-
Wu, Y., Guo, M., Moyle, M., Ardiel, E., Shroff, H., Bokinsky, A., Santella, A., McCreedy, E., Colon-Ramos, D., Karaj, N., Duncan, L., Bao, Z., Levin, M., Mohler, W., Lauziere, A., Harvey, B., Christensen, R.P.
[
International Worm Meeting,
2019]
The Caenorhabditis elegans embryo represents an excellent model system in which to study tissue formation. However, the onset of twitching and elongation makes data analysis during the second half of embryogenesis difficult. Previously, we developed software to enable computational untwisting of the C. elegans embryo, removing the effects of embryo movement and placing embryo images in a common reference frame for analysis. We have now improved our software suite to incorporate more user-friendly positional tracking and better segmentation, reducing clipping of images around the edges of the embryo. We also apply deep learning to segment nuclei in a semi-automated fashion. We apply our software to generate a map showing the position of 158 nuclei in the post-twitching worm embryo, as a partial step in the generation of a complete embryonic nuclear atlas. Tracked nuclei include 16 neurons and 81 body wall muscles. Our improved tools, combined with pre-twitching work from our collaborators on the WormGUIDES project, allow us to pursue the goal of developing a complete nuclear and neurite outgrowth atlas for the nematode embryo from the two cell stage until hatching.
-
Christensen, Ryan, Shroff, Hari, Colon-Ramos, Daniel, Harvey, Brandon, Guo, Min, Santella, Anthony, Xu, Stephen, Del Toro-Pedrosa, Daniel, Mohler, William, Wu, Yicong, Bokinsky, Alexandra, Moyle, Mark, Duncan, Leighton, Levin, Michael, Ardiel, Evan, Schwartz, Gabi, Lauziere, Andrew, Karaj, Nensi, McCreedy, Evan, Bao, Zhirong, Vazquez Martinez, Nabor
[
International Worm Meeting,
2021]
The limited number of cells and invariant cell lineage of the Caenorhabditis elegans embryo make it an excellent system for examining complex developmental events, such as tissue movement and neurodevelopment. Prior work from the WormGUIDES project has digitized the position of all nuclei for the first half of embryogenesis, creating a computational map of the embryo that can be used to overlay developmentally relevant information like gene expression or neurite outgrowth. Creating a similar map for the second half of embryogenesis is difficult due to embryo elongation and movement. We have developed software to computationally untwist the moving embryo, allowing for analysis of cell position during this period of development, and have begun expanding our computational map into the second half of embryogenesis. Our current map includes 202 nuclei across the embryo, including 32 neuronal nuclei, 81 body wall muscle nuclei, 20 intestinal, and 20 seam cell nuclei. We also include a tract-based model of the nerve ring, showing how it is positioned relative to neuronal and body wall muscle nuclei as the embryo elongates. In addition to our partial nuclear atlas, we describe improvements to our untwisting and tracking workflow, including a deep-learning image restoration capability which improves image quality during rapid embryo movements, and a semi-automated tracking upgrade to our untwisting software which improves tracking throughput. As we continue to add nuclei and neuronal morphology to the atlas, we plan to integrate our post twitching model with previous pre-twitching work to develop a digital atlas spanning the entirety of embryogenesis.
-
[
International Worm Meeting,
2013]
Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, these nematodes proliferate as they dine on an array of microbes. Interactions with microbes span a spectrum from constant confrontation (pathogens) to relative indifference (food) to perhaps even mutual benefit (symbionts). This study identifies these natural microbes and addresses whether microbiome composition influences proliferation of C. elegans in the wild.
To examine this question, we sequenced bacterial 16S (SSU) rDNA amplicons from habitats with wild C. elegans populations collected in France and Spain. Our results show that C. elegans encounters a broad array of bacteria in the wild-especially the divisions (phyla) of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. An abundance-weighted comparisons of phylogenetic differences (UniFrac) showed distinct clustering by habitat type, as rotting apples clustered separately from other habitats sequenced. Further, rotting apples clustered by large presence of proliferating or small non-proliferating (dauer) populations of worms. C. elegans appear to proliferate in apples with 'simpler' microbiomes (lower diversity, fewer species and Proteobacteria-rich). Specific alpha-proteobacteria were particularly enriched in apples with proliferating worms, while a number of genera were consistently found in apples with non-proliferating worms (e.g., Pseudomonas, several Bacteroidetes, etc.). Population size also correlated with apple rottenness, suggesting bacterial load is key to growth as well.
Similarly, Proteobacteria content does affect C. elegans (N2) growth rate in the lab, as worms grew faster on mixtures (and single isolates) with 80% Proteobacteria versus those with 40% Proteobacteria. Together, these studies define the microbial diet of C. elegans and implicate the natural microbiome as a key determinant of C. elegans' growth in the wild.
