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FIG. 1. Western blot analyses. Total embryo protein from stage 40
embryos was prepared and separated by SDS-polyacrylamide gel
electrophoresis and either stained with Coomassie brilliant blue (not
shown) or electrophoretically transferred to nitrocellulose paper and
probed with various antibodies. Each lane contains the proteins of
approximately four embryos. The anti-acetylated tubulin antibody
6-llB-1 (lane A) reacted with a single band (small arrow) of 55 kDa.
This band migrated slightly slower than P-tubulin, recognized by the
anti-P-tubulin antibody Ei� (lane B). The anti-NFm antibody RM044
reacted with a single band of approximately 175 kDa (lane C). The
positions of molecular weight markers (aa macroglobulin, 180 kDa;
j3-galactosidase, 116 kDa; bovine serum albumin, 66 kDa; ovalbulin, 45
kDa) are marked on the left side of lane A.
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WC. 2. Microtubules in the egg. Whole-mount, immunoperoxidase stained eggs stained with anti-acetylated tubulin antibody (A) or
anti-beta-tubulin (B) reveal that the arrested meiotic spindles of the eggs contain acetylated tubulin. Staining of eggs with anti-beta-tubulin (C-E) at
various times after fertilization reveals the appearance and growth of the sperm aster (C, 15 min postfertilization; D, 20 min postfertilization;
E, 25 min postfertilization). In early stages of sperm aster growth, a distinctive structure is visable at the center of the growing aster (arrows in
C and D; the outer boundary of the sperm aster is marked by arrowheads in D and E). Staining of eggs with anti-acetylated tubulin 20-25 min
postfertilization revealed only a small structure (F, marked by arrow). At higher magnification (F�) this structure appears irregularly
spherical. Cortical immunofluorescence staining of the fertilized egg 75 min after fertilization (first cleavage at 90 min) was used to visualize
the cortical microtubule system (G- anti-/3-tubulin antibody/fluorescein-conjugated, anti-mouse immunoglobulin secondary antibody; rotation
direction marked by double-headed arrow). This cortical microtubule array was not stained significantly by anti-acetylated tubulin (H). Bar in
A marks 20 pm for A and B, bar in C marks 50 pm for C-F, bar in F� marks 10 pm; bar in G marks 10 Frn for G and H.
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FIG. 3. Microtubules in the blastula and gastrula stage embryo. Stage 7 (64112%cell) blastulas were stained with anti-acetylated tubulin
antibody (A-G, I) or anti-fi-tubulin (H). The anti-acetylated tubulin antibody labeled mitotic midbodies (A, C, arrows in A) and spindles (B, E).
The midbodies were striking in that they appear cylindrical, with an unstained core (C, D, D was taken from a 16-cell embryo). Spindles
appeared either rectangular (B, E) or rhombodal (G), with discrete polar regions (arrowheads). While interphase arrays were difficult to
visualize with anti-acetylated tubulin, the spindle poles of prophase cells could clearly be seen (F, marked by arrowheads). The interphase
array of microtubules could be seen in blastulas using anti-P-tubulin antibodies (H, i marks interphase cells, p marks prophase cell). During
late blastula/early gastrula stages, anti-acetylated tubulin stained both spindles and interphase microtubule arrays clearly (I, m marks
metaphase cell, p marks prophase cell, unmarked arrowhead points out cortical staining of blastomere). Staining of a gastrula stage embryo
with anti-fl-tubulin revealed the extensive subcortical microtubule system (J, subcortical microtubules marked by arrows). The difference in
the image in J, compared with H and I reflects focusing on the deeper cortical microtubule system. Bar in A marks 50 pm for A and B; bar in C
marks 10 pm for C-F; bar in H marks 50 pm for H-J.
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FIG. 4. The appearance of ciliated cells. By late gastrulation (stage
12/13), acetylated tubulin staining was found only in midbody microtubdles
(A, two midbodies marked by arrows). At stage 15, a tangential
optical section of the surface of an embryo revealed the presence
of scattered epidermal cells stained by anti-acetylated tubulin antibody
(B, two cells marked by arrows). An optical cross section of the
same embryo (C) revealed that these anti-acetylated stained cells
were located beneath the surface cell layer (arrows). A physical seetion
of a stage 30 embryo stained with anti-acetylated tubulin (D)
revealed that the ciliated cells were now located in the surface layer of
the epidermis (arrows). Bar in A marks 50 pm; bar in C marks 20 ym
for B-D.
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FIG. 5. Acetylated tubulin in the forming neural tube. A view of the
surface of a late neural fold stage embryo stained with anti-acetylated
tubulin reveals the presence of large, heavily stained fibers in
the as yet unclosed anterior (head) region of the neural tube (A).
Similar staining was observed in the unclosed posterior region as well
(B, neural axis marked by long-tailed arrows). These anti-acetylated
stained fibers disappeared soon after the closure of the neural tube.
