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The maternal gene spn-4 encodes a predicted RRM protein required for mitotic spindle orientation and cell fate patterning in early C. elegans embryos

José-Eduardo Gomes, Sandra E. Encalada, Kathryn A. Swan^*, Christopher A. Shelton, J. Clayton Carter and Bruce Bowerman

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
^* Present address: Genomics, Exelixis Inc., San Francisco, CA, 94083, USA
Present address: Dept. of Comparative Genetics, GlaxoSmithKline, King of Prussia, PA 19406, USA

View larger version (48K): [in a new window]   Fig. 1. Nomarski photomicrographs of wild type (A-F) and spn-4(or80) (G-L) embryos. Photographs of wild-type and spn-4 embryos were taken at equivalent time points. Arrows point at centrosomes, and cell names are indicated. (M) Schematic drawings illustrate spindle angles observed at the one-cell and two-cell stages in wild-type and spn-4 embryos (see Materials and Methods). Each dash at the periphery of a quadrant represents a single scored embryo. In wild type, P0 spindle angles range from 4° to 14° (n=6), the AB spindle angles range from 74° and 89°, and the P1 spindle angles from 0° to 12° (n=11). In spn-4 mutant embryos, the following ranges were observed: or25 ‘P0’ varied from 3°-12° (n=5), or25 ‘AB’ and or25 ‘P1’ varied from 58°-89° and 32°-89° (n=11); or80 ‘AB’ and or80 ‘P1’ varied from 61°-89° and 7°-89° (n=8); or191ts ‘AB’ and or191ts ‘P1’ varied from 26°-90° and 7°-72° (n=8).

View larger version (54K): [in a new window]   Fig. 2. (A) Sequence alignment of full-length SPN-4 and related Drosophila, mouse and human proteins. Grey indicates regions of similarity. The mutations found in or25 and or191ts both result in G to E substitutions at highly conserved positions in the RRM (amino acids 213 to 300). (B) A molecular phylogeny based on the sequence similarities among SPN-4 related proteins. The length of a branch is proportional to the number of amino acid changes; SPN-4 is the most diverged member.

View larger version (72K): [in a new window]   Fig. 3. spn-4 is required for spindle rotation in two-cell stage par-3 mutant embryos. (A) Nomarski photomicrographs of par-3(it71) and par-3(it71);spn-4(RNAi) mutant embryos. Arrows point at the centrosomes. In par-3 embryos, both cells divide with longitudinally oriented spindles. In par-3; spn-4 double mutant embryos, both divide with transversely oriented spindles. In both par-3 and par-3; spn-4 embryos the two cells are similar in size and divide synchronously. (B) Schematic depiction of mitotic spindle orientations in two-cell stage in par-3 and par-3; spn-4 mutant embryos. In par-3 mutants, spindle angles range from 3° to 48° (n=7). Two allelic combinations of par-3; spn-4 double mutants were analyzed. In par-3(it71); spn-4(RNAi) spindle orientation ranges from 16° to 87° (n=7), in par-3(it71);spn-4(or191ts) from 20° to 89° (n=10).

View larger version (91K): [in a new window]   Fig. 4. Confocal photomicrographs of PAR-2 and P-granule immunofluorescence in wild type and spn-4 mutant embryos. P granules (green) are segregated to one daughter in the first two cell divisions in fixed wild-type (A,C,E) and spn-4(or80) embryos (B,D,F). In wild-type embryos and spn-4(or80) embryos, P granules are restricted to the posterior cortex during prophase in P1 (A,B). P granules are shifted laterally by anaphase in a spn-4(or80) mutant embryo (D). Note the linear arrays of P granules, particularly in the wild-type embryo (C). Thus, P granules may associate with astral microtubules, although most MTs appear not to have survived fixation in these embryos. During P1 prophase in wild-type and spn-4(or80) embryos, PAR-2 (green) is restricted to the posterior cortex of P1 (G,H). By metaphase, PAR-2 remains posterior in wild type (I). In spn-4(or80) embryos, PAR-2 shifts laterally to form a crescent centered around one pole of the ‘P1’ spindle (J). Microtubules (C,D) are red, DNA is blue.

View larger version (82K): [in a new window]   Fig. 5. (A) spn-4 embryos fail to produce intestinal and pharyngeal muscle cells, but make extra body wall muscle and germline. Fluorescence micrographs of terminally differentiated wild-type (left column) and spn-4 mutant embryos (right column). Intestinal cells were detected with the monoclonal antibody (mAb) J126; pharyngeal muscle cells with the mAb 3NB12; germline cells with polyclonal antibodies that recognize the P-granule component PGL-1; body wall muscle cells with a myo-3::GFP transgenic line. Micrographs of germline and body wall muscle cells in spn-4 embryos were obtained using confocal microscopy, the remaining micrographs were obtained using epifluorescence. See the text for numbers of embryos examined. (B) spn-4 is required for specifying pharyngeal muscle in par-3, mom-2 and pie-1 mutants. In par-3(it71) and par-3(it71);spn-4(or191ts) embryos, pharyngeal muscle cells were detected with the mAb 3NB12; in mom-2 and pie-1 RNAi mutant embryos pharyngeal muscle cells were detected using the GFP reporter ceh-22::GFP. In each case, only background staining is detected in the double mutant embryos. All micrographs obtained using epifluorescence microscopy. See the text for numbers of embryos examined.

View larger version (86K): [in a new window]   Fig. 6. PIE-1, SKN-1 and MED-1 expression in spn-4 embryos. PIE-1::GFP is segregated to the posterior daughter of P1 at the 4-cell stage in live wild-type embryos (A,C) and in live spn-4(RNAi) embryos (B,D). PIE-1::GFP localizes to centrosomes during mitosis (A,B) and to nuclei in older four-cell stage embryos (C,D). In wild-type embryos, SKN-1 protein was detected with a mAb at high levels in P1 and its daughters (E,G). In fixed 2-cell stage spn-4(or80) mutant embryos, SKN-1 is present at high levels in P1 in some embryos (F; see text). At the four-four cell stage, SKN-1 is present only in P1 descendants in fixed wild-type embryos (G). SKN-1 is present at high levels throughout a fixed spn-4(or80) embryo (H). In live wild-type embryos, MED-1::GFP is expressed zygotically in the two daughters of EMS. In live spn-4(RNAi) embryos MED-1::GFP in the two daughters of the anterior daughter of P1 (F). PIE-1 and SKN-1 micrographs were obtained using confocal microscopy, MED-1::GFP micrographs were obtained using epifluorescence microscopy.

View larger version (30K): [in a new window]   Fig. 7. P granules distribution in spn-4 mutant embryos. In wild-type embryos P granules are segregated to the germline precursors P1 (A), P2 (B), P3 (C), P4 (D) and finally the two daughters of P4, Z2 and Z3 (E). In spn-4(or80) embryos, P granules also are segregated to a single daughter during each of the first four divisions that normally produce the germline progenitor P4 (F-J). However, in most terminally differentiated spn-4(or80) embryos, at least six positively staining cells are detected (see Fig. 5A). During anaphase in two-cell stage embryos, P granules localize at the cortex near one pole of the posterior mitotic spindle in both wild-type embryos (A) and spn-4(or80) mutant embryos (F). Note the linear arrays of P-granules radiating out from the spindle pole in (A) and especially in (F), as in Fig. 4 (C,D). All images were obtained using epifluorescence microscopy.