Early, nonciliary role for microtubule proteins in left-right patterning is conserved across kingdoms

Abstract

Many types of embryos' bodyplans exhibit consistently oriented laterality of the heart, viscera, and brain. Errors of left-right patterning present an important class of human birth defects, and considerable controversy exists about the nature and evolutionary conservation of the molecular mechanisms that allow embryos to reliably orient the left-right axis. Here we show that the same mutations in the cytoskeletal protein tubulin that alter asymmetry in plants also affect very early steps of left-right patterning in nematode and frog embryos, as well as chirality of human cells in culture. In the frog embryo, tubulin α and tubulin γ-associated proteins are required for the differential distribution of maternal proteins to the left or right blastomere at the first cell division. Our data reveal a remarkable molecular conservation of mechanisms initiating left-right asymmetry. The origin of laterality is cytoplasmic, ancient, and highly conserved across kingdoms, a fundamental feature of the cytoskeleton that underlies chirality in cells and multicellular organisms.

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.

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.

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.

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.

Fig. 5.

Mutant tubulin disrupts LR asymmetry in C. elegans embryos and cultured HL-60 cells. (A) Wild-type C. elegans generate one AWC^ON olfactory neuron cell, which expresses the reporter gene str-2p::GFP, and one AWC^OFF 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 AWC^ON 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.)