Multiple Wnts Redundantly Control Polarity Orientation in C. elegans Epithelial Stem Cells.
Yamamoto Y, Takeshita H, Sawa H (2011) Multiple Wnts Redundantly Control Polarity Orientation in C. elegans Epithelial Stem Calls. PLoS genet 7(10): e1002308. Doi:10.1371/journal.pgen.1002308.
During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis (is the biological process that causes an organism to develop its shape).
In C. elegans, the Wnt/beta-catenin asymmetry pathway controls asymmetry in most somatic cell division occurring along the anterior-posterior axis (Mizumoto, 2007). For example, after asymmetric division, the beta-catenin homologs WRM-1 and SYS-1 accumulate in the posterior daughter nuclei, while POP-1.TCF localized more to the anterior than posterior nuclei. But how Wnts globally regulate the polarity of many cells is not known. (Figure. 1)
Figure 1. Pathways regulating the reciprocal asymmetry of POP-1 and SYS-1 in C.elegans. (Huang, 2007)
The Wnt pathway involves a large number of proteins that can regulate the production of Wnt signaling molecules, their interactions with receptors on target cells and the physiological responses of target cells that result from the exposure of cells to the extracellular Wnt ligands. Although the presence and strength of any given effect depends on the Wnt ligand, cell type, and organism, some components of the signaling pathway are remarkably conserved in a wide variety of organisms, from C. elegans to Humans.
To elucidate the mechanisms of polarity coordination, population of seam cells (epithelial stem cells) was focused.
Seam cells lie along the apical midline of the hypodermis, at the extreme left and right sides between nose and tail. During postembryonic development, they can act as stem cells to produce neurons of lumbar ganglia, tail spike neurons and phasmidial support cells, and sensory bristles along the sides and in the male tail. The seam cells are responsible for production of the cuticular alae in L1 stage, dauer larvae, and adults (Figure.2) (Altun, 2005).
Figure 2. . TEM(Transmission electron microscopy) transverse section of C.elegans during postembryonic development. (Altun, 2005)
For more information about seam cell, visit http://www.wormatlas.org/ver1/handbook/hypodermis/hypsupportseam.htm
At the L1 stage, the six seam cells V1-V6 are positioned on each lateral side of the animals, and repeatedly undergo self-renewing asymmetric cell divisions in each larval stage to produce anterior daughters that fuse with the hypodermal syncytium (hyp7) and posterior daughters that remain as seam cells (Sulston et al. 1977), (Figure 3).
Multiple Wnts control seam cell polarity.
To analyze the polarity of the seam cell division, elt-3::GFP was used because elt-3::GFP is expressed in hyp7 but not in seam cells (Gilleard,1999), (Figure 3).
Figure 1. Schematic drawing of seam cell divisions. Left: Anterior daughter of a seam cell fuses with hyp7 and becomes elt-3::GFP-positive. Right: seam cells (V1–V6) on the lateral sides of animals are polarized in the same orientation (represented by arrows) and divide asymmetrically. (Yamamoto et al. 2011)
About 1 hour after the division of seam cells at the L1 stage in wild-type animals, the anterior daughters fused with hyp7; their nuclei immediately began fluorescing like those of hyp7 cells because they incorporate GFP from the hyp7 cell. Therefore, daughter cell fates were determined from which they could deduce the division polarity type (normal, reverse, or loss of polarity) (Figure 4B) and the proportions of the polarity types of individual seam cells were mathematically converted to RGB. And this data was used to analyze the polarity of Wnt mutants.
Figure 4. Analyses of seam cell polarity(B)Examples of the polarity of seam cell (Vn) divisions (normal, reverse, or loss of polarity) as judged by the elt-3::GFP expression in the daughter cells. Merged differential interference contrast (DIC) and fluorescent images are shown. (C) An illustration of sample data for the polarity of seam cell divisions for various genotypes. Each colored box represents the polarity of an individual seam cell. Top, middle, and bottom numbers in the boxes are the percentages of individual seam cells with normal, reverse, and loss of polarity, respectively. An RGB color component was assigned for each polarity phenotype (normal = red; reverse = green; loss of polarity = blue), and the color of each box represents the combination of the calculated intensity of each RGB component per seam cell. Intensity was calculated as follows: 2552(% observed phenotype62.55), where 255 is the maximum intensity of each RGB component, and 2.55 is a factor for standardizing the phenotype percentage to the RGB scale. The colored boxes shown here represent the resulting four standard colors. Cells with 100% normal division-polarity are cyan (red 0, green 255, blue 255); cells with random division-polarity are lavender (red 128, green 128, blue 255); cells with 100% reverse division-polarity are magenta (red 255, green 0, blue 255); and cells with 100% loss of division-polarity are yellow (red 255, green 255, blue 0). In the similar illustrations in the following Figures, intermediate color compositions indicate relative tendencies towards a certain phenotype in relation to these four standards. (Yamamoto et al. 2011)
The C. elegans genome contains five Wnt genes, lin-44, cwn-1, cwn-2, egl-20, and mom-2. Before looking at the regulation of seam cell polarity, phenotypes of animals with mutations in one of the five Wnt genes were analyzed. Except for egl-20, the Wnt mutants showed weak phenotypes (Whangbo, 2000) and the hypothesized multiple Wnt genes will redundantly regulate seam cell polarity.
