Multi-tissue patterning drives anterior morphogenesis of the C. elegans embryo.

Complex structures derived from multiple tissue types are challenging to study in vivo, and our knowledge of how cells from different tissues are coordinated is limited. Model organisms have proven invaluable for improving our understanding of how chemical and mechanical cues between cells from two different tissues can govern specific morphogenetic events. Here we used Caenorhabditis elegans as a model system to show how cells from three different tissues are coordinated to give rise to the anterior lumen. While some aspects of pharyngeal morphogenesis have been well-described, it is less clear how cells from the pharynx, epidermis and neuroblasts coordinate to define the location of the anterior lumen and supporting structures. Using various microscopy and software approaches, we define the movements and patterns of these cells during anterior morphogenesis. Projections from the anterior-most pharyngeal cells (arcade cells) provide the first visible markers for the location of the future lumen, and facilitate patterning of the surrounding neuroblasts. These neuroblast patterns control the rate of migration of the anterior epidermal cells, whereas the epidermal cells ultimately reinforce and control the position of the future lumen, as they must join with the pharyngeal cells for their epithelialization. Our studies are the first to characterize anterior morphogenesis in C. elegans in detail and should lay the framework for identifying how these different patterns are controlled at the molecular level.

Physiological Starvation Promotes Caenorhabditis elegans Vulval Induction.

Studying how molecular pathways respond to ecologically relevant environmental variation is fundamental to understand organismal development and its evolution. Here we characterize how starvation modulates Caenorhabditis elegans vulval cell fate patterning - an environmentally sensitive process, with a nevertheless robust output. Past research has shown many vulval mutants affecting EGF-Ras-MAPK, Delta-Notch and Wnt pathways to be suppressed by environmental factors, such as starvation. Here we aimed to resolve previous, seemingly contradictory, observations on how starvation modulates levels of vulval induction. Using the strong starvation suppression of the Vulvaless phenotype of lin-3/egf reduction-of-function mutations as an experimental paradigm, we first tested for a possible involvement of the sensory system in relaying starvation signals to affect vulval induction: mutation of various sensory inputs, DAF-2/Insulin or DAF-7/TGF-ß signaling did not abolish lin-3(rf) starvation suppression. In contrast, nutrient deprivation induced by mutation of the intestinal peptide transporter gene pept-1 or the TOR pathway component rsks-1 (the ortholog of mammalian P70S6K) very strongly suppressed lin-3(rf) mutant phenotypes. Therefore, physiologically starved animals induced by these mutations tightly recapitulated the effects of external starvation on vulval induction. While both starvation and pept-1 RNAi were sufficient to increase Ras and Notch pathway activities in vulval cells, the highly penetrant Vulvaless phenotype of a tissue-specific null allele of lin-3 was not suppressed by either condition. This and additional results indicate that partial lin-3 expression is required for starvation to affect vulval induction. These results suggest a cross-talk between nutrient deprivation, TOR-S6K and EGF-Ras-MAPK signaling during C. elegans vulval induction.

Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction.

How cells coordinate their spatial positioning through intercellular signaling events is poorly understood. Here we address this topic using Caenorhabditis elegans vulval patterning during which hypodermal vulval precursor cells (VPCs) adopt distinct cell fates determined by their relative positions to the gonadal anchor cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1° vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1° cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how intercellular signaling reduces initial spatial variability in cell positioning to generate reproducible interactions across tissues.

Cryptic genetic variation uncovers evolution of environmentally sensitive parameters in Caenorhabditis vulval development.

Understanding the robustness of developmental systems requires insights into the sensitivity of underlying molecular and cellular parameters to perturbations, and how such sensitivity evolves. We address these issues using vulval cell fate determination--a reproducible and robust patterning process regulated by a cross-talk of EGF-Ras-MAPK and Delta-Notch pathways. Although the final vulval cell fate pattern is identical in all Caenorhabditis species, the patterning process underlies extensive cryptic genetic variation between and within species. Here, we tested whether this cryptic genetic variation translates into variation in developmental sensitivity to environmental perturbations. We disrupted vulval patterning using thermal perturbations to quantify and compare environmental sensitivity of different system parameters between distinct genotypes of C. elegans and C. briggsae. Thermal perturbations globally debuffered vulval development, triggering diverse pattering variants, whose frequency and spectra were strongly species- and genotype-dependent. This condition-dependent variation indicates that environmental sensitivity of different system properties, such as vulval competence or vulval induction, is subject to evolutionary change. High temperature induced a genotype-specific decrease of secondary fate induction and corresponding Notch pathway activity in the C. elegans N2 strain; in contrast, hypoinduction of the primary cell fate was never observed. Vulval precursor cells therefore differ in temperature sensitivity and such cell-specific sensitivity shows evolutionary variation. We further compared spectra of temperature-induced vulval variants to the ones induced by mutation accumulation in the same genotypes. In response to either perturbation, we observed similar genotype-dependence of variant production, allowing identification of distinct system features most sensitive to both mutation and environment. Taken together, we show how sensitivity of system parameters regulating Caenorhabditis vulval development depends on subtle interactions between perturbations and genetic background. Our results imply that cryptic genetic variation may reflect evolutionary variation in developmental robustness, therefore potentially contributing to the maintenance of phenotypic precision when facing perturbations.