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Fig. 1. Tubulin mutations affect LR asymmetry before the first cleavage event. (A–C) Organ situs of stage 45 embryos scored by observation. (A) A wild-type embryo, ventral view, showing the normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). (B) A heterotaxic embryo (ventral view) showing reversal of all three organs, i.e., situs inversus, induced by misexpression of the tubulin mutant. (C) A heterotaxic embryo (ventral view) showing reversal of the heart. (D) Statistical comparison of heterotaxia levels scored at stage 45 in embryos injected with mutated α-tubulin mRNA at various early cleavage stages. (E) Types of heterotaxia observed from embryos injected with mutated α-tubulin mRNA at the one-cell stage. (F) Statistical comparison of heterotaxia levels in embryos injected with mutated Tubgcp2 mRNA at various early cleavage stages. (G) Types of heterotaxia observed from embryos injected with mutated Tubgcp2 mRNA at the one-cell stage. **P < <0.01, Welch’s t test, sample sizes as noted in Table S2. For both constructs, it is only the presence before two-cell stage that allows these reagents to randomize laterality.
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Fig. 2. Tubulin mutations perturb sidedness of asymmetric gene expression in Xenopus. Embryos injected with either tub4a mutant or Tubgcp2 mutant were processed for in situ hybridization at stage 22 with an Xnr-1 probe. (A) Both tubulin mutants deviated significantly (denoted with double asterisks) from control embryos (Tub4a: 64.7% incorrect expression, n = 51, P < <0.01 Welch’s t test; Tubgcp2: 32.5% incorrect expression, n = 83; control: 7.33% incorrect expression, n = 150, P < <0.01 Welch’s t test). (B–D) Xnr-1 expression pattern (purple stain) characterized in tubulin mutant mRNA-injected embryos. (B) Left expression indicated by one red arrow and one white arrow. (C) Absence of expression as indicated by two white arrows. (D) Bilateral expression as indicated by two red arrows.
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Fig. 3. Tubulin mutations affect early microtubule-dependent motor protein transport. (A) Embryos were injected into the very top of the animal pole shortly after fertilization with either a control β-gal mRNA, mRNA encoding the β-gal:KHC motor protein fusion construct or a mixture of β-gal:KHC and mutated tubgc2. At the four-cell stage, embryos were fixed and processed for β-gal staining then embedded with consistent LR orientation, sectioned and scored for localization of the blue stain in the blastomeres as described (19). (B) Control embryos, injected with β-gal mRNA, displayed little LR bias (19% right, 25% left, 56% bilateral), whereas embryos that had been injected with β-gal:KHC displayed a significant rightward bias in β-gal localization (33% right, 23% left, 44% bilateral). Coinjections of tubgcp2 with the β-gal:KHC reversed this rightward bias (30% right, 38% left, 33% bilateral). *P < 0.05, **P < 0.01, χ2 test. (C–E) Typical β-gal expression patterns observed in sectioned four-cell embryos.
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Fig. 4. Tubulin mutations alter biased Cofilin-1 expression. (A) tdTomato:Cofilin-1a fusion mRNA was injected into Xenopus embryos either alongside tub4a mutant mRNA, tubgcp2 mutant mRNA or on its own. (B) Injections were made shortly after fertilization; embryos were reared to stage 45 before scoring for tdTomato fluorescent signal. (C) Control embryos, injected with solely the tdTomato fluorescent marker displayed virtually no bias for signal localization (left localized:right localized ratio, L:R = 0.89, n = 117), whereas tdTomato:Cofilin-1a injected embryos displayed a leftward bias (L:R ratio = 1.35, n = 192). Embryos that had been coinjected with the tdTomato:Cofilin-1a and a tubulin mutant (either tub4a or tubgcp2) resulted in reversals in this bias (0.91 L:R ratio in tub4a mutant, n = 127; L:R ratio = 0.69 in tubscp2 mutant, n = 208). Blue dashed line indicates embryo midplane. (D) tdTomato expression patterns observed in stage 45 Xenopus embryos. *P < 0.05; **P < 0.01.