-
[
International C. elegans Meeting,
1997]
We isolated zygotic mutants of a novel class, Hmp (humpback), specifically defective in embryo elongation. They identify three genes:
hmp-1,
hmp-2, and
hmr-1. Elongation of the embryo is mediated by cell shape changes in the hypodermis, the outermost cellular layer of the embryo. In Hmp mutants, the dorsal and lateral hypodermis do not elongate. The ventral hypodermis elongates slightly and extends toward the dorsal side of the embryo, forcing the dorsal hypodermis into bulges. In wild-type hypodermal cells, actin filament bundles form at the start of elongation and appear to contract to generate the force that elongates the embryo. These bundles are oriented circumferentially around the embryo and are linked to adherens junctions between hypodermal cells. In
hmp-1 mutants, circumferential bundles form normally but then disassociate from adherens junctions in the dorsal hypodermis during contraction. Germline mosaic analyses reveal that
hmp-1 is also required before elongation for migration of the leading hypodermal cells during hypodermal enclosure of the embryo.
hmp-1 encodes a protein homologous to the actin-binding protein a-catenin, a component of vertebrate adherens junctions. Antibodies against HMP-1 show the protein localizes to adherens junctions in all hypodermal cells.
hmp-2 gene activity is required to localize HMP-1 protein to adherens junctions.
hmp-2 can encode a protein homologous to the a-catenin-binding protein b-catenin/Armadillo. The
hmr-1 gene product appears similar to the homotypic cell adhesion protein cadherin, which binds b-catenin. We are investigating whether HMP-1, HMP-2, and HMR-1 form a complex that anchors circumferential actin filament bundles at adherens junctions and transmits contractile force between hypodermal cells.
-
[
International Worm Meeting,
2013]
We have investigated spatial gene positioning in the nuclei of the early C. elegans embryo. The developmental constancy of this model organism allows 1) to compare 3D gene positioning in cells that are equivalent in terms of history, developmental potential and gene expression profile; and 2) to identify changes associated with lineage commitment and cellular differentiation. We have used multi-color 3D FISH to determine whether gene positioning in the nucleus follows reproducible patterns during the development of C. elegans. Centrosomes were immunolabeled prior to FISH and served as extra-nuclear reference points to align embryonic nuclei both in time and space. The spatial distributions of 4 pairs of genomic segments (approx. 30-40kb in length) was assessed in the AB and P1 blastomeres of the 2-cell embryo. Comparisons were made between corresponding nuclei from different embryos. Our initial analysis failed to reveal reproducible geometrical patterns of 3D gene positioning at this early stage. The radial positioning of loci, given by the distance to the nuclear border in the spherical embryonic nuclei of C. elegans, was also analyzed before (2-cell embryo) and after embryonic genome activation (26-cell embryo). At these stages, no significant correlation was observed between nuclear localization and transcriptional activity or chromosomal localization. At later stages (~100-cell embryo), active genes were found to be preferentially localized in the nuclear interior. Altogether, these results suggest that the early worm embryo, despite fixed lineage commitment and gene expression programs, is characterized by a plastic architecture of its cell nuclei.
-
[
International C. elegans Meeting,
1995]
The morphogenesis of the external epithelium (hypodermis) of the Caenorhabditis elegans embryo drives ventral hypodermal enclosure and the subsequent elongation of the embryo. The presumptive ventral hypodermal cells arise on the dorsal surface of the embryo, and eventually spread laterally and then ventrally to establish junctional connections at the ventral midline. Time-lapse video microscopy as well as immunofluorescence has revealed that this enclosure process is led by the descendants of ABplaapp and ABpraapp. ABpraapppa and ABpraappap (the "leading" right-hand hypodermal cells) as well as ABplaapppa and ABplaappap (the "leading" left-hand hypodermal cells) appear to be pulling the rest of the ventral hypodermis along. We have investigated the role(s) of these leading cells during ventral enclosure using laser ablation. Ablation of the leading right-hand hypodermal cells (ABpraapppa and ABpraappap) causes the leading left-hand hypodermal cells (ABplaapppa and ABplaappap) to extend beyond the ventral midline, resulting in enclosure and elongation of the embryo. Likewise, ablation of the leading left-hand hypodermal cells has the same effect on the leading right-hand hypodermal cells. Ablation of all four cells early in enclosure causes the other ventral hypodermal cells to retract back towards the dorsal side of the embryo, resulting in interrupted elongation due to enclosure defects. Ablation of the anterior leading left and right-hand hypodermal cells still results in ventral enclosure, yet the embryo explodes during elongation (a similar result is seen when the two posterior cells are ablated). Ablation of one anterior leading left-hand hypodermal cell and one posterior leading right-hand hypodermal cell also results in wild-type enclosure with eventual irregular elongation. Ablation of ventral hypodermal cells which are posterior to the "leading" cells causes an immediate retraction of the hypodermis back towards the dorsal side of the embryo. We are currently recovering embryos after ablation and staining with the antibody MH27 in order visualize the apices of individual cells. We are also interested in analyzing the ventral enclosure process in the deficiency sDF23 (isolated by D. Baillie) because its "leading" cells do not migrate properly, while the rest of the hypodermis migrates around the embryo. Scanning electron microscopy is another technique which will be used to better elucidate the ventral enclosure process.
