Establishment of Signaling Interactions with Cellular Resolution for Every Cell Cycle of Embryogenesis

Abstract

Intercellular signaling interactions play a key role in breaking fate symmetry during animal development. Identification of signaling interactions at cellular resolution is technically challenging, especially in a developing embryo. Here, we develop a platform that allows automated inference and validation of signaling interactions for every cell cycle of Caenorhabditis elegans embryogenesis. This is achieved by the generation of a systems-level cell contact map, which consists of 1114 highly confident intercellular contacts, by modeling analysis and is validated through cell membrane labeling coupled with cell lineage analysis. We apply the map to identify cell pairs between which a Notch signaling interaction takes place. By generating expression patterns for two ligands and two receptors of the Notch signaling pathway with cellular resolution using the automated expression profiling technique, we are able to refine existing and identify novel Notch interactions during C. elegans embryogenesis. Targeted cell ablation followed by cell lineage analysis demonstrates the roles of signaling interactions during cell division in breaking fate symmetry. Finally, we describe the development of a website that allows online access to the cell-cell contact map for mapping of other signaling interactions by the community. The platform can be adapted to establish cellular interactions from any other signaling pathway.

Keywords: C. elegans; Cell contact; Notch signaling; cell lineage; division asymmetry.

Figure 1

Overview of a 350-cell stage C. elegans embryo. (A) Nomarski micrograph of a C. elegans embryo of approximately the 350-cell stage. (B) Epifluorescence micrograph showing superimposed nuclear expression patterns of lineaging markers (red) and a pharynx marker, PHA-4 (green). (C) Superimposed micrograph from (B) and (C). (D) Three-dimensional space-filling model of a 350-cell embryo. Cells are differentially color-coded based on their lineal origins. MS, cyan; E, green; Aba, red; ABp, blue; C, magenta; and D, peach. (E) Cell lineage tree up to the 350-cell stage. Cell lineages are differentially colored based on cell fate except the undifferentiated cell fates that are colored in black.

Figure 2

Modeling of cell–cell contact during C. elegans embryogenesis. (A) Demarcation of the ratio of contact areas between cells with and without a functional contact. Shown is the occurrence distribution of modeled contact areas between cell pairs that are known to have (green) or not to have (brown) the second Notch (see main text) interactions in 91 embryos. Percentage of contact area out of average cell surface area of all cells in the current time point is plotted on the x-axis and the number of embryos with a given ratio out of 91 wild-types on y-axis. (B) Distribution of any contacts (contact area > 0) in 91 wild-type embryos. The y-axis denotes the percentage of a given contact out of all observed contacts and the x-axis the observed times for a given contact out of 91 embryos. Contacts with > 95% reproducibility (i.e., observed in 87 out of 91 embryos) are shaded in red. (C) A diagram showing the definition of effective cell contact with cell A over three consecutive time points. A contact area that is bigger or smaller than 6.5% of the average surface areas of all the cells is differentially colored in red and blue lines, respectively. For a given cell pair, only a cell contact area that is > 6.5% for at least two consecutive time points (∼3 min) is defined as an effective cell contact. (D) Heat map of mutual Pearson correlation’s coefficient (r) of contact areas for all cells between 91 individual wild‐type embryos. Both the x- and y-axes denote the coefficient of an individual embryo against another. (E) An example of a 40-cell C. elegans embryo expressing GFP in nuclei and membrane marker PH (PLC1δ1) in the cell membrane (red).

Figure 3

Expression of Notch ligands and receptors in a C. elegans embryo. (A–F) Lineal expression (red) of two Notch receptors, glp-1and lin-12, and one ligand, apx-1, in the ABa and ABp lineage up to the 350-cell stage. (G–I) Spatial expression of the three genes differentially color-coded based on their lineal origins. (J) Combined spatial expression patterns of the three genes between the ligand, apx-1 (red), and the two receptors, glp-1 and lin-12 (green).

Figure 4

Refinement of third Notch signaling interaction in a 55-cell C. elegans embryo. (A–C) Epifluorescence micrographs of different focal planes of the same C. elegans embryo (dorsal view with anterior to the left) focusing on cells ABplaaa (plane 26), ABalapa (plane 51) and ABalapp (plane 72), respectively, as indicated by arrowheads. Cell membranes and nuclei are colored in red and green, respectively. (D) Three-dimensional (3D) projection of epifluorescence micrographs. (E) Cut-open view of the 3D projection in (D) showing cell boundaries. The projection is orientated to facilitate visualization of the cell boundaries. (F) Modeling of cell boundaries in an embryo at approximately the same stage as in (D). Nuclei of ABalapa and ABalapp are colored in red and indicated with an “a” and “p,” respectively, and the remaining nuclei colored in blue; ABplaaa nucleus is colored in green. (G–J) Lineal expression of Pref-1::mCherry in the “ABp” lineage of a wild-type embryo (G) or embryos with cell ablation (ablated cell indicated in parenthesis). Target cells of the third Notch signaling interaction are indicated with an arrowhead.

Figure 5

Refined fourth Notch signaling interaction in a C. elegans embryo. (A) Shown is a three-dimensional (3D) projection of an epifluorescence micrograph of an 87-cell embryo with cell membranes labeled by mCherry (red) and nuclei by GFP. One or both of the MSap daughters were previously proposed to signal excretory cell precursor, ABplpapp. The three cells are indicated with an arrowhead. (B) Cut-open view of the same embryo as in (A) showing cell boundaries. The embryo is oriented so that the boundaries of interest are most obvious. (C) Modeling of cell boundaries of the same embryo as in (B) with the same three cells indicated with an arrowhead. ABplpapp is colored in green, the two MSap daughters in red, and the remaining nuclei in blue. (D) 3D space-filling model of an embryo at the same stage as in (A). Red, ABa; dark blue, ABp; light blue, MS; green, E; pink, C; brown, D; and yellow, P4. The same three cells as in (A) are indicated with an arrowhead. (E) Lineal expression of a Notch ligand, lag-2, in MSapp (red) indicated with an arrowhead. Cell death is indicated with an “X.”

Figure 6

Identities of cells for fifth Notch signaling interaction in a C. elegans embryo. (A) Three-dimensional (3D) projection of a 96-cell embryo with cell membranes and nuclei colored in red and green, respectively. Signaling interaction was proposed to take place between two cells, MSappp (yellow arrowhead) and ABplpppp (white arrow head). (B) Cut-open view of the same embryo as in (A) showing cell boundaries. (C) 3D space-filling model of a 96-cell C. elegans embryo with cell pairs similarly color coded as in (A). (D) Modeling of cell boundaries in a 96-cell C. elegans embryo. Nuclei of MSappp and ABplpppp are colored in red and green, respectively, and the remaining nuclei in blue.