To test the idea, strain with mutations in all five Wnt genes (quintuple Wnt mutants) was constructed. The result showed that polarity of all the seam divisions was abnormal in quintuple Wnt mutants (Figure.5D, 5E).
Figure 5. Analyses of seam cell polarity. (D); lin-44(n1792); cwn-1(ok546); egl-20(n585) cwn-2(ok895); mom-2(ne874ts); vpIs1 (E); and lin-17(n3091) mom-5(ne12); cam-1(RNAi); vpIs1 (Yamamoto et al. 2011)
This result indicates that multiple Wnts are redundantly required for appropriately oriented seam cell polarity.
Most seam cells can be properly polarized by a single Wnt gene
Then what will happen to the polarity of seam cells with double, triple, or quadruple Wnt mutants?
The phenotype of quadruple (4x) Wnt mutants (mutation in mom-2) was quite similar to that of quintuple (5x) mutants (Figure.6). The result suggests that mom-2 has only minor functions in seam cell polarity
Figure.6 Seam cell polarity orientation is redundantly regulated by multiple Wnts. Polarity of seam cell division. The symbol (+M) indicates maternal contributions. In most cases, ‘‘loss of polarity’’ indicates divisions in which both daughter cells adopted the hyp7 fate, except for some divisions (indicated by asterisks in this and the following Figures; in all cases, one sample per cell) in which both daughters adopted the seam cell fate. (Yamamoto et al. 2011)
Analysis of triple Wnt mutants from these four Wnt mutations showed different result different cell type.
V1-V4: The phenotypes of V1-V4 Wnt triple mutants (cwn-1;egl-20;cwn-2) were similar to those of Wnt quadruple and quintuple mutants (Figure.7). The result suggests that polarity in these cells is regulated primarily by these three Wnt genes.
Figure 7. Seam cell polarity orientation is redundantly regulated by multiple Wnts. Phenotypes of V1-V4 Wnt triple mutants (Yamamoto et al. 2011)
V6: The most posterior seam cell, V6, was affected in quadruple Wnt mutants, but not in any triple or double combination analyzed. Therefore, the V6 cell polarity is redundantly regulated by the four Wnts.
So, V1-V4 and V6 cells are properly polarized by the presence of just one Wnt from among the three Wnts cwn-1, cwn-2 and egl-20 for V1-V4, or among the four Wnts lin-44, cwn-1, cwn-2 and egl-20 for V6.
Wnt genes control seam cell polarity through the Wnt/beta-catenin asymmetry pathway
To confirm that Wnt genes regulate the Wnt/beta-catenin asymmetry pathway, POP-1/TCF localization in triple Wnt mutants (cwn-1; egl-20 cwn-2) was analyzed because polarity of V1-V4 is disrupted in this triple Wnt mutants. The analysis showed that POP-1 asymmetry was abnormal in V1-V5 cells in the triple Wnt mutants (Figure 8A, 8D, 8E)
Figure 8. Wnts regulate the Wnt/ß-catenin asymmetry pathway in seam cells. (A) Each colored box represents the polarity of individual seam cell divisions as in Figure 1C, except that polarity was judged by the localization of GFP::POP-1 at the mid-L1 stage. (B–E) Examples of GFP::POP-1 localization in wild-type animals with qIs74 (GFP::POP-1) (B, C) and in cwn-1(ok546); egl-20(n585) cwn-2(ok895); qIs74 (D, E). Cell name colors indicate their polarity, as in Figure 1D-1F. GFP::POP-1 localization can be detected for about 1 hour after each seam cell division. Therefore, we can observe polarity of some but not all seam cells in individual animals. For example the V5 cell polarity in (D) could not be judged due to loss of the expression. A V1 cell is shown in (E) before its division. Scale bars: 10 mm. doi:10.1371/journal.pgen.1002308.g003 (Yamamoto et al. 2011)
As judged by elt-3::GFP expression, polarity reversal is more frequent than loss of polarity. Therefore, these Wnt genes control seam cell polarity via the Wnt/beta-catenin asymmetry pathway.
In C. elegans, asymmetric division and anterior-posterior polarization is regulated by a Wnt-signaling pathway.
As the results shown previously, seam cell polarity is redundantly regulated by multiple Wnt genes. In compound but not single Wnt mutants, most of the six seam cells, V1-V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes.
Strengths and Weaknesses
Very interesting method was used to illustrate the sample data for the polarity of seam cell division in Figure 6 and Figure 7. Assigned each polarity phenotype with different RGB color component based on their intensity and compared phenotypes of Wnt mutants
But we could question that would the result be the same for other species with high numbers of Wnt genes ?(Such as human. 19 Wnt genes). Because all metazoan species have multiple Wnt genes, their might be Wnt genes in any organism may have undiscovered functions that can contribute to the seam cell polarity. (Yamamoto et al. 2011)
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- Yamamoto Y, Takeshita H, Sawa H (2011) Multiple Wnts Redundantly Control Polarity Orientation in C. elegans Epithelial Stem Calls. PLoS genet 7(10): e1002308. Doi:10.1371/journal.pgen.1002308.