The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis

Abstract

Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation.

Figure 1

RNAi phenotype of mlc-4. In this and later figures, embryos are oriented with anterior pole to the left and dorsal pole up. All photomicrographs are reproduced at equal magnification. (A) Defects during the first embryonic mitosis in mlc-4(RNAi) embryos. Black triangles mark the two poles of the mitotic spindle in each embryo; white triangles indicate the cytokinetic furrow in each embryo. (a–d) First mitosis in a wild-type embryo. Note the posterior displacement of pronuclear meeting (a), the mitotic spindle (b), and the resulting formation of two unequally sized cells (d). (e–h) First mitosis in an mlc-4(RNAi) mutant embryo. Pronuclei meet in the center of the embryo (e), polar body extrusion fails (e–g), a symmetrically placed spindle is formed (f), and no attempt at a cytokinetic furrow is seen (the same defects were seen in all mlc-4(RNAi) embryos examined from pronuclear migration through the first mitotic cell cycle, n > 10). An abortive attempt to form a polar body is seen in the first frame. This polar body regresses during the first mitosis and results in the formation of a supernumerary nuclear structure, as seen in the last frame. (B) The position of pronuclear meeting and first mitotic spindle position in five different wild-type and mlc-4(RNAi) embryos are illustrated. Compared with wild type, the positions of pronuclear congression and of the first mitotic spindle is symmetric in mlc-4(RNAi) embryos. Bar, 10 μm.

Figure 2

Distribution of F-actin and nonmuscle myosin in wild-type and mlc-4(RNAi) embryos. Images were obtained by confocal microscopy (see Materials and Methods). (A) Distribution of NMY-2, a nonmuscle myosin II required for cytokinesis and polarity in the early embryo (Guo and Kemphues 1996). (first column) NMY-2 distribution in wild-type pronuclear, two-cell, and four-cell embryos. NMY-2 is uniformly localized to the cortex of early embryonic cells. (second column) NMY-2 distribution in mlc-4(RNAi) embryos. NMY-2 accumulates at the presumptive sites of cleavage furrow formation. At the pronuclear stage, a ring of NMY-2 localization is seen around the location of the polar body DNA (see third column for DNA localization; this localization was seen in 3/3 additional embryos scored at this stage). At the equivalent of the two-cell stage (middle), a surface focal plane image of the embryo shows a band of NMY-2 localization that extends around the circumference of the embryo (7/7 additional embryos at this stage showed similar localization). In later stages, shown in the bottom, NMY-2 is localized to bands that appear as patches in a confocal section. (third column) DNA localization in the same embryos as in column 2, as revealed by staining with DAPI. In the middle, note the extra, smaller, nuclear structure formed from the polar body chromosomes. (B) Distribution of F-actin revealed by rhodamine-conjugated phalloidin staining. (first column) F-actin distribution in wild-type pronuclear, two-cell, and four-cell embryos. F-actin appears uniformly distributed in the cortex of the embryonic cells. (second column) F-actin distribution in mlc-4(RNAi) embryos equivalent in age to pronuclear, two-cell, and four-cell embryos. As in wild type, F-actin is enriched at the cortex of the embryo. (third column) DNA localization in the same embryos as in column 2, as revealed by DAPI staining. Despite the localization of NMY-2 and F-actin to presumed regions of the cytokinetic furrow, no membrane ingression is seen in mlc-4(RNAi) embryos during mitotic cell cycles. Bars, 10 μm.

Figure 3

Cortical movement of yolk granules in wild-type, mlc-4 (RNAi), and nmy-2(RNAi) mutant embryos. Individual yolk droplets were followed by time-lapse digital photomicroscopy (see Materials and Methods) and were traced onto an outline of the respective embryos. Each dot along a line represents the position of the same yolk droplet at consecutive time intervals; the total elapsed time is shown below each embryo. The large shadowed circles represent the position of the paternal pronucleus in each embryo. (left) Cortical flow in a wild-type embryo. Note the cortical movement away from the paternal pronucleus. Analysis of 20 droplets in four different embryos yields an average speed of cortical yolk droplets at 4.5 ± 0.6 μm/min. (middle and right) Cortical yolk droplet movement in mlc-4(RNAi) and nmy-2(RNAi) mutant embryos, respectively. In these and three other embryos for each mutant, no directed yolk droplet movement was detected (20 granules for each mutant). Bar, 10 μm.

