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MEX-3 interacting proteins link cell polarity to asymmetric gene expression in Caenorhabditis elegans

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

View larger version (49K): [in a new window]   Fig. 1. PAL-1 immunolocalization in par and mex-3 single and double mutant embryos. Strains mutant for the gene indicated above each column were injected with dsRNA as indicated to the left of each row (see Fig. 2A for quantitative summary of staining experiments). (A) Wild-type four-cell embryos express PAL-1 in the two posterior blastomeres. (B) par-3 embryos express PAL-1 in variable patterns, including in all four blastomeres, as shown. (C) par-1 and (D) par-4 embryos do not express PAL-1. (E-H) PAL-1 is expressed in all blastomeres of all double mutants that include mex-3(RNAi). We observed that par-4; mex-3 embryos often failed to complete early cell cycle events normally, including nuclear division and cytokinesis. (I) par-1; par-3(RNAi) embryos and (J) par-4; par-3(RNAi) embryos do not express PAL-1. (K) Working model for control of PAL-1 expression. par-3 restricts par-1 and par-4 activities to the posterior, where they inhibit mex-3 repression of PAL-1. In this and all subsequent figures, embryos are oriented anterior left and dorsal upwards when it is possible to determine polarity. Scale bar: 10 µm.

View larger version (1K): [in a new window]   Fig. 2. Quantitative summary of PAL-1 and MEX-3 localization data. (A) PAL-1 localization patterns in four-cell embryos. Wild-type, par-1, par-4 or par-3 hermaphrodites, as indicated below the graphs, were injected with dsRNA, as indicated on the left. PAL-1 was scored as being present in none of the nuclei, all of the nuclei or patterned (see key). Patterned embryos express PAL-1 only in posterior blastomeres (see Fig. 1A), with the exception of par-3(it71) and par-3(RNAi) embryos, which express PAL-1 in two of four blastomeres but do not have an obvious polarity. Data for uninjected par-4(it57ts) and uninjected par-3(it71) embryos are taken from Bowerman et al. (Bowerman et al., 1997). (B) PAL-1 localization in embryos at different stages. Genotypes are indicated to the left and embryo stage is indicated below the graphs. In all cases in which PAL-1 was scored as patterned, PAL-1 was expressed only in posterior blastomeres (see Fig. 3B-D). (C) MEX-3 localization in wild-type and mutant embryos at different stages. Genotypes are indicated to the left and embryo stage is indicated below the graphs. In all two- and four-cell embryos in which MEX-3 was scored as patterned, MEX-3 was clearly brighter in the anterior blastomere(s) than in the posterior blastomere(s), as in the wild-type four-cell embryo in Fig. 5A2. In two- and four-cell embryos that were scored as ‘all’, MEX-3 was detected in all blastomeres with no clear asymmetries in signal intensity between anterior and posterior. Eight-cell and 14- to 18-cell embryos were not scored for MEX-3 patterning and were scored only for the presence or absence of MEX-3 in the majority of blastomeres. In this way, wild-type embryos expressing MEX-3 in P2 blastomere descendants (see Fig. 5A4) were scored as ‘none’ because they do not express MEX-3 in the majority of blastomeres. The data presented in these graphs reflect only the presence or absence of MEX-3 and do not reflect the intensity of MEX-3 signal, with the exception of clearly ‘patterned’ embryos, as described above. The total number of embryos scored is indicated above each bar in each case.

View larger version (44K): [in a new window]   Fig. 3. PAL-1 immunolocalization in wild-type, mex-6(RNAi) and spn-4(RNAi) embryos. Genotypes and embryo stages are indicated (see Fig. 2B for quantitative summary of staining experiments). (A-D) Wild-type embryos express PAL-1 only in posterior blastomeres starting at the four-cell stage. (E-H) mex-6(RNAi) embryos express PAL-1 at equally high levels in all blastomeres starting at the four-cell stage. (F) In the four-cell mex-6(RNAi) embryo shown, the two anterior cells are dividing before the posterior cells, demonstrating that mex-6(RNAi) embryos can maintain asymmetries in cell cycle time through the four-cell stage. For all 22- to 26-cell embryos, some nuclei are out of the plane of focus. (I-L) spn-4(RNAi) embryos express PAL-1 in all blastomeres at the four-cell stage; PAL-1 is often detected at lower levels in anterior blastomeres than posterior blastomeres, as evident in the eight-cell embryo shown (K). Approximately half of (L) spn-4(RNAi) 22- to 26-cell embryos express PAL-1 in all blastomeres as shown, the remainder express PAL-1 only in posterior blastomeres, similar to (D) wild-type embryos (see Fig. 2B for quantitation). spn-4(RNAi) embryos characteristically maintain asymmetries in cell cycle time as evident in the (K) eight-cell embryo shown, where the anterior blastomeres have proceeded to prophase while the posterior blastomeres are still in interphase.

View larger version (115K): [in a new window]   Fig. 4. Terminal phenotypes and cell type abundance in mip(RNAi) embryos. The DIC micrographs in the top row show that mex-3, mex-6 and spn-4 RNAi embryos produce differentiated cell types but do not undergo morphogenesis. In the remaining rows, body wall muscle, epidermis, intestine, pharynx and neurons were visualized as described in Materials and Methods. mex-3, mex-6 and spn-4 RNAi embryos produce excess body wall muscle and epidermal cells. mex-3 and spn-4 RNAi embryos produce a normal amount of intestine but reduced pharynx, while mex-6(RNAi) embryos produce no intestine and excess pharynx. By the time the neural GFP marker was expressed, neural tissue was dispersed throughout the embryo, making it difficult to judge the amount of neural tissue. Thus, we conclude only that neural tissue is present in mex-3, mex-6 and spn-4 RNAi embryos and do not make any conclusions about the relative amount. For all embryos, some fluorescent signal is out of the plane of focus. This is most noticeable in the intestine micrographs, where the wild-type intestine has elongated but the mex-3 and spn-4 RNAi intestine has not.

