Bias and evolution of the mutationally accessible phenotypic space in a developmental system

Abstract

Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change.

Figure 1. Caenorhabditis elegans vulval cell fate patterning.

The different cell fates of P3.p to P8.p are characterized by their cell lineage, i.e. number and orientation of cell divisions. Only three precursor cells, P5.p, P6.p and P7.p, adopt a vulval fate: P6.p adopts the central 1° vulval fate, and P5.p and P7.p the outer 2° vulval fate. P4.p and P8.p adopt a non-vulval 3° fate. P3.p shows high variability in its cell fate: it may divide once (3° fate) or it directly fuses with the syncytium without division (4° fate). For the reference isolate N2, the ratio of individuals adopting the 3° versus the 4° fate is approximately 1:1 ,. The canonical C. elegans pattern for P3.p to P8.p is thus defined as follows: 3°/4°−3°−2°−1°−2°−3°. The vulval cell fate pattern is conserved in Caenorhabditis. The lineages and competence properties of P3.p, P4.p and P8.p, however, may vary within and between species. Specifically, P3.p divides less frequently and is less competent (to adopt a vulval fate) in C. briggsae; in addition, P4.p and P8.p do not always divide in C. briggsae, i.e. they do sometimes adopt a 4° instead of a 3° fate ,,,. (A) L1-L2 stages: Competence establishment and maintenance of the vulval competence group. (B) Early L3 stage: Specification of vulval precursor cell fates involving primarily EGF/Ras/MAPK and Delta/Notch pathways. (C) Late L3 stage: Cell lineages. Each vulval fate corresponds to an invariant cell division pattern executed during the late L3 stage, resulting in a total of 22 vulval cells. The cell lineages of P5.p to P7.p are identical in all Caenorhabditis species . Vulval morphogenesis takes place during the L4 stage and the complete vulval organ develops by the final moult to the adult. AC: anchor cell, T: transverse (left-right) division, L: longitudinal (antero-posterior) division, U: undivided, SS: fusion to the epidermal syncytium (hyp7) after single division (3° fate); S: fusion to the syncytium in the L2 stage with no division (4° fate) (3° and 4° fates: non-vulval fates).

Figure 2. Variant cell fate patterns of vulval precursor cells (P3.p to P8.p).

We distinguish three classes of variant vulval patterns in decreasing order of vulva pattern disruption: Variants with disrupted 2°−1°−2° pattern (Class A: “defects”); variants with complete 2°−1°−2° pattern and altered vulval vs. non-vulval fates for the remaining cells (Class B), variants with complete 2°−1°−2° pattern and variable adoption of 3° versus 4° fate by P4.p and P8.p (Class C). 13 non-canonical subcategories of variants are further defined by their deviant cell fate pattern in P(4–8).p (see Material and Methods). Finally we present results on a highly variant trait, P3.p fate: 4° versus 3° (Class D), yet do not include this trait in the analysis of vulva precision. The reference (canonical) pattern for this figure (top) is arbitrarily shown with P3.p adopting a 3° fate. Note that not all variant patterns are mutually exclusive, so that a given individual may adopt multiple variants. (A) Variants with disrupted 2°−1°−2° vulval pattern (Class A). This class groups variant patterns that cause defects in the final vulval structure, likely leading to a reduction in offspring production . (1) Hyperinduction: more than three vulval precursor cells adopt a vulval cell fate (1° or 2° fate), preventing the formation of a complete vulva. For example, P8.p is induced and displaces P7.p progeny from the main invagination. Such cases of hyperinduction are often observed in the presence of an additional anchor cell. (2) Hypoinduction due to adoption of 3° or 4° non-vulval fates: fewer than three cells adopt a vulval cell fate (1° or 2° fate) because of a fate change from vulval to non-vulval. Example: P7.p adopts a 3° non-vulval fate instead of a 2° vulval fate. (3) Hypoinduction due to missing cells: Fewer than three cells adopt a vulval cell fate because one or several Pn.p cells are missing. Example: P7.p and P8.p are missing and only two cells, P5.p and P6.p, adopt vulval cell fates. (4) Misspecification of vulval fates (other than hyper- and hypoinduction): three cells adopt vulval fates but their cell lineages deviate from the canonical pattern. Example: P7.p is misspecified (in green) and adopts the lineage LLTU instead of UTLL. Such a defect in lineage orientation causes a ventral protrusion and is referred to as Bivulva phenotype . Although this specific case of fate misspecification need not always disrupt functionality of the vulva, it eliminates the capacity to mate with males . (B) Variants with complete 2°−1°−2° pattern and altered vulval versus non-vulval fates for the remaining cells (Class B). Such variant patterns do not obviously disrupt the formation of a functional vulval organ; however, whether certain variants negatively impact egg laying or other functions is unclear . (5) Hyperinduction: more than three cells adopt vulval cell fates. Example: P4.p adopts a 2° vulval cell fate instead of a 3° non-vulval cell fate. (6–7) Vulval centering shifts: the three cells adopting vulval fates are shifted to the anterior (centering on P5.p) or posterior (centering on P7.p). Example: P5.p adopts a 1° vulval fate while P4.p and P6.p adopt a 2° fate, with the anchor cell being attached to P5.p progeny. (8–9) Missing cells: One or more vulval precursor cells are missing. Example: P7.p is missing and P8.p adopts a 2° vulval fate instead. Note that in our experiments we could not distinguish whether this variant was due to a missing P7.p or P8.p cell. Therefore, we distinguish only whether one or more anterior cell (P3.p to P5.p) or a posterior cell (P7.p and P8.p) was missing. (10–11) Supernumerary cell divisions: Anterior (P3.p or P4.p) or posterior (P8.p) cells divide more than once, generating three or four cell progeny that fuse with the hypodermis. Example: P4.p (shown in pink) divides twice instead (lineage “ssss” instead of “SS”). (C) Adoption of 3° versus 4° fate of P4.p and P8.p (Class C). This class includes: (12) P4.p adopts the 4° fate or F fate, fusing with the hypodermis without prior division. (13) P8.p adopts the 4° fate. (D) Adoption of 3° versus 4° fate by P3.p (Class D). (14) P3.p adopts the 4° fate (frequent in the wild type).

Figure 3. Per-generation change in frequency R_m for variant vulval phenotypes.

Mean per-generation change in variant frequency R_m in mutation accumulation lines started from four C. elegans and C. briggsae isolates (colour-coded). Variants are numbered and placed in four classes (A–D) as in Figure 2. Note the different vertical scales of the graphs. Sample Sizes: HK104 (44 MA lines, 17 control lines), PB800 (53 MA lines, 17 control lines), PB306 (51 MA lines, 17 control lines) and N2 (52 MA lines, 17 control lines). For each MA and control line, 50 individuals were scored for their vulval phenotype. Error bars indicate standard errors of the (line) mean.

Figure 4. Comparison of Ras pathway activity in isolates of C. elegans (N2 versus PB306).

Quantification of wild genetic background effects on Ras reporter activity in animals carrying an integrated Ras pathway reporter transgene (egl-17::cfp-lacZ), at three distinct developmental stages during vulval induction. Bars labelled with the same letter did not show significant differences in expression levels (Tukey's HSD, P<0.05). Error bars indicate standard error of the mean. For ANOVA results, see Table S7.