Occasional mitotic spindles could be found within the neural tube
itself (arrowhead). Anterior (Ant) and posterior (Pos) ends of the
embryo are marked. Bar in A marks 20 pm for A and B.
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FIG. 6. Acetylated tubulin as a marker of neural differentiation. Following neural fold closure (stage 19) the staining of both the ciliated cells
of the epidermis (short arrow) and the neuronal processes and cell bodies within the neural tube (long arrow) can be seen in this oblique coronal
optical section (A) of a stage 19 embryo stained with anti-acetylated tubulin (POS marks posterior end of embryo, ANT marks anterior end of
embryo). In a coronal optical section of an anti-acetylated tubulin-stained stage 20 embryo (B), out-of-focus ciliated epidermal cells are visible
above the neural tube (short arrow). Within the neural tube lateral tract axonal processes are clearly stained (large arrow) and punctate
structures (arrowheads) are also visible. In a parasagittal optical section of an anti-acetylated tubulin-stained stage 23 embryo(C), both lateral
tract axons (arrowheads) and dorsal/ventral processes (arrows) can be clearly seen. An oblique coronal optical section through the dorsal
region of the spinal cord of a stage 30 embryo stained for acetylated tubulin (D) reveals the large Rohon-Beard cells located on either side of the
neural tube (short arrows) as well as motoneurons, interneurons, and neural processes located in the ventral region of the spinal cord (long
tailed arrow). An extramedullary sensory neuron is marked with an arrowhead. In a slightly oblique coronal optical section of another stage 30
embryo (E) extramedullary neurons (long arrow), cilated cells (thin arrows), and neuronal cell bodies associated with the lateral tracts (short
arrows) are all clearly stained by anti-acetylated tubulin. Numerous neurite tracts projecting into the periphery could be resolved at this stage
(arrowheads). Bar in A marks 80 km for A and B; the bars in C-E mark 30 pm.
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FIG. 7. Muscle-specific actin and myosin in the embryo. Embryos were stained with either an anti-muscle-specific aetin antibody (A, stage 16)
or an anti-muscle-specific myosin antibody (B,B�, stage 20). Both proteins appear to be present in the segmented myotome and within
prospective myotome, prior to its segmentation and rotation. Regions of axial mesoderm prior to rotation are marked by P, those caught in the
midst of segmentation and rotation are marked by R. In B�, an arrow marks what appears to be ends of the rotating myotomal cell mass. To
determine the relationship between the myotomal differentiation and the neuronal differentiation, we double stained embryos with anti-acetylated
tubulin and anti-muscle myosin (C). The lateral fibers stained by anti-acetylated tubulin (arrows) clearly run past the region of
segmented myotome (marked by arrowhead). The anterior (Ant) and posterior (Pos) ends of the embryo are marked. Bar in A marks 100 pm for
A and B; bar in C marks 100 mm; bar in B� marks 50 Wm.
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FIG. 8. Acetylated tubulin and NFm expression in the embryo. In a montage of parasagittal optical sections of a stage 32 embryo stained with
anti-acetylated tubulin (A) the ciliated epidermal cells can be clearly seen scattered over the embryo. The nervous system is also strongly
stained. In particular, cell bodies in the dorsal portion of the spinal cord can be easily resolved (arrow) along with fine processes extending into
the periphery (arrowheads). In B, a stage 32 embryo stained with anti-NFm reveals a similar pattern of staining within the neural tube (arrow
points to neuronal cell bodies in the dorsal region of the neural tube). Periphery motor axons can be clearly resolved (arrowheads). The pattern
of motoneurites in the periphery is diagrammed in C. In the tail/trunk region (marked TT) motor axons typically emerge from the neural tube
and run posteriorly toward the somite boundary. The positions of the somite boundaries were determined by examining the embryo under
polarization optics (see Fig. 9) and marked on the diagram with arrowheads and thin dashed lines at the somite boundaries. In the anterior
trunk region (marked AT), motor axons project both anteriorly and posteriorly. In the anterior-most region (marked Ant), motor axons project
primarily toward the anterior boundary of the somite. However, some motor axons clearly project caudally. The position of the eye, eye; otic
vesicle, OtV; and cement gland, cg, are marked in C. In B and C, a, b, and c correspond to the same regions shown in Fig. 9. Bar in B marks 100
pm for all parts.
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FIG. 9. Relationship between myotome and motoneurites. The relationship between the myotome and motoneurites can be better appreciated
in this image in which the myotomes have been visualized using polarization optics while the peripherally projecting motoneurites are
visualized by anti-NFm staining. This region is taken from Fig. 8B and the letters a, b, and c mark the similarly marked regions in Figs. 8B and
8C. It is clear that the motoneurites do not run down the intermytomal boundary (the myocommata, marked mc) but rather emerge from the
neural tube at a more midmyotomal position.
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