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First published online 8 October 2003
doi: 10.1242/dev.00839

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Embryonic handedness choice in C. elegans involves the G protein GPA-16

¹ Department of MCD Biology, University of Colorado, Boulder, CO 80309, USA
² Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA

View larger version (37K): [in a new window]   Fig. 1. Handedness of embryonic and adult asymmetries. Colored letters A, P, D, V, R and L indicate anterior, posterior, dorsal, ventral, right and left, respectively. (A) A dextral (normal) adult hermaphrodite viewed ventrally, showing asymmetric placement of the gonad (g), intestine (i), excretory cell (xc), coelomocytes (cc), and the traverse around the vulva (v) of the ventral nerve cord (nc). (B) Cell positions and spindle orientations before, during, and after the second AB cleavage. AB cells are colored gray. No LR asymmetry is apparent at the four-cell stage. In ventral view, the EMS cell (broken circle) lies above the ABa and ABp cells. As ABa and ABp enter mitosis, their spindles (arrows) are oriented parallel to the LR axis. The spindles then skew as shown, always in a counter-clockwise direction (from this vantage point), so that the resulting daughter cells ABal and ABpl are shifted anteriorly relative to their respective sister cells ABar and ABpr, giving the embryo LR asymmetry with the dextral handedness that is maintained throughout development.

View larger version (11K): [in a new window]   Fig. 4. Map position and predicted structure of the gpa-16/spn-1 gene. (A) Map of the left arm of LGI, showing selected markers and the telomere (red square). Numbers above the line indicate map units. Regions deleted by the hDf10 mutation and duplicated by the sDp2 duplication are indicated by horizontal lines below the map. gpa-16(it143) was positioned close to bli-3 by two- and three-factor mapping. (The src-1 locus, used for mapping but not shown, is located one map unit to the left of unc-35.) (B) Structure of the gpa-16 gene as determined by comparing cDNA sequence to the genomic sequence. The extent of the deletion pk481 and the position of the point mutation it143 are indicated in blue. PCR primers designated 1F, 5R and 6R (sequences available on request) were used to assay presence of the wild-type and the deletion sequence in single mutant animals by direct PCR sequencing (see Fig. S2 at http://dev.biologists.org/supplemental/ and text). The sequence around the GA substitution (bold) in the mutant is TTTTCGACGTTGACGGACAGCGATCC.

View larger version (131K): [in a new window]   Fig. 2. Spindle orientations during cleavage of ABa and ABp in wild-type and it143 mutant embryos. All embryos are viewed ventrally just prior to and during second AB cleavage (see Materials and methods). Double-headed arrows indicate orientations of the ABa and ABp spindles. (A-C) Normal spindle orientations in a wild-type embryo. (D-L) The aberrant spindle orientations in three abnormal it143 mutant embryos at 20°C. The embryo in G-I developed into a sinistral adult, while the other two mutant embryos (D-F and J-L) were inviable.

View larger version (63K): [in a new window]   Fig. 3. Cleavage of blastomeres in culture from wild-type and it143 mutant embryos. (A) The regular helical pattern of cleavages exhibited by AB cells separated from a cultured four-cell de-vitellinized embryo (egg shell removed) (Edgar and Wood, 1993). (B,C) DIC images of such an embryo (arrows indicate AB cells) at the four- and six-cell stages, respectively. The EMS and P2 (arrowhead) cells have been left in place to provide positional markers. In C, images at two focal planes have been superimposed to show positions of the four AB blastomeres. (D) Example of the more random cleavage patterns seen in cultured it143 mutant AB cells. (E) DIC image of AB cells from a mutant embryo. Two AB cells isolated from a de-vitellinized four-cell embryo have divided with their spindles oriented in approximately the same direction as those in the previous cleavage, to give an almost linear arrangement of four AB cells. Similar results were obtained in 10 independent experiments (see text).

View larger version (67K): [in a new window]   Fig. 5. The gpa-16(it143) mutation reduces centrosomal association of the Gß protein GPB-1 in early cleavages at 25°C. Embryos were stained with DAPI (blue), rabbit anti-GPB-1 visualized using an FITC-conjugated secondary antibody (green), and mouse anti-tubulin visualized with a TRITC-labeled secondary antibody (not visible). Mitotic cells were identified by their chromosomal (DAPI) and spindle (anti-tubulin) morphologies (not shown), recorded prior to examination of anti-GPB-1 staining. (A) Wild-type embryo showing cortical (white arrows) and centrosomal (red arrowheads) GPB-1 staining in a prophase nucleus. (B) A gpa-16 embryo at 25°C shows cortical but not centrosomal staining; red arrowheads indicate a metaphase and a prophase nucleus. (C) gpa-16(it143) embryo at 16°C showing both cortical and centrosomal staining. See text for quantitative data.

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