Asymmetric p38 activation in zebrafish: its possible role in symmetric and synchronous cleavage

Abstract

Cleavage is one of the initial steps of embryogenesis, and is characterized by a series of symmetric and synchronous cell divisions. We showed that p38 MAP kinase (p38) is asymmetrically activated on one side of the blastodisc during the early cleavage period in zebrafish (Danio rerio) embryos. When a dominant negative (DN) form of p38 was uniformly expressed, blastomere cleavage was impaired on one side of the blastodisc, resulting in the formation of blastomeres with a large mass of cytoplasm and an enlarged nucleus on the affected side. The area affected by the DN-p38 expression did not correlate with the initial cleavage plane, but coincided with the side where dharma/bozozok, a dorsal-specific zygotic gene, was expressed (Yamanaka et al. 1998). Furthermore, UV irradiation and removal of the vegetal yolk mass before the first cleavage, both of which inhibit the initiation of the dorsalizing signals, abolished the asymmetric p38 activation. Our findings suggest that asymmetric p38 activation is required for symmetric and synchronous cleavage, and may be regulated by the same machinery that controls the initiation of dorsalizing signals.

Figure 1

Functional activity of zebrafish p38 and MKK3. (A) Expression levels of zebrafish p38a, p38b, and MKK3 throughout development. RT-PCR for zebrafish p38a, p38b, and MKK3 cDNAs are shown. pBLSK, positive control for specific primer pairs for zebrafish p38a, p38b, and MKK3 cDNA clones. max, loading control. (B) Activity of zebrafish p38 and MKK3 mutants expressed in 293T cells. Western blot of cell lysates from 293T cells transfected with expression vectors for WT-p38a, DN-p38a, WT-p38b, DN-p38b, DN-MKK3, CA-MKK3, or WT-JNK and human CA-MKK4 are as indicated. Activation of WT-p38a was detected in 293T cells expressing WT-p38a and CA-MKK3 by the anti–dp-p38 antibody (anti–ACTIVE p38™ antibody; lane 1) or in the cells treated with UV light (lane 4). No activation of WT-p38a was detected in the cells overexpressing either DN-p38a (lane 2) or DN-MKK3 (lane 3). DN-p38a and DN-MKK also suppressed UV-induced activation of WT-p38a (lanes 5 and 6). Activation of WT-p38b was also detected in 293T cells expressing WT-p38b and CA-MKK3 (lane 7) or in the cells treated with UV light (lane 10), but not in the cells expressing DN-p38b or DN-MKK3 (lanes 8, 9, 11, and 12). p38 (lanes 1–12), the amount of WT-p38a and WT-p38b loaded that was detected with the anti-myc antibody. Myc-tagged WT-JNK, which was coexpressed with human CA-MKK4 in 293T cells, did not react with the anti–dp-p38 antibody (bottom panel, left). Endogenous p38 in 293T cells expressing CA-MKK3 reacted with the anti–dp-p38 antibody, but not with anti-myc antibody (bottom panel, right). (C) Activation of ATF2 in cells expressing MKK3. 293T cells were transfected with the GAL4-ATF2 (pFA-ATF2) expression vector and GAL4-dependent luciferase reporter construct together with the expression vectors for WT-p38a, DN-p38a, CA-MKK3, or DN-MKK3 constructs, or with the empty vector to control for endogenous p38 activity. Luciferase activity was measured in cell extracts and normalized against β-galactosidase activity as an internal control. The relative value of the luciferase activity was plotted. Error bars represent the SD (n = 3). (D) Endogenous p38 activation in zebrafish embryos. Embryos were injected with RNA (125 pg) for DN-MKK3, DN-p38a, WT-p38a as indicated. Protein extracts of the embryos at the early blastula stage were immunoblotted with anti–ACTIVE p38™ antibody. Amounts of endogenous p38, DN-p38a, or WT-p38a were detected by anti-p38 antibody (bottom).

