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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Dev Biol. 2010 Sep 16;348(1):58–66. doi: 10.1016/j.ydbio.2010.09.005

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Julie E Gleason 1, David M Eisenmann 1
PMCID: PMC2976807  NIHMSID: NIHMS238183  PMID: 20849842

Abstract

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates; Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem-cell like divisions in the L2–L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem-cell like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell - like behavior is conserved in nematodes.

Keywords: C. elegans, Wnt, stem cells, asymmetric cell division, seam cells

INTRODUCTION

Multipotent stem/progenitor cells function during metazoan development and adult life to increase the number of differentiated cells present in a developing tissue, while regenerating and maintaining the stem cell population. To accomplish this, stem/progenitor cells divide asymmetrically to generate one daughter that adopts a differentiated fate and one daughter that retains the stem cell fate (Knoblich, 2008). The nematode C. elegans is a short-lived organism that does not use stem cells for somatic tissue maintenance during its adult life. However, the ten lateral hypodermal cells known as the seam cells (H0 – H2, V1 – V6, T) are multipotent somatic cells that divide in a stem cell-like manner in each of the four larval stages to generate seam cells, other hypodermal cells, neurons and neuronal support cells (Figure 1A; Sulston and Horvitz, 1977; reviewed in Hall and Altun, 2008). For most seam cell divisions the posterior daughter retains the seam cell fate while the anterior daughter adopts a non-seam cell fate. Six seam cells (V1 – V6) also undergo a symmetric, proliferative division in the early second larval stage to generate two seam daughters, increasing the number of seam cells to sixteen. After their final division in the fourth larval stage, the 16 seam cells on each side exit the cell cycle and terminally differentiate as hypodermal cells.

Figure 1. Seam cell lineages and C. elegans Wnt signaling pathways.

Figure 1

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A) The division patterns of the seam cells during larval life (one side only). Hours post hatching and larval stages are indicated on the left. Cell fates: ‘S’ – seam cell; ‘H’ – hypodermal cell; ‘N’ – neuron; ‘X’ – programmed cell death. Not all cell fates adopted by seam cell descendants are indicated. B) Model for function of the ‘canonical’ Wnt signaling pathway that uses the beta-catenin BAR-1. In the absence of Wnt signal, the ‘destruction complex’ components APR-1, PRY-1, GSK-3 and KIN-19 act to facilitate destruction of BAR-1 by the proteasome. Wnt signals, acting through one or more Frizzled receptors and Disheveled homologs, inhibit the function of this complex. Stabilized BAR-1 translocates into the nucleus where it interacts with POP-1 to activate target gene expression. C) Model for function of the ‘Wnt/beta-catenin asymmetry’ pathway that uses the beta-catenins WRM-1 and SYS-1. The two daughters of a recent cell division are indicated; one receives Wnt signal, the other does not. In the cell that does not receive Wnt (left), high nuclear levels of POP-1 and low nuclear levels of SYS-1 lead to repression of target genes via interaction of POP-1 with transcriptional repressor proteins. In the cell that receives a Wnt signal (right), activation of Wnt and MAPK signaling pathways causes SYS-1 nuclear levels to increase and POP-1 nuclear levels to decrease, resulting in formation of a POP-1/SYS-1 heterodimeric transcription activator, and upregulation of target gene expression.

Several factors and pathways are known to regulate the cell division patterns and differentiation of the seam cells. For example, the temporal pattern of seam cell division is under the control of the well-studied ‘heterochronic’ gene pathway which functions in diverse cell types to specify specific cell behaviors at each larval stage (Moss, 2007; Vella and Slack, 2005), while the proliferation of the seam cells is regulated by the Runx and CBFβ homologs RNT-1 and BRO-1 (Kagoshima et al., 2007; Kagoshima et al., 2005; Nimmo et al., 2005b; Xia et al., 2007). Finally, the Wnt signaling pathway has been implicated in regulating the asymmetric cell division of two seam cells, V5.p and T, during early larval life (Eisenmann, 2005; Mizumoto and Sawa, 2007b).

Wnt signals play a prominent role in regulating stem cell behavior in vertebrates (Blanpain et al., 2007; Grigoryan et al., 2008; Reya and Clevers, 2005). A major pathway activated by Wnt ligands is the β-catenin-dependent Wnt signaling pathway (reviewed in (Clevers, 2006; Reya and Clevers, 2005)). In the absence of Wnt ligand, cytoplasmic β-catenin is targeted for ubiquitylation and degradation by the ‘destruction complex’ consisting of the scaffolding proteins Axin and APC and the kinases CK1 and GSK-3, which phosphorylate an amino terminal domain of β-catenin. Binding of Wnt to a Frizzled/LRP5/6 coreceptor complex disrupts the ‘destruction complex’ thereby stabilizing β-catenin, which translocates into the nucleus and interacts with LEF/TCF transcription factors to increase expression of Wnt pathway target genes.

C. elegans has four β-catenin genes, and two β-catenin-dependent Wnt signaling pathways have been characterized (reviewed in (Eisenmann, 2005; Mizumoto and Sawa, 2007b)). The ‘canonical’ or BAR-1 dependent Wnt pathway, which functions in several processes during larval life, utilizes the β-catenin BAR-1 and is similar in most aspects to the Wnt pathway described above (Figure 1B). The ‘Wnt/β-catenin asymmetry pathway’ utilizes the β-catenins WRM-1 and SYS-1 and components of a MAP kinase cascade and functions in many asymmetric cell divisions during C. elegans embryonic and larval life (Figure 1C; reviewed in (Eisenmann, 2005; Mizumoto and Sawa, 2007b)). Wnt pathway components such as the Frizzled receptor are asymmetrically localized in mother and daughter cells that utilize this pathway (Mizumoto and Sawa, 2007b). Ligand binding leads to an increase in nuclear SYS-1 and a WRM-1 dependent nuclear export of POP-1, the sole C. elegans TCF family member (Lin et al., 1995). Together, this results in an asymmetry between non-signaled cells, which have high levels of nuclear POP-1 and lower levels of SYS-1, and signaled cells, which have low levels of nuclear POP-1 and high levels of SYS-1. The altered SYS-1/POP-1 nuclear ratio shifts POP-1 from a transcriptional repressor to a transcriptional activator (via preferential interaction with SYS-1) and leads to upregulation of Wnt target genes (Kidd et al., 2005; Lo et al., 2004; Maduro et al., 2002; Phillips et al., 2007; Shetty et al., 2005).

