Investigating the role of the fusogen eff-1 and natural genetic variation in Caenorhabditis elegans seam cell development

Robustness is the ability of biological systems to produce invariant phenotypes despite perturbations. Development is especially robust to internal perturbations, like stochastic gene expression or mutations, and external perturbations, such as changes in environmental factors including temperature and nutrition. The highly invariant developmental patterning in Caenorhabditis elegans offers an ideal system to study the genetic and molecular mechanisms underlying developmental robustness. This work describes an experimental paradigm to discover the mechanistic basis and consequences of developmental robustness using the C. elegans seam cells as a model. Seam cells are lateral epidermal cells that are stem cell-like in their ability to produce differentiated cells and maintain proliferative potential. Through a forward genetic screen, I describe a novel role for the fusogen gene eff-1, which was previously known to drive cell fusion events, in the robustness of seam cell patterning. Furthermore, I show that eff-1 is not required for differentiation of seam cells, therefore I demonstrate that fusion is uncoupled from the differentiation programme. In another set of experiments, I show for the first time that the terminal number of seam cells in C. elegans is robust to standing genetic variation. A consequence of developmental robustness is the acquisition of cryptic genetic variation that does not modify the phenotype under normal conditions but manifests phenotypically upon perturbation. I demonstrate that the genetic background affects seam cell number at a higher developmental temperature of 25 C or upon mutations in the GATA transcription factor and target of the Wnt pathway, egl-18. CB4856 (Hawaii) suppressed the effect of temperature on the seam cell number compared to the lab reference N2 (United Kingdom), as well as lowered the expressivity of egl-18 mutations. Multiple regions of the genome were found to interact epistatically to modify egl-18 mutation expressivity, suggesting that a complex genetic architecture underlies seam cell development. Taken together, this work increases our knowledge on the robustness of seam cell patterning to various sources of variation.Open Acces

Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens

DELAYED OUTCROSSING IN CAENORHABDITIS ELEGANS BY MATING AVOIDANCE BEHAVIOR

The species Caenorhabditis elegans has a mating system of androdioecy, which consists of hermaphrodites and males. The evolutionary pressures on the two sexes are different. C. elegans hermaphrodites make self-sperm during larval development and therefore are self-fertile, whereas the males have to mate with hermaphrodites to reproduce. Behaviors that increase the reproductive success of each sex may evolve to maximize fitness. Prior studies indicate that self-sperm exhausted hermaphrodites are more receptive to mating while a recent study suggests that males have a preference for sperm-depleted hermaphrodites. These observed behaviors are confounded with receptivity of hermaphrodites, male preference, and the effects of age. In this study, I present mating assays that attempt to disentangle the effects of age, receptivity of hermaphrodite, and male preference on mating success. In the mating assays, a higher proportion of sperm-depleted hermaphrodites mate compared to hermaphrodites that have sperm. During their self-fertile period, hermaphrodites actively avoid mating with males by sprinting away, thus, delaying outcrossing by mating avoidance. Hermaphrodites that are paralyzed due to mutations in their genes do not show mating avoidance behavior. Therefore, mating avoidance is an active behavior of hermaphrodites, which requires locomotion. The velocities of older hermaphrodites that are sperm-depleted are significantly higher than velocities of young hermaphrodites that have sperm. Therefore, older hermaphrodites are capable of mating avoidance but do not avoid mating because they are sperm-depleted. I conclude that sperm-status of the hermaphrodite is a strong predictor of mating avoidance behavior. The sperm-sensing pathway of the hermaphrodites mediates the mating avoidance behavior by dynamically changing the behavior of hermaphrodites.Biology and Biochemistry, Department o

Natural Infection of C. elegans by an Oomycete Reveals a New Pathogen-Specific Immune Response

International audienc

Context-dependent gain and loss of variability in <i>lin-22(icb38)</i> mutants.

<p>(A) Quantification of brood size in wild-type (<i>n</i> = 13) and <i>lin-22(icb38)</i> mutants (<i>n</i> = 15). Black bars show mean ± SEM. (B) Quantification of P3.p division frequency in wild-type and <i>lin-22(icb38)</i> animals. Note that almost all mutant animals show division of P3.p. (C) Model showing <i>lin-22</i> interactions discovered in this study (orange), while previously known interactions are shown with dashed grey lines. These new interactions may not be direct. Seam cell number variability is increased in <i>lin-22</i> mutants due to loss and gain of symmetric divisions. Stochastic loss of symmetric divisions at the L2 stage generates more neuroblasts at the expense of seam cells. Stochastic gain of symmetric divisions towards the seam cell fate mostly at the L3/L4 stage generates more seam cells. Cell-to-cell variability in Wnt pathway activation correlates with phenotypic variability. Numerical data used for Fig 7A, B can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. L2, second larval stage; L3, third larval stage; L4, fourth larval stage.</p

Gene expression changes associated with loss and gain of daughter cell fate symmetry in <i>lin-22</i> mutants.

