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The dorsoventral axis of Xenopus embryos is specified by a rotation of the egg cortex relative to the underlying yolky cytoplasm. This cortical rotation, which occurs during the first cell cycle after fertilization, is dependent upon an array of parallel microtubules in the subcortical cytoplasm. We have used confocal immunofluorescent microscopy and microinjection of affinity-purified anti-XMAP230 antibody to address the role of XMAP230, one of three high-molecular-weight microtubule-associated proteins (MAPs) in Xenopus eggs, in the assembly and organization of the cortical microtubule array and specification of the dorsoventral axis. Confocal immunofluorescence microscopy revealed that XMAP230 was associated with cortical microtubules shortly after their appearance in the subcortical cytoplasm. XMAP230 staining became more prominent as microtubules were aligned and bundled during the cortical rotation. Loss of XMAP230 appeared to precede disassembly of cortical microtubules at the end of the first cell cycle. Deeper within the cytoplasm, XMAP230 was associated with microtubules early in the assembly of the sperm aster. However, later in the first cell cycle, XMAP230 was associated with microtubules (MTs) of the first mitotic spindle, spindle asters, and the cortical MTs, but not with microtubule remnants of the sperm aster. Microinjection of anti-XMAP230 antibody locally disrupted the assembly and organization of microtubules in the cortex of activated or fertilized eggs and resulted in defects in the dorsoventral patterning of embryos. These results indicate that the assembly and/or organization of cortical microtubules in fertilized Xenopus eggs and subsequent specification of the dorsoventral axis are dependent upon XMAP230.
FIG. 1. XMAP230 is associated with cortical microtubules during the first cell cycle after fertilization. Fertilized eggs were fixed in FGT
at the indicated times after fertilization (all times are normalized to the length of the first cell cycle, see text) and stained with antibodies
to a-tubulin (A, D, G, J, and M) and XMAP230 (B, E, H, K, and N). Merged images are shown in C, F, I, L, and O. (A–C) At 0.35 NT,
anti-a-tubulin reveals a few MTs in the vegetal cortex (A, red channel in C). Only a few of these MTs are stained by anti-XMAP230 (B, green
channel in C; arrows in B and C). (D–F) By 0.40 NT, both anti-a-tubulin (D, red channel in F) and anti-XMAP230 (E, green channel in F)
stain a sparse population of cortical MTs. The merged image (F) reveals that most MTs are stained by both antibodies. (G–I) At 0.50 NT,
a dense, but disordered, network of MTs is apparent in the vegetal cortex. All MTs are brightly stained by both anti-a-tubulin (G, red
channel in I) and anti-XMAP230 (H, green channel in I). (J–L) During the cortical rotation, MTs become aligned into wavy bundles (shown
at 0.70 NT). All MTs are brightly stained by both anti-a-tubulin (J, red channel in L) and anti-XMAP230 (K, green channel in L). (M–O)
Disassembly of the cortical MTs occurs just prior to first cleavage (here shown at 0.95 NT). Anti-a-tubulin (M) reveals that disassembly
occurs in a wave (here from upper right to lower left). The remaining MT bundles stain brightly with anti-XMAP230 (N). Individual MTs
in the zone of disassembly are only weakly stained by anti-XMAP230 (arrows in N) and thus appear red in the merged image (arrows in O).
All images are the projection of three to four optical sections. All scale bars are 25 um.
FIG. 2. XMAP230 is not uniformly distributed along cortical MTs. (AâC) Close examination of the cortical MTs fixed at 0.45 NT and
stained with anti-a-tubulin (A, red channel in C) and anti-XMAP230 (B, green channel in C) reveals that XMAP230 is not uniformly
distributed, but appears in small spots or foci along the MTs (arrows in B and C). (DâF) The punctate distribution of XMAP230 is also
apparent along individual MTs in eggs fixed at 0.70 NT and stained with anti-a-tubulin (D, red channel in F) and anti-XMAP230 (E, green
channel in F; arrows in E and F). All scale bars are 10 um.