Previous results have indicated a role for Wnt signaling in regulating the asymmetric cell division of two individual seam cells, V5.p and T: Wnt signaling is required for the cell V5.p to generate the postdereid neuronal structure in the L2 stage, and Wnt signaling also regulates the polarity of the cell divisions of the most posterior seam cell T and its progeny during the L1 and L2 stages (Goldstein et al., 2006; Herman, 2001; Herman and Horvitz, 1994; Mizumoto and Sawa, 2007a; Takeshita and Sawa, 2005; Whangbo et al., 2000). We asked if Wnt signaling might play a more general role in seam cell development, and found that the ‘Wnt/β-catenin asymmetry’ pathway, but not the BAR-1-dependent ‘canonical’ pathway, is required for the proper stem cell-like asymmetric division of most or all of the seam cells. Alterations in the stem-cell like division of the seam cells in the L2 – L4 stages was seen in animals in which Wnt signaling was perturbed: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. These results suggest that the regulation of asymmetric stem cell-like divisions by Wnt signaling is an evolutionarily conserved phenomenon conserved in nematodes as well as flies and vertebrates.

MATERIALS and METHODS

Standard methods

Standard methods were used for RNAi by feeding, observation of GFP reporter expression, heat shock induction of proteins from transgenic arrays, and immunostaining. Specific methods, along with a list of genes and alleles used in this work, are described in Supplemental Materials.

Seam cell division analysis

We determined the fates adopted by the daughters of seam cell divisions during larval development for three strains; scmp::gfp animals treated for RNAi with vector control, scmp::gfp animals treated for RNAi of pop-1, and wrm-1(ne1982ts); scmp::gfp animals grown at the restrictive temperature (26.5° C). Embryos from gravid adults from strains to be examined were isolated by bleach treatment, allowed to hatch in the absence of food, then newly hatched L1 larvae were placed on plates containing the appropriate bacteria (OP50 or HT115; see below) and incubated at either 20° C or 26.5° C. After eight hours of feeding, GFP+ animals that had completed the first larval seam cell division and that had ten seam cells per side were chosen for analysis (because of this we were unable to evaluate the effects of these treatments on the L1 seam cell division). For cell division analysis of pop-1(RNAi) and vector control animals, GFP+ L1 larvae were picked to new NGM plates seeded with either HT115 expressing pop-1 dsRNA, or HT115 with empty feeding vector, and allowed to develop at 20°C. Seam cell divisions were monitored at approximately three hour intervals using a Leica MZ16 F dissecting stereomicroscope until the late L4 stage when divisions ceased. For wrm-1(ts); scmp::gfp and wild-type scmp::gfp control lineage analysis, individual GFP+ L1 larvae larvae were picked to NGM plates seeded with OP50 and allowed to develop at 26.5°C, which results in faster development. No differences were observed in seam cell lineages for wild-type animals grown at 26.5° C. Seam cell divisions were monitored at approximately two hour intervals until the early L3 stage, at which point no further seam cell divisions were observed. All animals were later observed as young adults to verify that the terminal seam cell number was the same as that last seen by direct observation (indicating no additional divisions had occurred). Constant observation of all seam cells, as would be carried out in traditional lineage analysis based on observation under Nomarski microscopy conditions, could not be carried out due to sickness and lethality caused by GFP expression, however in many cases the division of individual seam cells into two daughters was directly observed.

The pattern of GFP expression after division was used to deduce the division pattern of the mother seam cell. When an scm-1::GFP+ seam cell divides, the two daughters initially express GFP, however GFP expression persists in the daughter adopting the seam cell fate, but is lost in the daughter adopting the non-seam fate. Individual seam cells were observed in division, and then the number of GFP+ cells in the position of the daughters of that division was determined at the next observation time. One GFP+ daughter was taken as evidence of a normal asymmetric division (except for the L2 proliferative division), while zero or two GFP+ cells were taken as evidence that the seam cell division had become symmetric and generated two non-seam daughters, or two seam daughters, respectively. Ectopic GFP+ daughters almost always went on to divide again, consistent with them having adopted the seam cell fate.

The increased penetrance of seam cell division defects (symmetric divisions, more than one division per larval stage) later in larval life is likely due to the fact that L1 stage RNAi larvae were used in these experiments, such that the RNAi effect may have been stronger after more time had passed.

RESULTS

Reduction of pop-1 activity leads to an excess of seam cells

It was previously shown that reducing the levels of the single C. elegans TCF family member POP-1 by RNA interference disrupts the asymmetric cell divisions of the seam cell T and its progeny (Herman, 2001). To determine if Wnt signaling plays a role in the asymmetric stem cell-like divisions of other seam cells, we used RNAi to reduce pop-1 function in a strain carrying the seam cell reporter scmp::GFP, which expresses GFP in all seam cells at all developmental stages (Koh and Rothman, 2001). GFP is expressed in 10 cells per side in the L1 larva, expands to 16 cells in the L2 after the symmetric proliferative division of six seam cells, and persists in young adults after seam cell divisions are completed (Figure 2A). To bypass the embryonic requirement for pop-1, we fed newly hatched L1 larvae on bacteria expressing pop-1double stranded RNA (which leads to a modest reduction in pop-1 transcript levels; Supplemental Figure 1) and examined GFP expression in these animals as young adults. The number of GFP+ seam cells per side in pop-1(RNAi) animals was greatly increased over RNAi controls, with an average of 50 GFP+ seam cells per side in adults (Figure 2B; Table 1). As POP-1 is the downstream transcriptional effector of Wnt signaling in C. elegans, this indicates that Wnt signaling likely regulates the fate of seam cells other than T.