<p>(A-B) Representative smFISH images and quantification of wild-type and <i>lin-22(icb38)</i> animals at the L2 symmetric division stage using an <i>elt-1</i> probe. (A) Comparable amounts of <i>elt-1</i> spots are detected in wild-type V cell daughters and more spots in the posterior H2 daughter. In <i>lin-22(icb38)</i> animals, more spots are detected in the posterior V daughters than the anterior (marked by arrowheads) and even numbers in the 2 H2.p daughters (arrow points to anterior H2.p daughter cell). (B) Quantification of <i>elt-1</i> expression in H2.p daughters (<i>n</i> > 9) and pools of V.p daughter cells (<i>n</i> > 41) of wild-type and <i>lin-22(icb38)</i> animals at the L2 symmetric division stage. (C) <i>mab-5</i> expression expands to posterior daughters of V1–V4 cells (arrowheads) in <i>lin-22(icb38)</i> animals, reminiscent of the expression in the posterior V5 cell in wild-type (arrow). (D-F) <i>egl-18</i> smFISH images and quantification of wild-type and <i>lin-22(icb38)</i> animals. (D) Quantification of <i>egl-18</i> smFISH spots in the H2.p cell daughters at the L2 stage (<i>n</i> ≥ 21). (E) Images depicting <i>egl-18</i> expression at the L3 stage. Note expression in anterior daughter cells in <i>lin-22(icb38)</i> animals (arrowheads). (F) Quantification of <i>egl-18</i> smFISH spots in V1-V4 cells (<i>n</i> ≥ 68) of wild-type and <i>lin-22(icb38)</i> animals at the L2 asymmetric division stage. (G) Representative <i>eff-1</i> smFISH images of wild-type and <i>lin-22(icb38)</i> animals at the L3 asymmetric division stage. Note absence of signal in the most anterior of the 4 daughter cells in <i>lin-22(icb38)</i> animals (arrowheads). H2 consists of 4 cells that have arisen due to symmetric division at the L2 stage. (H) Quantification of seam cell number in wild-type (<i>n</i> = 39), <i>lin-22(icb38)</i> (<i>n</i> = 29), <i>eff-1(hy21)</i> (<i>n</i> = 31), and <i>lin-22(icb38)</i>; <i>eff-1(hy21)</i> (<i>n</i> = 35) animals. Note that the <i>eff-1(hy21)</i> does not show a significant difference in seam cell numbers compared to wild-type, but the double mutant does in comparison to both parental strains. Black stars show statistically significant changes in the mean with a <i>t</i> test or one-way ANOVA /Dunnett’s test. Scale bars in A, C, E, and G are 10 μm; black spots correspond to mRNAs and green labels the seam cell nuclei. Error bars in B, D, F, H show mean ± SD. Numerical data used for Fig 5B, D, F, H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. GFP, green fluorescent protein; L2, second larval stage; L2sym, symmetric first division at the L2 stage; L3, third larval stage; SCM, seam cell marker; smFISH, single molecule fluorescent in situ hybridization.</p

The developmental basis of variability in <i>lin-22</i> mutants.

<p>(A) The upper panel shows wild-type seam cell lineages, while the bottom panel indicates the most frequently occurring errors in <i>lin-22(icb38)</i> mutants (<i>n</i> = 14 independent complete lineages). The developmental errors are grouped for simplicity in 4 main classes and presented as a function of the developmental stage. The percentages refer to occurrence of these errors within the total number of relevant cell lineages. Note that the errors described are not independent, so they can occur within the same animal and even within the same lineage (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s010" target="_blank">S4 Fig</a>). (B) Heat map showing the frequency of errors per cell lineage and developmental stage (<i>n</i> = 14 lineages). Blue depicts errors leading to gain of terminal cell number due to gain of symmetric division, and red depicts errors leading to loss of symmetric division. (C-D) Quantification of seam cell number (C) and number of PDE neurons (D) in wild-type (<i>n</i> ≥ 36) and <i>lin-22(icb38)</i> animals (<i>n</i> ≥ 30) treated with control or <i>lin-28</i> RNAi. Black stars show statistically significant changes in the mean with a <i>t</i> test (<i>P</i> < 0.0001). Error bars show mean ± SD (C) or mean ± SEM in (D). Numerical data used for Fig 4B, C, D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. L2, second larval stage; L3, third larval stage; L4, fourth larval stage; PDE, post-deirid; RNAi, RNA interference.</p

The <i>icb38</i> mutation represents a loss of function mutation in <i>lin-22</i>.