FIG. 3. Association of XMAP230 with microtubules is temporally and spatially regulated during the first cell cycle. (AâC) Early in the first
cell cycle, MTs of sperm aster are stained by both anti-a-tubulin (A and B) and anti-XMAP230 (C) (fixed at 0.45 NT). Arrows in A mark the
pronuclei. (D) During the first mitosis after fertilization, MTs (here stained with anti-a-tubulin) of the sperm aster disassemble from their
proximal ends (arrowheads), creating a MT-free zone in which the mitotic spindle is assembled (arrow) (fixed at 0.75 NT). (E) Higher
magnification reveals remnants of the sperm aster stained with anti-a-tubulin (fixed at 0.80 NT). (F) MT remnants of the sperm aster are
not detectably stained with anti-XMAP230 during mitosis, although MTs in the vegetal cortex are brightly stained (arrows) (fixed at 0.80
NT). (G, H) The first mitotic spindles are brightly stained by both anti-a-tubulin (G) and anti-XMAP230 (H) (fixed at 0.75 NT). Scale bars
are 100 um (A and D) and 25 um (B, C, EâH).
FIG. 4. Microinjection of anti-XMAP230 into the vegetal cortex of oocytes and fertilized eggs locally disrupts the assembly and
organization of cortical microtubules. Anti-XMAP230 (A & B and D & E) or nonimmune rabbit IgG (C and F) was injected into the vegetal
cortex of stage VI Xenopus oocytes (AâC) or fertilized eggs (DâF). Oocytes were matured in vitro and artificially activated (see text). Cortical
MTs in activated oocytes and fertilized eggs were visualized with monoclonal anti-a-tubulin and Texas red anti-mouse IgG (red channels).
Injected anti-XMAP230 was detected with fluorescein anti-rabbit IgG (green channels). (A) A low-magnification view of anti-XMAP230-
injected oocyte after maturation and activation reveals substantial reduction in staining with anti-a-tubulin (red channel) and numerous
aggregates of the injected antibody (green channel) surrounding the injection site (asterisk). (B) At higher magnification, staining with
anti-a-tubulin (red channel) reveals the absence of MTs in the region occupied by the injected antibodies, which are detected as numerous
aggregates brightly stained with fluorescein anti-rabbit IgG (green channel, arrows). Assembly of cortical MTs in adjacent regions (lower
right in B) appears unaffected. Fixed at 60 min after activation, corresponding to 0.70 NT. (C) Injection of nonimmune rabbit IgG intooocytes had no effect on assembly of cortical microtubules. (D) A low-magnification view reveals similar disruption of MTs in the vegetal
cortex of a fertilized egg injected with anti-XMAP230 (fixed at 0.70 NT). An asterisk marks the injection site (red channel shows
anti-a-tubulin; green channel shows the injected antibody). (E) At higher magnification, a few MTs are visible near the injection site of this
fertilized egg fixed at 0.70 NT (arrowheads). Microtubules outside the zone of disruption (arrows in D and E) appear aligned, suggesting that
cortical rotation occurred. (F) Injection of nonimmune rabbit IgG had no effect on cortical microtubule assembly in fertilized eggs (fixed at
0.70 NT; asterisk marks the injection scar). Scale bars are 100 mm (A, C, and F), 50 mm (B and D), and 25 um (E).
FIG. 5. Embryos developed from eggs injected with anti-XMAP230 exhibit defects in dorsoanterior structures. (A) Embryos developing
from eggs injected with anti-XMAP230 exhibit a wide range of defects in dorsoanterior patterning, ranging from microcephaly (top right)
to complete lack of dorsoanterior structures (lower left). (B) Sister embryos developing from fertilized eggs injected with nonimmune rabbit
IgG (at stage 30) exhibit no axis defects.