Figure 2. Reduction of pop-1and wrm-1 activity alters the number of seam and hypodermal cells in opposite ways.

Figure 2

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Representative young adult hermaphrodites expressing GFP or YFP on one side are shown. Worms in (A–C) contain an integrated array with scm-1p::GFP, which expresses GFP in the seam cells at all developmental stages. Worms in (D–F) contain an integrated array with dpy-7p::YFP, which expresses YFP in hypodermal cells, including the hypodermal daughters of the seam cell divisions. Worms were grown on bacteria containing RNAi feeding vectors targeting the indicated genes: (A, D) empty vector control; (B, E) pop-1; (C, F) wrm-1.

Table 1.

The Wnt/β-catenin asymmetry pathway regulates terminal seam cell number.

strain n ave. # adult seam cells range
wild-type(FV RNAi) 20° C 410 16.0 ± 0.6 15–18
wild-type (FV RNAi) 24.5° C 362 16.3 ± 0.7 15–19
wild-type (FV RNAi) 26.5° C 99 16.2 ± 0.8 14–18
pop-1(RNAi) 20° C 90 50.6 ± 8.4* 33–67
pop-1(RNAi) 24.5° C 111 50.3 ± 11.5* 22–82
pop-1(RNAi) 26.5° C 50 43.9 ± 7.3* 29–59
wrm-1(ne1982ts) 15° C 50 15.8 ± 0.6 14–17
wrm-1(ne1982ts) 24.5° C 346 15.8 ± 1.9 11–21
wrm-1(ne1982ts) 26.5° C 101 7.2 ± 2.2* 3–12
wrm-1(RNAi) 26.5° C 50 9.1 ± 2.6* 4–15
sys-1(RNAi) 24.5° C 80 16.3 ± 0.8 15–19
sys-1(RNAi) wrm-1(ne1982ts) 24.5° C 76 10.4 ± 3.3** 3–17
sys-1(RNAi) pop-1(RNAi) 24.5° C 60 25.9 ± 11.1*** 15–61
wrm-1(ne1982ts) pop-1(RNAi) 26.5° C 50 30.4 ± 7.0*** 18–45
lit-1(RNAi) 20° C 50 12.9 ± 2.7* 6–16
mom-4(or11) 20° C^ 50 15.3 ± 1.0* 13–16
lit-1(RNAi) mom-4(or11) 20° C^ 50 7.8 ± 2.3**** 4–13
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All strains analyzed contained the integrated scmp::GFP reporter construct. ‘FV’ indicates that the control strain was fed on bacteria containing the ‘feeding vector’ with no inserted C. elegans gene fragment.

^

homozygous mom-4(or11) escapers were scored from unc-13(e1091) mom-4(or11)/hT2 heterozygous animals. Results from t tests:

*

p < 0.01 compared to wild type (FV);

**

p < 0.01 compared to wrm-1(ts) 24.5°;

***

p < 0.01 compared to pop-1(RNAi) at appropriate temperature;

****

p < 0.01 compared to lit-1(RNAi) and to mom-4(or11).

The ‘Wnt/β-catenin asymmetry pathway regulates seam cell number

To determine which C. elegans Wnt signaling pathway was involved in regulating seam cell fate, we examined which beta-catenin functions in this process. Worms carrying a putative null allele of bar-1 had no significant difference in terminal seam cell number (Supplemental Table 1), however, wrm-1(RNAi) animals and wrm-1(ts) animals grown at 26.5° C had fewer seam cells (averages of 9.1 and 7.2 seam cells, respectively) (Figure 2C; Table 1). Also while sys-1(RNAi) had no effect in a wild-type background, we observed an effect in two sensitized backgrounds. First, when grown at 24.5° C, wrm-1(ts) animals have no defect in seam cell number; however, when sys-1(RNAi) was performed on wrm-1(ts) animals at 24.5° C, the average number of seam cells dropped from 15.8 to 10.4 (Table 1). Second, the average number of seam cells dropped from 50.3 in pop-1(RNAi) animals to 25.9 in sys-1(RNAi);pop-1(RNAi) animals.

The involvement of WRM-1 and SYS-1 in regulating terminal seam cell number implicates the ‘Wnt/β-catenin asymmetry pathway’ in control of this process. Consistent with this, mom-4(or11) and lit-1(RNAi) animals had reduced numbers of seam cells (Table 1). As reported previously using the general hypodermal reporter ajm-1::GFP (Takeshita and Sawa, 2005), animals treated with both mom-4 and lit-1 RNAi had even fewer seam cells, with an average of 7.8 per side, similar to the effect seen with reduction of wrm-1 function. In addition, RNAi against three factors that function in POP-1 nuclear export in the embryo, CRM-1, IMB-4 and PAR-5 (Nakamura et al., 2005), lead to fewer terminal seam cells in a wrm-1(ts) sensitized background (Supplemental Table 2). Finally, reduction of function of PRY-1/Axin, APR-1/APC and KIN-19/CK1, but not GSK-3, led to an increase in seam cell number in an otherwise wild type background. The increase in seam cell numbers caused by reduction of apr-1 and kin-19 function was dependent on wrm-1activity but not bar-1activity (Supplemental Table 3). An increase in seam cell numbers upon apr-1RNAi was previously reported (Mizumoto and Sawa, 2007a).