View larger version (79K): [in a new window]   Fig. 5. MEX-3 immunolocalization in wild-type and mutant embryos. Genotypes and embryo stages are indicated (see Fig. 2C for quantitative summary of staining experiments). (A) In wild-type embryos, MEX-3 is present through the eight-cell stage and essentially disappears by the 14- to 18-cell stage, except for weak staining in the P2 blastomere descendants. (B) In mex-6(RNAi) embryos, MEX-3 is undetectable by the eight-cell stage. (C,D) In spn-4(RNAi) embryos and mex-6(RNAi); spn-4(RNAi) embryos, MEX-3 persists through to the 14- to 18-cell stage. (E) In par-1 embryos, MEX-3 persists through the eight-cell stage but is undetectable by the 14- to 18-cell stage. (F,G) Defects in par-1 do not effect the MEX-3 phenotype of mex-6(RNAi) embryos or spn-4(RNAi) embryos. (H) In par-4 embryos, MEX-3 persists through the eight-cell stage but is undetectable by the 14- to 18-cell stage. (I) par-4 is required for the premature degradation of MEX-3 seen in mex-6(RNAi) embryos. (J) Defects in par-4 do not affect the MEX-3 phenotype of spn-4(RNAi) embryos. Slight variations in the intensity of MEX-3 signal in the micrographs are often the result of the embryo position in the focal plane, and may not reflect actual asymmetries in protein distribution. Only clear asymmetries that are visible in all focal planes, as in A2, were scored as patterned (see Fig. 2C). Representative embryos are shown. Owing to the high intensity of the signal, (C,D,G,J) spn-4(RNAi) two- and four-cell embryos are generally underexposed relative to other embryos. All eight-cell embryos are shown at the same exposure, with the exception of C3 and G3, which are underexposed relative to the other eight-cell embryos. All 14- to 18-cell embryos are shown at the same exposure with the exception of C4, G4 and J4, which are underexposed relative to the other 14- to 18-cell embryos. In two-cell (B1) mex-6(RNAi) and (C1) spn-4(RNAi) embryos, the anterior cell is typically larger than the posterior cell as in (A1) wild type, demonstrating that mex-6 and spn-4 RNAi embryos can maintain asymmetries in cell size. mex-6(RNAi) disrupts P-granule localization as monitored by MEX-3 localization to P-granules (Draper et al., 1996). Ten out of 10 mex-6(RNAi) embryos mis-segregate P-granules to all blastomeres of the four-cell embryo, while 10/10 of spn-4(RNAi) embryos segregate P-granules normally. (D) mex-6(RNAi); spn-4(RNAi) double mutant embryos show persistent MEX-3 expression characteristic of spn-4(RNAi) embryos, while 8/10 four-cell embryos mislocalize P-granules like mex-6(RNAi) embryos, indicating that both injected dsRNAs were effective in RNAi.

View larger version (1K): [in a new window]   Fig. 6. MEX-3 intensity and working model. MEX-3 intensity in ABa and EMS from individual (A) wild-type or (B) spn-4(RNAi) embryos. The average ratio between ABa and EMS intensities is given. (C) Comparison of MEX-3 intensity in wild-type and spn-4(RNAi) blastomeres. Units are arbitrary for MEX-3 intensity. (D) Working model for regulation of PAL-1 expression. MEX-3 exists in active and inactive (*) forms. The active form is normally associated with mex-5 and mex-6 and is competent to repress PAL-1. In the absence of mex-5 and mex-6, the inactive form is targeted for degradation by spn-4. In the absence of spn-4, the inactive MEX-3 accumulates and interferes with active MEX-3.

View larger version (53K): [in a new window]   Fig. 7. PAL-1 immunolocalization in par; mip(RNAi) double mutant embryos. Strains mutant for the gene indicated above each column were injected with dsRNA (left) (see Fig. 2A for quantitative summary of staining experiments). (A) par-3 mutants express PAL-1 in variable patterns, including in all four blastomeres, as shown. (B,C) par-1 and par-4 mutants do not express PAL-1. (D-F) All double mutants that include mex-6(RNAi) express PAL-1 at a high level in all four blastomeres. (G,H) par-3; spn-4(RNAi) embryos and par-1; spn-4(RNAi) embryos express PAL-1 in all four blastomeres. The characteristic par-3 cell arrangement (evident in A,D) was suppressed by spn-4(RNAi) in 9/10 (G) par-3; spn-4(RNAi) embryos. (I) par-4; spn-4(RNAi) embryos do not express PAL-1. We observed that par-4; spn-4(RNAi) embryos, like par-4; mex-3(RNAi) embryos (see Fig. 1), often failed to complete early cell cycle events normally, including nuclear division and cytokinesis.

View larger version (30K): [in a new window]   Fig. 8. Summary and proposed model. (A) Summary of PAL-1 (red) and MEX-3 (green) localization data. (B) Working model for regulation of PAL-1 expression. Anteriorly localized PAR-3 (blue) restricts cortical PAR-1(orange) to the posterior. par-1 then restricts MEX-5 and MEX-6 (purple) to the anterior. mex-5 and mex-6 protect MEX-3 from degradation in the anterior, enabling the continued repression of PAL-1. In the absence of mex-5 and mex-6 in the posterior, par-4 inactivates MEX-3 (*), and subjects it to rapid spn-4-dependent degradation. In the absence of spn-4, inactive MEX-3 can interfere with active MEX-3, resulting in ectopic PAL-1 expression.

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