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Fig. 5. Mutant tubulin disrupts LR asymmetry in C. elegans embryos and cultured HL-60 cells. (A) Wild-type C. elegans generate one AWCON olfactory neuron cell, which expresses the reporter gene str-2p::GFP, and one AWCOFF cell, which does not (32). (B) C. elegans bearing mutations in aspartic acid (256th) and glutamic acid (259th) residues in α-tubulin exhibit a 2 AWCON phenotype at a frequency significantly higher than that caused by expression of wild-type TBA-9. These frequencies are quantified in C. Differentiated HL-60 cells were transiently cotransfected with GFP-Arrestin-3 (as marker of MTOC) and wild type tub-a6 (D) or mutant tub-a6 (E), and then exposed to uniform fMLP (100 nM), which induced polarization. The red arrow is drawn through the center of the nucleus, pointing to the centrosome, at 0 s as described (35). Final centrosome positions are indicated by the blue dots, relative to all red arrows coaligned. Whereas wild-type tub-a6 does not affect the leftward bias, mutant tub-a6 abolishes it (χ2 test, P < 0.01). (Scale bar, 20 μm.)
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Fig. S1. α-Tubulin and the γ-tubulin associated protein complex, Tubgcp2, are highly conserved among organisms. (A) Amino acid sequence alignment of the alpha/beta domain interface in various alpha tubulin orthologs. The conserved Ala180 (indicated with an arrowhead) was substituted with Phe in Xenopus laevis α-Tubulin mutants, as in the lefty2 Arabidopsis mutants. (B) Amino acid sequence alignment of the Grip motif 1 in various GCP2 orthologs (known as Tubgcp2 in Xenopus). The conserved Gly453 was substituted with Arg in Tubgcp2 mutants, as in spr3 Arabidopsis mutants.
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Fig. S2. Early mRNA injection gives rise to proteins by the first cleavage. (A) Injections of mRNA encoding tdTomato:cofilin fluorescent protein fusions were performed shortly after fertilization into one side of the egg (off-center). Embryos were allowed to develop to the late two-cell stage. (B) Embryos were processed for immunohistochemistry with an anti-RFP antibody (Rockland 600â401-379) showing expression of cofilin protein is already strong by the two-cell stage, consistent with our ability to exert functional changes at the two-cell by injections made soon after fertilization.
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Fig. S3. Injection into one of two early blastomeres allows targeting of the left or right side of embryo. Embryos were injected in one blastomere of the two- cell frog embryo with mRNA encoding the enzyme β-galactosidase. At stage 45, the embryos were developed using a chromogenic reaction, illustrating that mRNA injected in this manner targets cells on one side of the embryo. The first cleavage plane is indeed usually aligned with the prospective LR midline of the embryo; as in numerous studies, where one blastomere injection at the two-cell stage allows the other side to serve as an unaffected contralateral control, this confirms our ability to target early injections of cofilin to one side or the other.
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Fig. S4. Heterotaxia rates in tdTomato:Cofilin-a1 coinjections. Embryos injected with cofilin constructs were scored for positioning of the heart, gut, and gall bladder at stage 45. All of the cofilin constructs, but not the controls (injected with the fluorescent protein tdTomato alone) exhibited low but significant levels of heterotaxia. N’s are given in the figure; Student’s t test was used for significance calculations; *P < 0.05, **P < 0.01.
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Fig. S5. A model for tubulinâs role in LR asymmetry in Xenopus embryos. (A) In unperturbed embryos, the right-biased cytoskeleton (1, 2) is likely to derive from the orientation of the microtubule organizing center (centriole) with respect to the dorso-ventral and animal-vegetal axis, as proposed originally for a chiral âF-moleculeâ (3). Correct function of both α-tubulin (determining the structure of the microtubules along the animal-vegetal axis, and those involved in the dorso-ventral axis induction at cortical rotation shortly after fertilization) and γ-tubulin (mediating anchoring of LR-oriented microtubules to the MTOC) results in the proper linkage of the MTOC to the two axes, allowing intracellular transport of LR determinant cargo molecules (4) to the right side, which ultimately results in correct organ situs. (B) Mutations in the GTPase-activating domain of tubulin suppress correct microtubule dynamics and promote po- lymerization (5). Thus, the previously described (1) subtle right-ventral bending of the cytoskeleton would be altered when mutated α-tubulin and γ-tubulin subunits were introduced at the early one-cell stage. In the absence of rightward intracellular transport of maternal proteins important for subsequent LR patterning steps, the downstream steps are randomized, resulting in a mixture of wild-type and heterotaxic embryos.
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