The function of wrm-1, sys-1, pop-1, mom-4, lit-1, pry-1, apr-1 and kin-19 in the regulation of terminal seam cell numbers indicates that the ‘Wnt/beta-catenin asymmetry’ pathway regulates seam cell fate and/or terminal differentiation. Although this pathway was previously shown to regulate asymmetric cell division by the T seam cell (Goldstein et al., 2006; Herman, 2001; Herman and Horvitz, 1994; Mizumoto and Sawa, 2007a; Takeshita and Sawa, 2005; Whangbo et al., 2000), the large change in seam cell number we observed, and the distribution of GFP+ cells along the anterior-posterior axis, suggest that this pathway acts in many seam cells.

Wnt signaling regulates the choice between seam and non-seam fate

In the asymmetric divisions of cells in the early embryo as well as the somatic gonad precursors in the larva, Wnt signaling lowers nuclear POP-1 levels and raises nuclear SYS-1 levels in one of the daughter cells, such that a POP-1/SYS-1 complex activates expression of Wnt target genes that are otherwise repressed when POP-1 levels are high (Mizumoto and Sawa, 2007b). The seam cell daughters have been show to display nuclear POP-1 asymmetry at several larval stages, with anterior daughters having higher POP-1 levels than posterior daughters (Herman, 2001; Kanamori et al., 2008; Lin et al., 1998). These results and our data suggest a model in which the ‘Wnt/β-catenin asymmetry pathway’ functions to lower nuclear POP-1 levels in the posterior seam cell daughters, causing these cells to adopt a seam cell fate, while anterior seam cell daughters retain high nuclear POP-1 levels, causing them to adopt a non-seam/hypodermal cell fate.

This model makes three predictions. First, the model predicts an inverse correlation between the number of cells adopting the hypodermal fate and the number adopting the seam cell fate. We examined this using dpy-7::YFP, a reporter expressed in hypodermal cells but not seam cells (Myers and Greenwald, 2005)(Figure 2D). As predicted, pop-1(RNAi) animals, which have too many seam cells, have a decrease in hypodermal cell number from 53.8 in control animals to 41.7 in pop-1(RNAi) animals (Figure 2E and data not shown), while wrm-1(RNAi) animals, which have fewer seam cells, show an increase in hypodermal cell number from 53.8 to 58.4 (range 54–65; Figure 2F).

Second, the model predicts that if daughter cells are undergoing a seam-to-hypodermal fate transformation in Wnt pathway mutants, then the posterior daughters should have higher nuclear POP-1 levels than in wild-type. Indeed, antibody staining for POP-1 in the lateral hypodermis of L3 stage larvae showed that in wild-type animals 69% of seam cell daughter pairs showed a higher level of nuclear POP-1 in the anterior daughter (hypodermal fate) than the posterior daughter (seam cell fate), while in wrm-1(ts) animals grown at 26.5° only 16% of seam cell daughter pairs showed this pattern, while 82% of the pairs had equal brighter POP-1 staining in both daughters (Figure 3).

Figure 3. Reduction of WRM-1 levels results in a loss of POP-1 nuclear asymmetry in the seam cell daughters.

Figure 3

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The top panels show midbody photos of L3 stage animals grown at 24.5° C and stained with antibodies against POP-1 (stains nuclei) and AJM-1 (stains cell junctions). (A) wild-type; (B) wrm-1(ne1982). White lines indicate seam cell daughters. Analysis of multiple stained animals scored for nuclear POP-1 asymmetry in seam cell daughters is shown in the table below. The percentage of daughter pairs showing greater staining in the anterior daughter (A>P), equal staining in the two daughters (A=P), or greater staining in the posterior daughter (A<P) is indicated.

Third, this model predicts that ectopically activating Wnt-dependent gene expression in anterior daughters should cause them to adopt the seam cell fate, while inhibiting Wnt-dependent transcriptional activation in the posterior daughters should cause a decrease in adoption of the seam cell fate. To ectopically increase Wnt-dependent transcription activation we used a previously characterized gain-of-function variant of BAR-1 expressed from the heat shock promoter. It is known that mutation or deletion of the amino terminal GSK-3 phosphorylation sites in beta-catenin creates a stabilized protein that can activate Wnt target gene expression in the absence of Wnt signaling (Polakis, 2000). Expression of such a delNTBAR-1 variant from the heat shock promoter leads to Wnt gain-of-function phenotypes in C. elegans (Gleason et al., 2002). Since BAR-1 and SYS-1 interact with the same domain on POP-1 (Kidd et al., 2005), we reasoned that excess BAR-1 might bind to POP-1 already present at Wnt target genes in the seam cells and activate expression of genes normally activated by SYS-1/POP-1. Consistent with this idea, microarray and qRT-PCR experiments indicate delNTBAR-1 can activate expression of genes that are dependent on SYS-1 for full expression (B. Jackson and D. M. Eisenmann, unpublished results). We found that animals containing the hsp::delNTbar-1 transgene had a dramatic increase in terminal seam cell number (≥ 40 seam cells per side) when given a single heat shock at any time from before the L1 molt through the mid L3 stage (Figure 4). This indicates that increasing the amount of beta-catenin transcriptional activator and presumably activating Wnt target gene expression leads to an increase in seam cell number. To decrease Wnt-dependent transcriptional activity in the seam cells we used a heat shock inducible dominant negative version of POP-1 truncated at the amino terminus to remove the β-catenin binding domain (Korswagen et al., 2000). This dominant negative POP-1 has been shown to cause Wnt loss-of-function mutant phenotypes and to alter the asymmetric division of the T seam cell when overexpressed (Herman, 2001; Korswagen et al., 2000). Animals containing the hsp::delNTpop-1 transgene had a decrease in terminal seam cell number when given a single heat shock at any time from the mid L1 through the early L4 stages, with the strongest effects seen around the L1 and L2 molts (Figure 4). Taken together, these data suggest that Wnt-dependent transcription activation induces the seam cell fate, and that lack of Wnt-dependent gene activation causes cells to adopt the hypodermal cell fate.

