XB-ART-26411Dev Biol. November 1, 1989; 136 (1): 104-17.
The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus.
Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.
PubMed ID: 2680681
Article link: Dev Biol.
Genes referenced: act3 actl6a alb nefm tuba4b tubb
Antibodies referenced: Act3 Ab2 Fast Skeletal Muscle Ab1 Nefm Ab1 Tuba4b Ab11 Tubb Ab1
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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.|
|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.|
|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.|
|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.|
|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.|
|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.|
|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.|
|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.|
|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.|