The Wnt/beta-catenin asymmetry pathway patterns the atonal ortholog lin-32 to diversify cell fate in a Caenorhabditis elegans sensory lineage

Abstract

Each sensory ray of the Caenorhabditis elegans male tail comprises three distinct neuroglial cell types. These three cells descend from a single progenitor, the ray precursor cell, through several rounds of asymmetric division called the ray sublineage. Ray development requires the conserved atonal-family bHLH gene lin-32, which specifies the ray neuroblast and promotes the differentiation of its progeny. However, the mechanisms that allocate specific cell fates among these progeny are unknown. Here we show that the distribution of LIN-32 during the ray sublineage is markedly asymmetric, localizing to anterior daughter cells in two successive cell divisions. The anterior-posterior patterning of LIN-32 expression and of differentiated ray neuroglial fates is brought about by the Wnt/β-catenin asymmetry pathway, including the Wnt ligand LIN-44, its receptor LIN-17, and downstream components LIT-1 (NLK), SYS-1 (β-catenin), and POP-1 (TCF). LIN-32 asymmetry itself has an important role in patterning ray cell fates, because the failure to silence lin-32 expression in posterior cells disrupts development of this branch of the ray sublineage. Together, our results illustrate a mechanism whereby the regulated function of a proneural-class gene in a single neural lineage can both specify a neural precursor and actively pattern the fates of its progeny. Moreover, they reveal a central role for the Wnt/β-catenin asymmetry pathway in patterning neural and glial fates in a simple sensory lineage.

Figure 1.

Postembryonic development of the male tail sensory rays. A, The adult male tail bears nine pairs of sensory rays. Each ray (numbered 1–9) has characteristic position and morphology. B, Each ray is composed of three cell types: two sensory neurons, called RnA and RnB, and an associated glial-like structural cell, Rnst. C, The three cells of each ray are derived through a stereotyped pattern of division and differentiation called the ray sublineage. In the L3 larval stage, each side of the male tail bears nine ray precursor cells, R1–R9. These cells undergo several rounds of division to generate one hypodermal cell (Rn.p) as well as the three cell types of each mature ray and one apoptotic cell.

Figure 2.

Loss of lin-32 function significantly disrupts but does not abolish ray development. A, The structure of the lin-32 locus, with point mutations and deletion alleles indicated. The portion of the lin-32 ORF that encodes the bHLH domain is shown underneath the lin-32 exon–intron structure. B, Ray frequency, in rays per side, in wild-type males as well as lin-32 mutants. Asterisks indicate statistical significance compared with lin-32(tm2044), as assessed by one-way ANOVA and Dunnett's multiple comparison test. ***p < 0.001. Figures below each bar indicate the number of sides scored. C, Wild-type males have nine rays per side. Most of the ray RnA neurons express the marker trp-4, whereas eight of the nine RnB neuron pairs express the marker pkd-2. In lin-32(tm2044) and lin-32(fs6) mutants, stochastic but generally severe ray loss is observed, as seen in both the ray process itself (which is generated by the Rnst ray structural cell) and the expression of RnA and RnB markers.

Figure 3.

lin-32 is expressed asymmetrically during ray development. A, An individual male followed from late L3 to early L4, ∼10 h. Expression of LIN-32::GFP is shown on the left of each panel. The R5 (ray 5) lineage is indicated in each panel and enlarged in the bottom right corner of the fluorescence image. The right side of each panel shows a DIC image of the same animal shortly after the fluorescence image was captured. B, The ray sublineage, indicating the asymmetric expression of LIN-32::GFP with green vertical lines. i–vi indicate the six time points shown in A.

Figure 4.

lin-17 regulates the patterning of the ray sublineage. A, POP-1::GFP is expressed asymmetrically during ray development. Shown is an Rn.aa-stage animal; each Rn.aa/ap pair is indicated with a bracket. B, The Wnt receptor LIN-17 is widely expressed during the ray sublineage. Expression of the rescuing LIN-17::GFP reporter mhIs9 was examined in a lin-17(n671) background. An Rn.aa-stage animal is shown; LIN-17::GFP fluorescence is shown above the corresponding DIC image. C, Three anterior–posterior asymmetries in the ray sublineage could be regulated by Wnt signaling. Dashed arrows indicate possible cell-fate transformations in lin-17 mutants. D, The frequency of rays, RnA and RnB neurons in wild-type and lin-17 mutant males. trp-4 and pkd-2 expression marks most RnA and RnB neurons, respectively. dat-1 expression marks the RnA neurons of rays 5, 7, and 9. Asterisks indicate statistical significance compared with wild type, as assessed by one-way ANOVA and t test with Bonferroni's correction: **p < 0.01; ***p < 0.001. E, F, Adult male tails in wild-type and lin-17 mutants carrying trp-4 and pkd-2 markers (E) or a dat-1 marker (F) to visualize ray neurons.

Figure 5.

pop-1(RNAi) results in defects in ray cell-fate specification and lin-32 expression. A, A typical adult pop-1(RNAi) male carrying reporters for RnA (tba-9::YFP) and RnB (tbb-6::mCherry) fate. Left, Many additional tba-9-expressing cells are apparent (in a wild-type male, ∼8–9 are typically seen) as is a loss of expression of tbb-6. Right, Many rays are missing (bracketed area); in addition, a large fused ray can be seen (arrowhead). B, A typical L4 pop-1(RNAi) male. At the Rn.aaa stage, many cells ectopically expressing lin-32::GFP are visible (compare with Fig. 3Avi, a wild-type male at this stage).

Figure 6.

The Wnt/β-catenin asymmetry pathway regulates lin-32 expression. A, Early (above) and late (below) Rn.axx-stage wild-type and lin-17 mutant L4 males. lin-17(n698) animals harbor extra LIN-32::GFP-positive cells. In early Rn.axx animals, clusters of four strongly GFP-positive cells can be seen, as opposed to the asymmetric pairs seen in wild type. B, In 45 of 49 wild-type ray lineages examined (in 11 animals), LIN-32::GFP was asymmetrically distributed at the Rn.a[a/p] stage. All of these lineages gave rise to a normal ray. In contrast, only 31 of 65 lineages in lin-17(bx109) males (16 animals) showed LIN-32 asymmetry at the Rn.a[a/p] stage, whereas 34 of 65 appeared to be symmetric. A significantly lower fraction of these symmetric lineages generated rays than did the asymmetric lineages.

Figure 7.

Overriding lin-32 asymmetry disrupts RnB development. Representative control (nontransgenic) and transgenic hsp::LIN-32::GFP adult males are shown as adults after a transient L3 heat shock. Control males express the normal complement of pkd-2::GFP-expressing RnB neurons. In contrast, transgenic males generally have normal rays but exhibit a marked decrease in the extent of pkd-2::GFP expression.

Figure 8.

Model: the Wnt/β-catenin asymmetry pathway patterns LIN-32 expression during ray development to diversify ray cell fates. Shading of nuclei indicates LIN-32 expression; “SYS/POP” refers to the SYS-1/POP-1 ratio that determines the output of the Wnt/β-catenin signaling pathway. In addition to regulation by lin-17 and lin-44, our findings also indicate a possible role for cell-intrinsic polarity-generating mechanisms. For details, see Discussion.