Figure 4. Activation of Wnt signaling leads to an increase in seam cell number and inhibition of Wnt signaling leads to a decrease in seam cell number.

Figure 4

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Populations of worms were given a single heat shock at the indicated times post-hatching and allowed to develop at 20° C. The number of GFP+ cells per side was determined when animals reached the young adult stage. Triangles indicate control animals. Diamonds indicate animals expressing a dominant, activated BAR-1 (ΔNTBAR-1) from an integrated transgenic array. Squares indicate animals expressing a dominant-negative POP-1 (ΔNTPOP-1) from an integrated transgenic array. The approximate durations of the four larval stage at 20° C are indicated above.

Perturbation of Wnt signaling alters the asymmetric stem cell-like divisions of the seam cells

To more precisely determine the effect of compromising Wnt pathway function on seam cell fate, we determined the division pattern of seam cells during larval development in individual live pop-1(RNAi) animals, control RNAi animals, and wrm-1(ts) animals raised at 26.5° (Figure 5 and Supplemental Figure 2A–J). All strains contained scmp::GFP. From each strain, individual L1 animals that had undergone the first seam cell division correctly and had 10 seam cells were examined every few hours on a fluorescent stereomicroscope from the L1 stage to a point when no further seam cell divisions were observed, and the division pattern of individual seam cells in the L2, L3 and L4 stages was deduced from the pattern of GFP expression observed in the seam cells and their daughters (see Materials and Methods).

Figure 5. The ‘Wnt/β-catenin asymmetry pathway’ regulates the stem cell-like divisions of seam cells.

Figure 5

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Lineage of the seam cell V3.p from three single worms: A) scmp::GFP worm treated with RNAi empty vector control at 20° C; B) scmp::GFP worm treated for pop-1RNAi at 20° C; C) wrm-1(ts) scmp::GFP worm grown at 26.5°. Time post-hatching is indicated on the left. Observation began after the seam cells had undergone their first (L1) division and continued at 2–3 hour intervals until the mid L4 stage. A solid line indicates expression of GFP; a grey line indicates loss of GFP expression. A dotted line indicates that the exact time of GFP loss was not observed. The presumptive fates based on GFP expression are shown as ‘H’ for hypodermal and ‘S’ for seam. wrm-1(ts) animals were grown at 26.5°C, which results in faster development. No lineage defects were observed in wild-type animals grown at 26.5°C. Complete cell lineages are shown in Supplemental Figure 2.

In pop-1(RNAi) animals, the asymmetric stem cell-like divisions of the seam cells often became symmetric with both daughters adopting the seam cell fate (Figure 5B and Supplemental Figure 2B-E). For example, in the V3.p lineage shown in Figure 5B, the cells V3.pap, V3.papa, V3.papp, V3.pppp, V3.ppppa and V3.ppppp all divided symmetrically to give two daughters that retained scmp::GFP expression. The cells undergoing hypodermal to seam cell fate transformation often went on to divide again, as expected for a seam cell (V3.pap, V3.pppp). Symmetric seam cell divisions were most often seen in the L3 and L4 stage, although a few symmetric divisions were observed in the L2 stage (V1.pp, V2.pa, V2.pp in Suppl. Fig. 1C; V3.pp in Suppl. Fig 1E). We also observed cases where a seam cell appeared to divide more than once per larval stage (eg., the V3.ppp lineage in Figure 5B), a phenomenon restricted to the L2 stage in wild-type. Finally, the H0 cell, a seam cell that does not normally divide, was seen to divide in pop-1(RNAi) animals (Supplemental Figure 2B, C and E). Taken together, these phenomena account for the higher number of seam cells in pop-1(RNAi) adults compared to wild type.

In wrm-1(ts) animals raised at 26.5°, the normally asymmetric stem cell-like divisions of the seam cells also became symmetric; however, in this case both daughters adopted a non-seam cell fate and did not divide further (Figure 5C and Supplemental Figure 2F-J). For example, the V3.p cell shown in Figure 5C underwent a normal symmetric proliferative division, but the daughter cells V3.pa and V3.pp both divided to give two daughters that lost scmp::GFP expression. Therefore in this animal the seam cell V3 generate no adult seam cell progeny. In general, in wrm-1(ts) animals at 26.5° the seam cells divided two times in the L2 stage as in wild type, but the second division was often symmetric generating no seam cells, and in those animals which did generate seam cells from these L2 divisions, the seam cells did not divide further during larval life, suggesting they were defective in execution of the seam cell fate. These defects account for the decreased number of seam cells in wrm-1(ts)animals at restrictive temperature.

Together these results indicate that perturbation of the ‘Wnt/β-catenin symmetry pathway’ disrupts the asymmetric stem cell-like division of the seam cells, so that the divisions become symmetric, either generating two daughters that adopt the seam cell fate, or two daughter that adopt the non seam fate.

Mutations in Wnt genes and Wnt receptor genes have little effect on terminal seam cell number

To attempt to identify the Wnt ligand(s) and Wnt receptor(s) regulating the asymmetric stem-cell like division of the seam cells, we looked at terminal seam cell numbers in animals containing one or more mutations in Wnt genes or Wnt receptor genes. Although we were unable to examine all possible combinations of mutations in the five Wnt genes due to previously described embryonic lethality (Gleason et al., 2006), surprisingly, we did not find any combination of Wnt mutations that caused even a modest change in terminal seam cell number (Supplemental Table 4). Even a lin-44; cwn-1; egl-20 cwn-2 quadruple mutant strain had an average of 16.6 cells per side expressing the seam cell GFP reporter, and RNAi of mom-2 in this background did not substantially change this number. In addition, a mutation affecting MIG-14/Wntless, which acts in Wnt sending cells and is required for many Wnt-dependent processes in C. elegans (Banziger et al., 2006; Eisenmann and Kim, 2000), did not alter adult seam cell number (Supplemental Table 4). As with the Wnt genes, no single mutation in any of the five Wnt receptor genes had any effect on terminal seam cell number on its own (Supplemental Table 5). We did find that introduction of either a lin-17 mutation or mom-5 RNAi treatment in the sensitized background of a wrm-1(ts) mutation at 24.5° caused a statistically significant decrease in terminal seam cell number, from an average of 15.8 cells per side to 12.9 and 13.8 respectively. However, the lin-17; mom-5(RNAi); wrm-1(ts) animals were no worse than lin-17; wrm-1(ts) alone. This suggests that at least two of the Wnt receptors, LIN-17 and MOM-5, may have some function in seam cell asymmetric division; however to date we have not identified a Wnt gene or combination of Wnt genes that appear to affect the larval stem-cell like division of any seam cell other than T.