Figure 2

Asymmetric activation of zebrafish p38 during the early cleavage period. Top view of zebrafish embryos after fluorescent immunostaining with the anti-p38 antibody at the 2-cell (A), 4-cell (D), 8-cell stage (G), and the 16-cell stage (J), or with anti–dp-p38 antibody (anti–ACTIVE p38™ antibody) at the 2-cell (B and C), 4-cell (E and F), 8-cell (H and I), and the 16-cell stage (K and L). The distribution of p38 or activated p38 (red) was determined using a confocal microscope. Images were taken at a depth of 100 μm from the animal pole surface. Bar, 100 μm.

Figure 3

Impaired symmetric and synchronous cleavage by DN-p38. (A–D) Phenotypes after microinjection of the WT-p38a RNA (125 pg): 2-cell (A), 4-cell (B), 8-cell (C), and 16-cell stage (D). (E–L) Phenotypes after microinjection of the DN-p38a RNA (125–250 pg); DN-p38a RNA-injected embryos corresponding to the 2-cell (E and I), 4-cell (F), 8-cell (G and J), and 16-cell (H, K, and L) stage. (F–H) Incomplete cleavages were preferentially located on one side (right) of the first cleavage plane. (I, K, and L) Blastomeres with incomplete cleavages were preferentially located on one side (bottom portion) of the second cleavage plane. (I) Shortened cleavage furrow formation (arrowheads). Incomplete cleavages (arrows, the end of the cleavage furrow ingression) were observed in the intermediate region between the first and second cleavage planes (J). Localization of Myc-tagged DN-p38a (F′; see text). (M) Cross-sections of control embryos at the sphere stage (4 hpf). (N) Magnification of area outlined in M. (O) Cross-sections of the DN-p38a RNA-injected embryos at 4 hpf. (P) Magnification of outlined area in O. (Q) Schematic representation of cleavage formation. Each number denotes the order and sites of the cleavage furrow formation, and the letters in parenthesis correspond to the phenotypes shown in A–P. Asterisks mark each blastomere. Bars, 100 μm.

Figure 3

Impaired symmetric and synchronous cleavage by DN-p38. (A–D) Phenotypes after microinjection of the WT-p38a RNA (125 pg): 2-cell (A), 4-cell (B), 8-cell (C), and 16-cell stage (D). (E–L) Phenotypes after microinjection of the DN-p38a RNA (125–250 pg); DN-p38a RNA-injected embryos corresponding to the 2-cell (E and I), 4-cell (F), 8-cell (G and J), and 16-cell (H, K, and L) stage. (F–H) Incomplete cleavages were preferentially located on one side (right) of the first cleavage plane. (I, K, and L) Blastomeres with incomplete cleavages were preferentially located on one side (bottom portion) of the second cleavage plane. (I) Shortened cleavage furrow formation (arrowheads). Incomplete cleavages (arrows, the end of the cleavage furrow ingression) were observed in the intermediate region between the first and second cleavage planes (J). Localization of Myc-tagged DN-p38a (F′; see text). (M) Cross-sections of control embryos at the sphere stage (4 hpf). (N) Magnification of area outlined in M. (O) Cross-sections of the DN-p38a RNA-injected embryos at 4 hpf. (P) Magnification of outlined area in O. (Q) Schematic representation of cleavage formation. Each number denotes the order and sites of the cleavage furrow formation, and the letters in parenthesis correspond to the phenotypes shown in A–P. Asterisks mark each blastomere. Bars, 100 μm.

Figure 4

Cell lineage tracing. (A) DN-p38a RNA (125–250 pg) was injected into the yolk cell at 10 mpf, followed by the injection of a cell-tracing dye into one of the blastomeres at the two-cell stage. (B) Tracing dye (rhodamine/biotin-dextran, 2,000 kD) labeled one of the blastomeres of the two-cell stage embryos (red). (C) The labeled blastomere and its daughter cells (red) were located on one side of the blastodisc at the 1,000-cell stage. (D) Cross-section of DN-p38a RNA-injected embryo at the 1,000-cell stage (C, arrow, 100 μm from the animal pole surface). (E–G) Outlined area as in C. The blastomeres with cleavage defects were always located on the labeled side (red). (E–G) Marginal area (demarcated by dotted line) between normally divided (N) and enlarged blastomeres (EN). The blastomeres containing a large amount of cytoplasm and enlarged nucleus were strictly located on either the labeled (E) or the unlabeled side (G), or the labeled side included both enlarged and normal blastomeres (F). The red area in D–G is a superimposition of the tracing dye staining. Bars: (B and C) 110 μm; (D) 80 μm; (E–G) 4 μm.