-
[
International Worm Meeting,
2019]
Cells need to be positioned correctly during embryogenesis for achieving important processes like body axis formation and organ development. The mechanisms by which cells reposition in the early developing embryo are still not completely understood. Recently, Naganathan et al 2014, showed that the gradient of myosin in the actomyosin cortex generates chiral flows and these flows are important for breaking left-right symmetry in a developing C. elegans embryo. We here show that chiral flows arise in the AB lineage only, and that the presence of these flows correlates with cellular repositioning in the embryo. Using reverse genetics approach and temperature sensitive mutants we demonstrate that cellular rearrangements in the AB lineage are driven by chiral actomyosin flows. Thus, we conclude that chiral actomyosin flows drive cellular rearrangement in early development.
-
[
European Worm Meeting,
2002]
Nematodes were considered to be important paradigms for determinate development, where fates are assigned autonomously by descent and cells are placed by cell cleavages. After it is now generally accepted, that many inductions occur during early embryogenesis of C. elegans, it was now also shown that cells in the worm embryo are not placed by cell cleavages but by cell migrations into their terminal positions. Thus the worm is also suited to study the mechanisms of cell migration in the early embryo. It appears that the C. elegans embryo is highly regulative not only in cell fate determination but also in positioning cells during pattern formation. Initial experiments showed that the fate of a cell determines its position in the terminal embryo. If the fate of a cell is altered by a manipulation it redirects its movement and migrates to the same position a cell with this specific fate occupies in the normal embryo. To uncover the mechanisms by which cells find their position in the early embryo, we tested, whether a global signal, guiding posts or specific local cell-cell interactions, corresponding to the normal neighbourhood, guide cells to their positions. Surprisingly neither of the mechanisms alone or any redundant combinations appear to be involved in navigating cells. Removing the eggshell showed, that it does not appear to supply any essential information to guide migrations. Also altering the neighbourhood relations of cells and the combination of cell fates to configurations, which normally never occur in normal embryos, does not affect the sorting by migrations. These manipulations are achieved in either mutant and/or laser ablated embryos. We also combined cells from different embryos in an in vitro culture system under a 4D-microscope. We must conclude that a general sorting mechanism exists in the early embryo, which guides cells using information provided by the direct surrounding. Since cells orient themselves properly in any cellular environment, the system appears to be extremely robust. A model for such a general guiding mechanism is discussed.
-
[
International Worm Meeting,
2021]
The first division of the C.elegans embryo is asymmetric; this requires forming an anterior and posterior domain along the long axis of the embryo. The only signaling component of the anterior domain is the kinase, aPKC. Our lab has found that making an anterior domain of active aPKC requires its interaction with two complexes: one is responsible for aPKC's localization (the localization complex) and the other to activate aPKC (the activation complex). Intriguingly, aPKC's kinase activity is required for these interactions. Due to the kinase dependency of these interactions, we hypothesize that they require a substrate or interactor of aPKC, which we will identify. To identify aPKC's substrates and interactors, we plan to use a combination of analogue sensitive kinase assays, phosphoproteomic profiling, and BioID. Once we have generated a list of candidate substrates and interactors, we shall confirm that they have roles in embryo polarization by RNAi knockdown. For candidates with roles in the embryo, phosphomimetic and non-phosphorylatable mutants will be generated for selected substrates. We will analyze these mutants for alterations to aPKC's interaction with the localization and activation complexes. Identification of aPKC's substrates and interactors will find mechanisms responsible for the dynamic interaction between the localization and activation complexes, which is key for the polarization of the embryo. Also, we will identify aPKC substrates and interactors governing other aspects of polarity and embryonic development, providing a rich resource for future study.
-
Zegarek, Matthew H, Velarde, Nathalie V, Ohm, Jon, Parry, Jean M, Grant, Barth D, Hang, Julie, Druzhinina, Marina, Piano, Fabio, Lefkovith, Ariel J, Singson, Andrew, Kelly, Lindsay K
[
International Worm Meeting,
2009]
Oogenesis produces an egg that once fertilized, must transition to a developing embryo. The molecular underpinnings of this oocyte-to-embryo transition are poorly understood. After fertilization, major events of this transition to a developing embryo typically include the resumption of meiosis, formation of polar bodies, remodeling of the egg surface, rearrangements of the cytoskeleton and the block to polyspermy. These events must also be tightly coordinated with cell cycle progression.We have identified a group of psudo-phosphatases (EGG-3, EGG-4, EGG-5) that define an emerging protein complex that is required for sensing sperm entry and directing many of the events required to convert the egg into a developing embryo. In animals lacking EGG-3 or EGG-4 and EGG-5 function, we observe defects in meiosis, polar body formation, F-actin dynamics, eggshell deposition and/or the block to polyspermy. During oogenesis, EGG-3, EGG-4 and EGG-5 assemble at the oocyte plasma membrane with the egg activation effectors MBK-2 and CHS-1. All of these molecules share a complex interdependence with regards to their subcellular localization during oogenesis. Furthermore, shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the plasma membrane at meiotic anaphase I. Therefore EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle. Finally, we have identified candidate molecules that could be additional components of this "EGG complex" and we are working to understand how they could be involved in regulating the events of the oocyte-to-embryo transition.