The early C. elegans embryo is well suited for the study of microtubule function as it undergoes a variety of microtubule dependent processes during its first cell division. These processes include meiosis, centrosome duplication, establishment of embryonic polarity, maternal pronuclear migration, centrosome/pronuclear complex centration and rotation, mitosis, cytokinesis, and daughter nuclei positioning. We have used RNAi and genetic alleles of a beta tubulin gene to investigate the requirements for specific beta and alpha tubulin isoforms in the early C. elegans embryo. We initially isolated a temperature sensitive mutant,
qt1, which fails to undergo centrosome rotations in the P0, P1, and EMS blastomeres of the early embryo and determined that the
qt1 mutation affects
tbb-2, a beta tubulin gene. Interestingly,
tbb-2(
qt1) complements another allele of
tbb-2,
t1623 (Gonczy et al., 1999).
tbb-2(
t1623) embryos also fail to undergo centrosome rotation in P0 but have a higher rate of defects in pronuclear migration and meiosis than
tbb-2(
qt1). To define the null phenotype, we used RNAi to deplete
tbb-2 gene function from wild-type embryos.
tbb-2(RNAi) embryos divide normally except for an exaggerated spindle rocking in the first cell division. Two
tbb-2 deletion alleles obtained from the C. elegans Knockout Consortium show a similar early embryonic phenotype. Using RNAi to deplete
tbb-2 in
qt1 and
t1623 mutant embryos rescues the centrosome rotation defects indicating that
qt1 and
t1623 are gain-of-function alleles. Staining
qt1 and
t1623 embryos with an anti-tubulin antibody reveals additional differences in their phenotypes. In both mutants, spindle orientation in P0 is disrupted due to centrosome rotation failure, but some
t1623 embryos have stunted microtubules and microtubule kinetochore attachment often fails. In contrast,
qt1 microtubules look wild type. Thus, these two beta tubulin mutations appear to be affecting microtubule structure in different ways, perhaps by altering the dynamic instability of the microtubules or interfering with microtubule binding partners. To further investigate tubulin function, we examined the role of other tubulins present in the early embryo. Microarray analysis indicates that two beta tubulin genes and two alpha tubulin genes are expressed abundantly in the early C. elegans embryo. To determine if any of these tubulin subunits have specific functions, we used RNAi to deplete these gene products individually and in various combinations in wild-type embryos.
tbb-1(RNAi),
tba-1(RNAi), and
tba-2(RNAi) embryos have no early embryonic phenotypes, while
tbb-2(RNAi) embryos show the vigorous spindle rocking described above. As expected,
tbb-1(RNAi)
tbb-2(RNA-1) and
tba-1(RNAi)
tba-2(RNAi) embryos make no spindle structures, verifying the lack of cross-reactivity of our RNAi. We also used RNAi to remove all four pair-wise combinations of the alpha and beta tubulins to investigate whether different alpha and beta tubulin heterodimers might have specific functions. This experiment was suggested by the observation that, like
tbb-2(RNAi),
tba-2 (RNAi) rescues the
tbb-2(
qt1) phenotype indicating that alpha and beta tubulins might have specific partners. We found embryos missing the four combinations of alpha and beta microtubules divide like wild-type embryos with a low level of meiotic and centrosome rotation defects. These results indicate that the alpha and beta tubulins are functionally redundant in the early embryo.