Control of stem cell self-renewal and differentiation by the heterochronic genes and the cellular asymmetry machinery in Caenorhabditis elegans

Abstract

Transitions between asymmetric (self-renewing) and symmetric (proliferative) cell divisions are robustly regulated in the context of normal development and tissue homeostasis. To genetically assess the regulation of these transitions, we used the postembryonic epithelial stem (seam) cell lineages of Caenorhabditis elegans. In these lineages, the timing of these transitions is regulated by the evolutionarily conserved heterochronic pathway, whereas cell division asymmetry is conferred by a pathway consisting of Wnt (Wingless) pathway components, including posterior pharynx defect (POP-1)/TCF, APC related/adenomatosis polyposis coli (APR-1)/APC, and LIT-1/NLK (loss of intestine/Nemo-like kinase). Here we explore the genetic regulatory mechanisms underlying stage-specific transitions between self-renewing and proliferative behavior in the seam cell lineages. We show that mutations of genes in the heterochronic developmental timing pathway, including lin-14 (lineage defect), lin-28, lin-46, and the lin-4 and let-7 (lethal defects)-family microRNAs, affect the activity of LIT-1/POP-1 cellular asymmetry machinery and APR-1 polarity during larval development. Surprisingly, heterochronic mutations that enhance LIT-1 activity in seam cells can simultaneously also enhance the opposing, POP-1 activity, suggesting a role in modulating the potency of the cellular polarizing activity of the LIT-1/POP-1 system as development proceeds. These findings illuminate how the evolutionarily conserved cellular asymmetry machinery can be coupled to microRNA-regulated developmental pathways for robust regulation of stem cell maintenance and proliferation during the course of development. Such genetic interactions between developmental timing regulators and cell polarity regulators could underlie transitions between asymmetric and symmetric stem cell fates in other systems and could be deregulated in the context of developmental disorders and cancer.

Keywords: asymmetric division; heterochronic; lit-1/Nlk; pop-1/Tcf; stem cell.

Fig. 1.

Stage-specific asymmetric and symmetric divisions in postembryonic seam cell divisions in the heterochronic mutants. (A) Asymmetric cell division yields one anterior (a) differentiated cell (D, red square) and one posterior (p) stem (seam) cell (S, green circle); the asymmetric division can transit to symmetric division generating either two stem cells or two differentiating cells. (B) Arrangement of lateral hypodermal seam stem cells along one side of an L1 and L2 larva in C. elegans. (C) Genetic relationship of lit-1, wrm-1, and apr-1 on the pop-1 regulation and cell fate of seam cells in C. elegans. (D) Normal stage-specific division pattern of V1–V4/V6 cell lineages during postembryonic development of WT C. elegans; seam cells divide once at the beginning of each larval stage (or twice in the L2) until the final molt, when they terminally differentiate and secrete adult specific cuticular alae (triple bars). These cell lineage patterns include stage-specific asymmetric cell divisions [yielding one differentiated cell (red square) and one stem (seam) cell (green circle) and (specifically in the L2 stage) symmetric divisions (generating two stem cells)]. The normally L2-specific symmetric division occurs precociously (promoted) in L1 stage in the lin-14(lf) mutant, is prevented in the lin-14(gf) and lin-4(lf) mutants, is skipped in lin-28(lf), and is reiterated in lin-46(lf), lin-28(gf), or let-7-family(lf) larvae.

Fig. 2.

lit-1/Nlk promote seam cell division asymmetry and oppose differentiation. (A) lit-1(RNAi) (Right) causes decrease in seam cell number, assayed in young adults (YA) using scmp::gfp, compared with empty vector RNAi controls (Left). (B) Animals doubly marked with scmp::gfp and ajm-1::gfp were used to monitor fusion (differentiation) of seam cells to syncytium during the L2 developmental stage. In the WT, four sisters for each V seam cell lineage are evident just after the L2 asymmetric divisions. The posterior sisters remain seam cells, whereas the anterior sisters differentiate by fusing to the syncytium. (C) lit-1(RNAi) caused premature fusion (differentiation) of seam cells (yellow D) in L2 stage asymmetric division. S, seam cell; D, differentiated cell. (D) lit-1(RNAi) seam cell number phenotype is enhanced in combination with the lit-1(ne1991) hypomorphic mutation. (E) Schematic representation of seam cell division pattern in WT, lit-1(RNAi) and lit-1(RNAi);lit-1(n1991) animals. Seam cell fate: green circles; differentiation fate: red squares. Dotted line, variable fates (seam or differentiation). Anterior is to the left. Error bars indicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. (Scale bar, 50 μm.)

