hpf. (A,E,I) Untreated embryos; (B,F,J,N,O) embryos injected with control-MO; (C,J,K,P,S) PSR-MO-injected embryos; (D,H,I,R,S) embryos injected with PSR-MO and psr mRNA. (A-D) By 12 hpf, PSR-MO-injected embryos had accumulated corpse cells in whole embryo (E, arrows); these were not observed in the rescued embryos (D), or in controls (A,B). (E-H) At 48 hpf, PSR-MO morphants were severely defective (G, long arrow), whereas embryos in the rescued group were only weakly defective (H, short arrow). The morphology of the control-MO-injected embryos (F) is comparable to that of wild type (E). (I-L) At 3 dpf, rescued embryos (L) were compared with PSR-MO-injected embryos (K), wild-type variants (I), and control-MO-injected embryos (J). In contrast to the severely-defective morphant (K), the trunk and heart cavity of rescued embryos (L, arrows) resembles those of wild-type (I) and control-MO (J) embryos. (M) Estimation of the protection ability of differing doses of psr mRNA. D, dose; N, number of embryos. (N-S) At 3 dpf, rescued embryos (O,R) reveal normal morphology and a normal psr expression pattern in the kidney (arrows), as shown in control-MO embryos (N,Q), when compared to the PSR-MO-injected embryos (P,S). Scale bars: A-H, 250 µM; I-L, 200 µM; N-P, 100 µM; Q-S, 50 µM.

View larger version (28K): [in a new window]   Fig. 8. Depiction of the hypothesis that PSR plays a crucial role in engulfing apoptotic cell corpses that affect normal embryonic development and organogenesis. The zygotic embryo is featured as a newly-fertilized entity through to the completion of the first zygotic cell cycle at zero hours, then at the cleavage stage (0.75 hpf), and cell cycles 2-7, which occur rapidly and synchronously. Embryos enter the blastrula at 2.25 hpf, at which time the metasynchronous cell cycles rapidly give way to a lengthening at the 8-10 cell-cycle stage, when the asynchronous cell cycles at the midblastula transition and epiboly commence. From 5.25 hpf, the gastrula begins development of the three germ layers, when all cells require the facility of cell movement in order to achieve their developmental goals, such as the morphogenetic movements of involution, convergence, and extension from the epiblast, hypoblast and embryonic axis through to the end of epiboly. During the early gastrula stage (6 hpf), apoptotic death can occur during the entrance to the shield stage, if the apoptotic cell corpses are not removed promptly. Inhibited removal can result from an absence of a specific engulfing receptor, such as the PSR. The accumulated corpses gradually and progressively impede the cell movement and cell-cell interaction necessary for the triggering of signaling to activate the downstream developmental events. At the onset of organ development, cells in the embryo are associated with one of three germ layers, the ectoderm, mesoderm and endoderm, from the time of segmentation (10 hpf), when somites, pharyngeal, primordia and neuromeres develop, for primary organogenesis, and for the proper appearance of the tailbud. This influences the morphogenesis of organs at the pharyngula (24 hpf), hatching (48 hpf) and early larval (72 hpf) stages.

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