Figure 4

First mitotic spindle morphology and PAR protein distribution in wild-type and mlc-4(RNAi) embryos. (A–D) Spindle morphology in wild-type and mlc-4(RNAi) embryos. (A and B) Spindle morphology at telophase of the first mitosis as visualized by immunolocalization with antibodies recognizing tubulin. A shows the morphology of wild-type spindle poles. The centrosome at the posterior pole splits and migrates well ahead of the anterior centrosome, giving a barlike morphology to the posterior spindle pole. B shows an mlc-4(RNAi) mutant embryo at the equivalent stage of the cell cycle. The morphology of both the anterior and posterior spindle poles resemble the anterior spindle pole in a wild-type embryo. (C and D) DAPI staining of the same embryos in A and B. Both embryos are in telophase of the first mitosis. (E–H) Distribution of PAR-2 and PAR-3 proteins in wild-type and mlc-4(RNAi) embryos. Images were obtained by confocal microscopy (see Materials and Methods). (E and F) Distribution of PAR-2. In the wild-type embryo (E), PAR-2 is localized to the posterior cortical hemisphere of the embryo. In the mlc-4(RNAi) mutant embryo (F), cortical PAR-2 is restricted to a small, laterally displaced, patch. (G and H) Distribution of PAR-3. PAR-3 is enriched in the cortex at the anterior of a wild-type embryo (G), but is uniformly distributed to the cortex in the mlc-4(RNAi) mutant embryo (H). Similar distributions of PAR proteins were seen in all mlc-4(RNAi) embryos at these early stages (5/5 for par-2, 7/7 for par-3). Bar, 10 μm.

Figure 5

P granule localization in wild-type, mlc-4(RNAi), and nmy-2(RNAi) mutant embryos. Three pairs of columns are presented: the left-hand column of each pair shows P granule localization, whereas the right-hand column shows DNA localization in the same embryos as revealed by DAPI staining. (top) Localization of P granules in wild-type, mlc-4(RNAi), and nmy-2(RNAi) mutant pronuclear stage embryos. Note the posterior bias in P granule localization in the wild-type embryo but not in the mlc-4(RNAi) or nmy-2(RNAi) mutant embryos. (middle) P granule localization in two-cell stage embryos. In the wild-type embryo, P granules are seen associated with the posterior most cortex in the posterior cell. In the mlc-4(RNAi) and nmy-2(RNAi) mutant embryos, P granules are localized to the posterior end of the embryos. (bottom) P granule localization in four-cell stage embryos. In the wild-type embryos, P granules are distributed to one cell of the four-cell embryo. Intriguingly, in the equivalently staged mlc-4(RNAi) and nmy-2(RNAi) mutant embryos, we note that P granules appear to have coalesced into a patch that is similar in size to that in the wild-type embryo. Bar, 10 μm.

Figure 6

Schematic of the or253 deletion allele in mlc-4. Exon/intron structure is shown as predicted by Genefinder (Consortium, 1998) and confirmed by sequence from cDNA clones; boxes represent the exon sequences and the black shading indicates the protein coding regions. The mlc-4(or253) deletion removes 1,007 bp of sequence from the 29 codon of the first exon to beyond the stop codon in the last exon.

Figure 7

Embryonic elongation phenotype of mlc-4(or253). (A) Nomarski photomicrograph of a wild-type embryo just before hatching (12 h at 22°C). (B) Photomicrograph of an mlc-4(or253) embryo of similar age as in A. Note that the mutant embryo has only elongated to the twofold stage, whereas the wild-type embryo in A has elongated to the threefold stage (stages as in Wood 1988). mlc-4(or253) embryos do not elongate further and hatch as shortened, thicker larvae. (C) Fluorescence micrograph of phalloidin staining of actin in a wild-type larva shortly after hatching. Two of four bands of bodywall muscles are shown (micrograph is the same magnification as A, but the larva is no longer folded into the eggshell). (D) Phalloidin staining of actin in an mlc-4(or253) larva. Two of four bands of bodywall muscles are shown. Although the muscles appear structurally similar to wild type, the larva is shorter and thicker than wild-type larva. (E) Fluorescence micrograph of MH27 antibody staining in a wild-type larva of similar age as A. The epitope recognized by MH27 marks the boundaries of hypodermal cells. The focal plane shows 7 of 10 seam cells present along one side of the larvae. (F) MH27 staining in an mlc-4(or253) embryo of similar age to A, B, and E. 8 of 10 seam cells along one side of the larva are shown. Note that the seam cell morphology in the mlc-4(or253) embryo lacks the thin, elongated, appearance of seam cells in the wild-type embryo. (G) MH27 staining in a bean stage embryo that harbors an mlc-4::GFP expression construct and is just beginning the elongation process. Shown are 8 of 10 seam cells. (H) Same embryo as in G, double stained with antibodies recognizing GFP. Note that the anti-GFP antibody stains the seam cells, as revealed by comparison to the MH27 staining in G. All photomicrographs are at equivalent magnification. Bar, 10 μm.