Figure 5

Cleavage disruption by DN-p38 is cell autonomous. (A) Cell-tracing dye (rhodamine/biotin-dextran, 2,000 kD) was injected together with DN-p38RNA (125–250 pg) into one of the blastomeres of two-cell embryos. (B) Cross-section of DN/dye-coinjected embryo (A) at 100 μm from the animal pole surface at the 1,000-cell stage. The marginal area, which is the same as that outlined in Fig. 4 D, is shown. Normally divided (N) and enlarged blastomeres (EN) are demarcated by a dotted line. Bar, 4 μm.

Figure 7

Expression of dharma/bozozok in DN-p38a–expressing embryos. Whole-mount in situ hybridization with dharma RNA probe of WT-p38a or DN-p38a RNA–injected embryos (125–250 pg) is shown. In control embryos, dharma transcripts were detected in a small group of the blastomeres and the YSL on the future dorsal side at the sphere stage (A, arrowhead). In DN-p38a RNA–injected embryos, dharma transcripts were detected both in the enlarged cells inside the blastoderm (B, arrowhead) and the dorsal YSL (arrow) at 4 hpf. (C) Sagittal section of A: (arrowhead) dharma-expressing blastomeres; and (arrows) margin of the YSL. (D) Sagittal section of B: (arrowhead) dharma expression inside the blastoderm; and (arrow) the dorsal YSL. Bars, 100 μm.

Figure 6

Inhibition of p38 activation perturbs cytokinesis on one side of the blastomere. (A) BrdU staining of WT-p38aRNA–injected (125 pg) embryo. (B) Phalloidin staining of A. (C) Merged view of A and B. (D) BrdU staining of DN-p38a RNA–injected(125 pg) embryo. (E) Phalloidin staining of D. (F) Merged view of D and E. Cleavage furrow only formed on one side of the DN-p38a RNA–injected embryos (arrows) because of an incomplete mitotic segregation or cytokinesis on another side of the blastomere (arrowhead). Bar, 100 μm.

Figure 8

Effects of UV irradiation on asymmetric p38 activation before the first cleavage formation. Immunostaining of the embryos by either anti-p38 antibody (A and C) or anti–dp-p38 antibody (B and D) after UV irradiation (312 nm) for 10 min from 10 mpf (A and B) or from 25 mpf (C and D) was viewed using a confocal microscope. Asymmetric p38 activation (see Fig. 1B and Fig. C) was totally abolished by UV irradiation at 10 mpf (B, dotted line, outline of the blastodisc). (D) Activation of p38 after UV irradiation at 25 mpf. White lines indicate the site of the first cleavage furrow formation at the two-cell stage. Images were taken as described in the legend for Fig. 1. Bar, 100 μm.

Figure 9

Effects of yolk removal on p38 activation and dharma/bozozok expression. (A) Whole-mount in situ hybridization by dharma RNA probe. Removal of the vegetal yolk hemisphere abolished the expression of dharma (A, left indicated as yolk-depleted; 100%, n = 30) at the sphere stage. dharma transcripts were detected in the dorsal YSL of the control on which no operation was performed (right). (B) Immunostaining of the yolk-depleted embryo at the two-cell stage by anti-p38 antibody to show the distribution of p38, and p38 activation was probed using the anti–dp-p38 antibody (n = 20) (C). Images were taken as described in the legend for Fig. 1. Bars, 100 μm.

Figure 10

The asymmetric p38 activation is required for symmetric and synchronous cleavage in zebrafish. “X,” unknown factor(s) transported from the vegetal pole through the vegetal microtubule array. DD, dorsal determinants; and “Z,” putative signal(s) transported from the vegetal pole through the vegetal microtubule array that compete with p38. V, ventral side; D, dorsal side.