<p>(A) Illustration of the <i>lin-22(icb38)</i> mutation, which is a 3,329 bp deletion removing part of the distal <i>lin-22</i> promoter (2,371 bp upstream of the <i>lin-22</i> ATG). The deletion also removes part of the third exon and 3′ UTR of the upstream gene Y54G2A.3. The deleted part is replaced by a 1,733 bp insertion consisting of exon 7 and parts of introns 6 and 7 of the downstream gene <i>mca-3</i>. The position of other <i>lin-22</i> alleles described in the manuscript is shown on the wild-type sequence. (B) Quantification of the number of PDE neurons (<i>dat-1</i>∷<i>GFP</i> foci) in the EMS-derived <i>lin-22(icb-38)</i> mutant and CRISPR-derived <i>lin-22</i> mutants (<i>n</i> ≥ 30). Reference sample is <i>egIs1</i> containing only the marker. (C) Quantification of seam cell number in <i>lin-22(icb38)</i> and other CRISPR-derived <i>lin-22</i> mutants (<i>n</i> ≥ 30). Note an increase in seam cell number variance in <i>lin-22</i> mutants depicted with red stars. Black stars show statistically significant changes in the mean with one-way ANOVA followed by the Dunnett test, and red stars depict changes in variance with a Levene’s median test (in both cases, **** corresponds to <i>P</i> value < 0.0001). Error bars show mean ± SEM (B) or mean ± SD (C). Numerical data used for Fig 2B, C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; EMS, ethyl methanesulfonate; GFP, green fluorescent protein; PDE, post-deirid; scm, seam cell marker; UTR, untranslated region.</p

Quantification of <i>lin-22</i> expression in the seam.

<p>(A) Transgenic animals carrying transcriptional reporters consisting of various fragments of upstream of <i>lin-22</i> sequences fused to GFP. From top to bottom: full <i>lin-22</i> endogenous promoter, distal <i>lin-22</i> promoter region that is deleted in <i>lin-22(icb38)</i>, proximal <i>lin-22</i> promoter present in <i>lin-22(icb38)</i>, distal <i>lin-22</i> promoter with deleted CR1, and CR1 only driving expression of GFP. White arrows indicate expression in the seam cells; white arrowheads expression in the hypodermis and green arrowheads expression in intestinal cells. (B) Quantification of expression pattern for each transcriptional reporter (<i>n</i> ≥ 35 animals). (C) Representative smFISH images showing <i>lin-22</i> expression (black spots correspond to mRNAs and seam cells are labelled in green due to <i>scm</i>∷<i>GFP</i> expression) in wild-type V cells after the symmetric L2 division (top), the L2 asymmetric division (middle), and late after the L2 asymmetric division (bottom). (D) Quantification of <i>lin-22</i> spots per seam cell in wild-type and <i>lin-22(icb38)</i> animals at the late L1 stage (<i>n</i> ≥ 10 cells per genotype). (E) Quantification of <i>lin-22</i> spots in wild-type, <i>lin-22(ot267)</i>, <i>lin-22(ot269)</i>, and <i>lin-22(icb49)</i> mutants in pools of H cells and V cells at the late L1 stage (<i>n</i> ≥ 41). (F-G) Comparison of number of <i>lin-22</i> spots between wild-type and the <i>elt-1(ku491)</i> mutant (F) or the <i>egl-18(ok290)</i> mutant, (G) both at the late L1 stage in pools of H and V cells (<i>n</i> ≥ 49). Black stars show statistically significant changes in the mean with a <i>t</i> test or one-way ANOVA as follows: * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, **** <i>P</i> < 0.0001. Reference samples for comparisons in E, F, G are the control samples depicted in black. Scale bars in A and C are 100 μm and 10 μm, respectively. Error bars show mean ± SEM (D, F, G) or mean ± SD (E). Numerical data used for Fig 3B, D, E, F, G can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. CR1, conserved region 1; GFP, green fluorescent protein; L1, first larval stage; L2, second larval stage; smFISH, single molecule fluorescent in situ hybridization.</p