Wnt signaling acts in parallel to RNT-1 and BRO-1

The transcription factors RNT-1(Runx) and BRO-1(Brother/CBF) also regulate seam cell division: reduction-of-function for rnt-1 or bro-1 causes a small decrease in seam cells numbers, while a strain carrying bro-1::GFP and rnt-1::GFP constructs has an increased number of seam cells (Kagoshima et al., 2007; Kagoshima et al., 2005; Nimmo et al., 2005a; Xia et al., 2007). RNAi of rnt-1 or bro-1 in a pop-1(RNAi) background reduced the terminal number of extra seam cells compared to pop-1(RNAi) (Supplemental Table 6), suggesting these factors function downstream of POP-1 or in a parallel pathway. However, there was no change in bro-1 or rnt-1 transcript levels in pop-1(RNAi) animals as assayed by qRT-PCR, and no change in the GFP expression from bro-1::GFP and rnt-1::GFP reporters in pop-1(RNAi) animals (data not shown), suggesting that the Wnt pathway may act by a different mechanism, in parallel to RNT-1/BRO-1, to regulate seam cell division.

DISCUSSION

The lateral epithelial seam cells of C. elegans are born embryonically and generally divide once during each larval stage in a stem cell-like division, generating one daughter that terminally differentiates as a hypodermal syncytial cell, neuron, or neuronal support cell (most often the anterior daughter) and one daughter that retains the stem cell fate (most often the posterior cell) (Hall and Altun, 2008). Previous work has implicated Wnt signaling in the control of the asymmetric division of the seam cells and/or their fate specification. First, several studies have indicated that the ‘Wnt/beta-catenin asymmetry’ pathway is required for asymmetric division of the most posterior seam cell (T) (reviewed in (Eisenmann, 2005 Mizumoto and Sawa, 2007b)). Second, the ‘Wnt/BAR-1′ pathway is required for ability of the seam cell V5.p to divide asymmetrically and generate a sensory structure called the postdereid (Austin and Kenyon, 1994; Hunter et al., 1999; Whangbo et al., 2000). Third, asymmetric nuclear localization of the TCF homolog POP-1 between the daughters of seam cell divisions has been noted in several larval stages (Herman, 2001; Kanamori et al., 2008; Lin et al., 1998). Fourth, asymmetric localization of Wnt pathway components has been observed in several seam cells (Herman, 2001; Kanamori et al., 2008; Mizumoto and Sawa, 2007a; Takeshita and Sawa, 2005). Finally, changes in terminal seam cell number had been noted in apr-1 and mom-4; lit-1 mutant strains(Huang et al., 2009; Mizumoto and Sawa, 2007a; Takeshita and Sawa, 2005).

We examined whether Wnt signaling is involved in the asymmetric cell divisions of seam cells other than T during larval life, and our results suggest that the stem cell-like division of all of the seam cells is globally regulated by the ‘Wnt/beta-catenin asymmetry’ pathway, utilizing the beta-catenins WRM-1 and SYS-1 and the TCF homolog POP-1. By examining markers of cell fate and following seam cell divisions in live animals, we found that when this Wnt pathway is perturbed, the normally asymmetric divisions of the seam became symmetric, often either generating two daughters with seam cell fate, or two daughters with the non-seam fate. More specifically, treatments which are predicted to increase the ratio of POP-1 to SYS-1 in the seam cell daughter nuclei led to a reduction in terminal seam cell numbers, while reduction of pop-1 function by RNAi caused a drastic increase in terminal seam cell numbers. In addition, gain-of-function reagents that should activate or inhibit expression of Wnt target genes caused increases or decreases in seam cell number respectively. Defects were seen in both the L2 proliferative division of the seam cells, and the stem cell-like asymmetric divisions in the L2, L3 and L4 stages. These results suggest that the asymmetric stem cell-like divisions of the seam cells along the anterior-posterior axis are regulated by the activation of the Wnt/beta-catenin asymmetry pathway in one of the two daughters, most often the posterior daughter. The asymmetric activation of this pathway is predicted to regulate the expression of a set of genes that causes a cell to retain the seam ‘stem cell’ fate, including the ability to divide further and protection from terminal differentiation.