Fig. 3.

lin-14 modulate asymmetric and symmetric cell fates. (A) Effect of pop-1(RNAi) on lin-14(n179) L1 larvae at permissive temperature 15 °C showing GFP-marked seam cells after control RNAi (Left) or pop-1(RNAi) (Right). S indicates a seam cell and D indicates a differentiated cell based on the location, size, and shape of the nucleus, using differential interference contrast (DIC) microscopy (9). (B) Representative of lin-14(n179) young adults (YA) stage at 20 °C showing GFP-marked seam cells after control RNAi (Upper) or pop-1(RNAi) (Lower). (C) Effect of pop-1(RNAi) and lit-1(RNAi) compare with empty vector (EV) control on seam cells numbers in lin-4(e912), lin-14(n355), lin-14(n179), and lin-4(e912);lin-14(n179) backgrounds at 20 °C. (D) Quantitative analysis of the effects of lin-14(n179) on the sensitivity of L2 V1-V4/V6 seam lineages to pop-1(RNAi) at 15 °C. In the second cell division in L2 stage, each V1–V4/V6 seam cell undergoes two rounds of division producing two seam cells and two differentiated cells per lineage. (Upper) Effect of pop-1(RNAi) on individual V1-V4/V6 seam cells in WT by changing the asymmetric to symmetric division producing three or four seam cells. The production of four cells is pronounced as the result of hypersensitivity of lin-14(n179) to pop-1(RNAi) (Lower). Anterior is to the left. Error bars indicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. (Scale bar, 50 μm.)

Fig. 4.

Heterochronic genes control the stem cell fate. The graph represents the level of sensitivity or insensitivity of the heterochronic mutants compare with WT based on the data reported in Table 1. The x axis shows the fold change of seam cell number decrease by lit-1(RNAi) or increase by or pop-1(RNAi) (Table 1). Strains are classified by the fold change (either decrease or increase, compared with the WT) in seam cell number that they displayed after pop-1(RNAi) or lit-1(RNAi) as follows: insensitive (blue bars) for less than twofold change, sensitive (yellow bars) for fold change between 2-fold and 3.5-fold, and hypersensitive (red bars) for fold change greater than 3.5. NT, not tested. *The fold decrease of seam cell number by lit-1(RNAi) for lin46(ma146);lin-28(n719) strain is −37.4.

Fig. 5.

A model of genetic interactions between heterochronic genes and LIT-1/POP-1/APR-1 in the developmental regulation of asymmetric and symmetric seam cell divisions. The stage-specific pattern of asymmetric and symmetric division of seam cells is orchestrated by parallel microRNA-regulated developmental timing pathways. (A) lin-14 acts in the L1 to enhance the activity of the LIT-1/POP-1/APR-1 polarity machinery and hence prevents early expression of the L2 symmetric seam cell division. (B) LIN-14 level is then partially down-regulated in the late L1 stage by the lin-4 microRNA, so that the early L2 symmetric division is permitted because of a below threshold level of LIN-14 together with a parallel activity of lin-28 that inhibits premature activation of lin-46. Our data tentatively place HBL-1 as acting independently of LIT-1/POP-1/APR-1 to promote the symmetric stem cell proliferative division. After the first L2 symmetric division, seam cells immediately switch back to asymmetric division because of the down-regulation of HBL-1 by the let-7-Fam microRNAs, as well as down-regulation of LIN-28, which permits the up-regulation of lin-46, and hence restoration of full LIT-1/POP-1/APR activity. Note that LIN-14 is understood to be partially down-regulated to a medium level in the L2 stage and to a lower level in the L3 (12), and our data from this study confirm that lin-14 activity contributes partially to regulating the activity of LIT-1/POP-1 in the second-L2 asymmetric division (Fig. 3D). The results of Ren and Zhang are incorporated into the model here by invoking feedback circuits wherein LIT-1/POP-1 asymmetry machinery confers robustness to the temporal dynamics of lin-4 and let-7-Fam microRNA activities by negatively modulating those microRNAs during the L2 and L3 stages (45).

Fig. 6.

lin-46 modulates asymmetric and symmetric cell fates. (A) Representative lin-46(ma164) adults showing GFP-marked seam cells after control RNAi (Left) or pop-1(RNAi) (Right). Anterior is to the left. (B) Effect of pop-1(RNAi) and lit-1(RNAi) on seam cells numbers in lin-46(ma164) and WT control. Error bars indicate ±SEM. *P < 0.05, **P < 0.01, ***P < 0.005, two-tailed t test. (Scale bar, 50 μm.)