A key question is how the Wnt pathway becomes asymmetrically activated in the posterior daughter? We can imagine three possible models that would explain asymmetric pathway activation in the seam cell daughters (Figure 6). First, Wnt ligands may be expressed in a gradient such that the posterior cell is exposed to more Wnt ligand than the anterior daughter (Figure 6A). We have suggested that the vulval precursor cells are exposed to a posterior to anterior Wnt gradient during larval life (Gleason et al., 2006), and a gradient of EGL-20/Wnt has been observed (Coudreuse et al., 2006). Others have also suggested a global system to orient the seam cells along the anterior-posterior axis may exist (Whangbo et al., 2000). Consistent with this idea, the division pattern of the T cell is sensitive to the location of the Wnt ligand in vitro and in vivo (Goldstein et al., 2006), and the polarities of the V seam cell L1 stage divisions can be reversed by overexpression of the Wnt EGL-20 (Whangbo et al., 2000). In a second model, the seam cell daughters might differ in their ability to respond to permissive Wnt signals in the environment. The amount, localization or activity of some Wnt pathway component (eg., receptor) or regulator of the pathway could be different between the daughters, biasing the posterior daughter to activate the pathway (Figure 6B). Such a model has been proposed for the differential response of two left/right symmetric neuroblasts to EGL-20/Wnt in the larva (Whangbo and Kenyon, 1999). The asymmetric localization of several Wnt pathway components observed in some seam cells in the L1 larval stage could mediate asymmetric activation of the Wnt pathway in the two seam cell daughters in such a model (Mizumoto and Sawa, 2007a; Park et al., 2004; Takeshita and Sawa, 2005). Finally, the asymmetric localization of pathway components themselves might be sufficient to activate the pathway in one of the two daughters in the absence of a Wnt ligand (Figure 6C). An initial Wnt ligand or other asymmetry present in the embryo or early larva could set up asymmetric localization of the signaling machinery in the seam cells. This asymmetry could then be propagated through each division in a ‘relay’ mechanism, leading to pathway activation in the posterior daughter at each division in the absence of Wnt ligand. Such a model is consistent with our inability to identify any of the Wnt genes as functioning in this process. However, functional redundancy and antagonistic interactions among Wnt genes have been reported (Deshpande et al., 2005; Gleason et al., 2006; Green et al., 2008; Pan et al., 2006; Zinovyeva and Forrester, 2005; Zinovyeva et al., 2008), which could complicate such genetic experiments. It should be noted that no Wnt ligand has been identified for the Wnt/beta-catenin asymmetry pathway acting in the asymmetric cell divisions in early somatic gonad development.

Figure 6. Models for asymmetric activation of the Wnt pathway in posterior seam cell daughters.

Figure 6

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A generalized mother seam cell (SC) divides in a stem cell-like manner to generate an anterior daughter that adopts a non-seam cell fate, and a posterior daughter that retains the seam cell fate. In the posterior daughter, activation of the Wnt pathway leads to formation of a POP-1/SYS-1 complex that promotes expression of target genes required for the seam cell fate. In the anterior daughter, POP-1 interacts with transcriptional repressors to keep these Wnt pathway targets off. The curvy line at the membrane indicates expression of Wnt receptor. A) An anterior-posterior gradient of Wnt ligands (higher in the posterior) leads to asymmetric activation of the pathway in the posterior daughter. B) The asymmetric localization of Wnt pathway components (such as the receptor, shown here) to the posterior cell allows only that daughter to respond to a permissive Wnt signal in the environment. Alternatively, the asymmetric distribution of a negative pathway regulator (shown by a triangle) to the anterior daughter could keep the pathway off in that cell, even in the presence of permissive Wnt signal and appropriate receptor. C) Asymmetric localization of some Wnt pathway components to the posterior daughter (such as the receptor) and other factors to the anterior daughter, leads to activation of the pathway in the posterior daughter in a Wnt-independent manner. For example, a non-Wnt ligand localized to the anterior daughter (shown by a star) could activate the Wnt receptor in the posterior daughter.

Conversely, we found that mutations in the Wnt receptor genes lin-17 and mom-5 caused a decrease in terminal seam cell number in a wrm-1(ts) sensitized background (Supplemental Table 5). This suggests that either a Wnt ligand may act on the seam cells at some point during their development, or that Wnt receptor(s) in the absence of ligand are required for this process. Both of these Wnt receptors asymmetrically localize to the posterior when they are expressed in the seam cells, and in the case of LIN-17 in the T cell, this localization is dependent on LIN-44/Wnt (Goldstein et al., 2006; Park et al., 2005; Sawa et al., 1996). Further work will be needed to elucidate the requirement for specific Wnt ligands and receptors in this process, and the time at which they are required, in order to distinguish between the models described above.

In summary, although C. elegans does not contain somatic stem cells that divide to replenish tissues during adult life, we have shown that the Wnt pathway, which regulates the stem cell niche in other species, acts to regulate the stem cell-like division patterns of certain somatic cells during C. elegans larval development. The epithelial seam cells divide asymmetrically during larval life to generate another seam cell, and a cell that terminally differentiates, and the C. elegans ‘Wnt/beta-catenin asymmetry’ pathway is necessary for many of these asymmetric cell divisions. A number of intriguing questions remain as to how this pathway is asymmetrically activated in only one daughter, whether specific Wnts are involved, whether they are required only initially or throughout larval life, and how this Wnt pathway interacts with the heterochronic pathway that controls the specific patterns of seam cell division observed in different developmental stages. Therefore, a further understanding of the mechanisms regulating the division patterns of the C. elegans seam cells should contribute to our understanding of metazoan stem cell biology.

Supplementary Material

01

Supplemental Figure 1. L1 feeding RNAi treatment modestly reduces pop-1 transcript levels. Synchronized L1 stage animals were fed for 24 hours on bacteria expressing either ‘feeding vector’ control (lanes 1–4), or pop-1 dsRNA (lanes 4–8). Semi-quantitative PCR analysis was performed on RNA samples prepared from these populations for either ama-1 (control; lanes 1, 3, 5, 7) or pop-1 (experimental; lanes 2, 4, 6, 8). The figure shows results after 30 cycles of PCR. The amount of ama-1 product in experimental samples (lanes 5 and 7) was normalized to the amount in the FV control samples (either lane 1 or lane 3), then the ratio of the pop-1 product in experimental (lanes 6 and 8) versus control strains (lane 2 or lane 4) was calculated after normalization based on ama-1. This ratio varied from 0.50 to 0.81, with an average of 0.74 (n=4), indicating a modest reduction of pop-1transcript (assayed in total worm lysate) is sufficient to give a seam cell phenotype. This may be related to low levels of pop-1 transcript and protein in mid-larval stage animals in general (K. Thompson, L. Gorrepati, J. Gleason, D Eisenmann; unpublished results). The same pop-1 RNAi treatment performed on hermaphrodites results in nearly 100% lethality in the F1 progeny.

Supplemental Figure 2. Observation of seam cell divisions on one side of individual animals using scmp::gfp. (A) scmp::gfp control(RNAi) animal treated with RNAi vector control at 20° C, (B–E) four scmp::gfp pop-1(RNAi) animals at 20° C, (F-I) four wrm-1(ts); scmp::gfp animals grown at the restrictive temperature (26.5° C). For pop-1(RNAi) and vector control animals, individual L1 larvae that had already completed the first L1 seam cell division correctly (had 10 GFP+ cells) were picked to NGM plates seeded with either HT115 expressing pop-1 dsRNA or HT115 with empty feeding vector and allowed to develop at 20°C. Seam cell divisions were monitored at approximately three hour intervals until the late L4 using a Leica MZ16 F dissecting microscope. For wrm-1(ts) and wild-type control animals grown at 26.5°, individual L1 larvae that had already completed the first L1 seam cell division were picked to NGM plates seeded with OP50 and allowed to develop at 26.5°C. Seam cell divisions were monitored at approximately two hour intervals until the early L3. No differences were observed in seam cell lineages for wild-type animals grown at 26.5° C. More frequent monitoring of the animals led to sickness, presumably due to GFP expression. Horizontal lines indicate observed cell divisions, solid vertical lines indicate cells expressing GFP, grey vertical lines indicate cells that lost GFP expression. Dotted vertical lines indicate the cells were not observed during this time, but their behavior has been inferred from the position of GFP+ nuclei before and after this time (as described in Materials and Methods). These inferences are by no means precise, and alternative division patterns are possible in many of these cases. An ‘H’ indicates that a cell lost scmp::gfp expression and is presumed to have adopted a hypodermal or non-seam cell fate. An ‘S’ at the bottom of the figure indicates that a cell that retained scmp::gfp expression was observed at that position along the anterior-posterior axis in young adult animals.

02

Acknowledgments

We thank Rachel Nimmo and Alison Woollard for the rnt-1 strain, Anna Zinovyeva and Wayne Forrester for the egl-20 cwn-2 double mutant, Joel Rothman for a strain containing wIs51 [scmp::GFP], Mike Krause for antibody P4G4, and Iqbal Hamza for several gene-specific RNAi plasmids. We thank Frank Slack, Anna Zinovyeva and Wayne Forrester for discussing unpublished results. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources (NCRR), and from the National BioResource Project of Japan. This work was supported by NSF grant IBN-0131485 and NIH grant GM65424.

Footnotes

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Supplementary Materials

01

Supplemental Figure 1. L1 feeding RNAi treatment modestly reduces pop-1 transcript levels. Synchronized L1 stage animals were fed for 24 hours on bacteria expressing either ‘feeding vector’ control (lanes 1–4), or pop-1 dsRNA (lanes 4–8). Semi-quantitative PCR analysis was performed on RNA samples prepared from these populations for either ama-1 (control; lanes 1, 3, 5, 7) or pop-1 (experimental; lanes 2, 4, 6, 8). The figure shows results after 30 cycles of PCR. The amount of ama-1 product in experimental samples (lanes 5 and 7) was normalized to the amount in the FV control samples (either lane 1 or lane 3), then the ratio of the pop-1 product in experimental (lanes 6 and 8) versus control strains (lane 2 or lane 4) was calculated after normalization based on ama-1. This ratio varied from 0.50 to 0.81, with an average of 0.74 (n=4), indicating a modest reduction of pop-1transcript (assayed in total worm lysate) is sufficient to give a seam cell phenotype. This may be related to low levels of pop-1 transcript and protein in mid-larval stage animals in general (K. Thompson, L. Gorrepati, J. Gleason, D Eisenmann; unpublished results). The same pop-1 RNAi treatment performed on hermaphrodites results in nearly 100% lethality in the F1 progeny.

Supplemental Figure 2. Observation of seam cell divisions on one side of individual animals using scmp::gfp. (A) scmp::gfp control(RNAi) animal treated with RNAi vector control at 20° C, (B–E) four scmp::gfp pop-1(RNAi) animals at 20° C, (F-I) four wrm-1(ts); scmp::gfp animals grown at the restrictive temperature (26.5° C). For pop-1(RNAi) and vector control animals, individual L1 larvae that had already completed the first L1 seam cell division correctly (had 10 GFP+ cells) were picked to NGM plates seeded with either HT115 expressing pop-1 dsRNA or HT115 with empty feeding vector and allowed to develop at 20°C. Seam cell divisions were monitored at approximately three hour intervals until the late L4 using a Leica MZ16 F dissecting microscope. For wrm-1(ts) and wild-type control animals grown at 26.5°, individual L1 larvae that had already completed the first L1 seam cell division were picked to NGM plates seeded with OP50 and allowed to develop at 26.5°C. Seam cell divisions were monitored at approximately two hour intervals until the early L3. No differences were observed in seam cell lineages for wild-type animals grown at 26.5° C. More frequent monitoring of the animals led to sickness, presumably due to GFP expression. Horizontal lines indicate observed cell divisions, solid vertical lines indicate cells expressing GFP, grey vertical lines indicate cells that lost GFP expression. Dotted vertical lines indicate the cells were not observed during this time, but their behavior has been inferred from the position of GFP+ nuclei before and after this time (as described in Materials and Methods). These inferences are by no means precise, and alternative division patterns are possible in many of these cases. An ‘H’ indicates that a cell lost scmp::gfp expression and is presumed to have adopted a hypodermal or non-seam cell fate. An ‘S’ at the bottom of the figure indicates that a cell that retained scmp::gfp expression was observed at that position along the anterior-posterior axis in young adult animals.

NIHMS238183-supplement-01.pdf (187.5KB, pdf)
02
NIHMS238183-supplement-02.pdf (342.8KB, pdf)

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