-
[
International C. elegans Meeting,
2001]
Cells within the animal embryo separate into distinct germ layers during gastrulation. Although the morphogenetic movements accompanying gastrulation have been described for many species, the molecular mechanisms used to drive these rearrangements are poorly understood. Gastrulation in C. elegans involves the movement, or ingression, of cells from the ventral surface of the embryo to its interior. Since these cells can be followed individually in live embryos, the C. elegans embryo provides a useful model for investigating gastrulation. In some types of animal embryos, gastrulation involves coordinated shape changes and movements by a sheet of surface cells. By contrast, gastrulation in C. elegans includes several examples of non-contiguous cells that ingress at different times. Since all of these cells enter from the ventral surface, we are interested in whether gastrulation is controlled by cell position or lineage. We present evidence indicating that the normal sequence of gastrulation events is not essential; at least some cells that normally ingress at relatively late stages of embryogenesis can do so even when earlier cells fail to ingress. We have found that some cells undergo a reproducible flattening of their exterior-facing surface as they ingress. Non-muscle myosin accumulates at the cortex underlying the flattened surfaces. This flattening does not occur in cells that fail to ingress in certain mutant backgrounds. We are interested in whether flattening is required for gastrulation, and whether the mechanism that results in flattening is controlled by factors that specify cell lineage.
-
[
International Worm Meeting,
2003]
The morphogenesis and migration of cells depends on the polar localization of proteins to specific membrane domains. The first cell migrations within the C. elegans embryo occur during gastrulation, when those cells destined to form internal tissues such as intestine or muscle move from the embryo's surface into a central cavity, the blastocoel. We are interested in learning how early embryonic cells are polarized to allow processes such as blastocoel formation and gastrulation to occur.There is a well-described requirement for a group of conserved proteins called PAR proteins in cell polarity, including in the one-cell embryo. PAR proteins that show anterior-posterior cortical asymmetries at the one-cell stage are redistributed at later stages to adopt an apical-basal asymmetry; 'anterior' PAR proteins (PAR-3, PAR-6, PKC-3) localize to the contact-free (apical) surfaces of cells, and 'posterior' PAR proteins (PAR-1, PAR-2) localize to surfaces (basolateral) in contact with other cells. By combining embryos in culture, we previously demonstrated that cell-cell contact is sufficient to establish this apical-basal PAR asymmetry.As an approach to studying the role of PAR proteins in apical-basal polarity, we have expressed 'anterior' PAR proteins (PAR-3 and PAR-6) tagged with a sequence from the PIE-1 protein (the ZF1 domain1) that promotes their degradation in early embryonic cells. We find that PAR::ZF1 proteins rescue the anterior-posterior defects of par mutant embryos, and are then degraded. After PAR::ZF1 protein degrades, PAR proteins normally restricted to basolateral surfaces spread to the apical surfaces of cells. par mutant embryos expressing PAR::ZF1 develop defects in the adhesion of early embryonic cells, and the ingression of cells during gastrulation is delayed. We propose that cell contact establishes the apical localization of PAR-3 and PAR-6, which in turn regulates the localization or activity of proteins involved in cell migration and/or adhesion.1.Reese, K.J., et al. (2000). Asymmetric segregation of PIE-1 in C. elegans is mediated by two complementary mechanisms that act through separate PIE-1 protein domains. Molecular Cell 6: 445-455.
-
[
West Coast Worm Meeting,
2002]
The directed movement of cells depends on the polar localization of proteins to specific membrane regions. The first cell migrations within the C. elegans embryo occur during gastrulation, when those cells destined to form internal tissues such as intestine or muscle move from the embryo's surface to its interior. We are interested in learning how these cells are polarized to allow such directed processes to occur.
-
[
International Worm Meeting,
2005]
During the four-cell stage of C. elegans development, cells polarize along the embryonic inner-outer, or apicobasal, axis. Apicobasal polarity is important for cellular asymmetries in adhesion that appear during blastocoel formation and cytoskeletal asymmetries that form during gastrulation. PAR-3 is required for these asymmetries and is itself asymmetrically localized. During the early four-cell stage PAR-3 is present around the entire cortex, but by the end of the four-cell stage PAR-3 disappears from regions of cell contact (basal and lateral surfaces) and becomes restricted to caps centered at the apex of the contact-free (apical) surface. Foci of cortical non-muscle myosin (NMY-2) in early embryonic cells move away from regions of cell contact and become enriched in apical caps similar to those of PAR proteins. We have identified a mutation in
nmy-2 that alters cell adhesion, producing an enlarged blastocoel. When myosin activity is reduced, PAR-3 still becomes restricted to the apical surface but fails to form apical caps. We are currently testing the model that myosin movements condense PAR proteins into an apical cap that regulates adhesion at the opposite, basal surface to control blastocoel size.
-
[
International Worm Meeting,
2015]
Organs are composed of various tubes of differing cellular architectures. Seamless tubes are a type of unicellular tube that lack junctions and are found in the mammalian and zebrafish vasculature, Drosophila trachea, and C. elegans excretory system. Lumen formation in seamless tubes involves cell hollowing, whereby intracellular vesicles coalesce to create a luminal space. Recent studies in the C. elegans excretory canal have shown that coordinated vesicle fusion is also required for lumen expansion, however polarity cues that target vesicles to the apical surface of the lumen remain poorly understood. Our lab recently showed that PAR polarity proteins are present on the luminal surface of the excretory canal and co-localize with components of the exocyst, a complex required for targeting vesicles to discrete cell surface sites, and for lumen formation in the excretory cell. In addition, a reduction-of-function mutant in the par gene
pkc-3/aPKC displays lumen expansion defects, suggesting that PAR proteins may coordinate vesicle fusion with the apical surface of the membrane. To determine the role of PAR proteins during tubulogenesis, we are combining CRISPR-Cas9 genome editing with a recently described protein depletion strategy in C. elegans to generate conditional loss-of-function alleles of par genes, allowing us to remove their function specifically in the excretory cell. To deplete protein function, we can exogenously express the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, enabling the rapid degradation of heterologous proteins tagged with a small zinc finger domain (ZF1). As PAR proteins are well known for their essential roles during embryogenesis, we characterized promoter constructs that express ZIF-1 specifically in the excretory canal from late embryonic stages through adulthood to enable PAR loss-of-function only in this cell type. Using this tissue-specific depletion strategy, we aim to identify the polarity cues that membrane-targeted vesicles receive during lumen expansion to better understand the initial steps of seamless tube formation.
-
[
International Worm Meeting,
2015]
Niche-derived signals have been shown to control stem cell quiescence, proliferation and differentiation. Because the niche is needed for stem cells to function, formation of the niche is a critical event in development. Yet, niche formation is poorly understood. This is because in most models, the cells participating in the niche are not well identified prior to niche assembly, and because formation is very difficult to observe in vivo. To understand niche formation, we are studying the gonad primordium in C elegans. The gonad primordium is a simple organ consisting of just four cells - two primordial germ cells (precursors to the germline stem cells), and two somatic gonadal precursor cells (niche cells). The primordium forms via a fascinating morphogenetic process in which the two niche cells migrate posteriorly along the endoderm, turn and recognize the germ cells, and finally wrap almost completely around the germ cells. This process recapitulates many key steps that must occur more generally in niche formation including cell recognition, adhesion with the niche, and activation of the quiescent stem cells. Using transgenic analysis, fluorescent imaging and embryological manipulations including laser ablations we are investigating the early signals exchanged between stem and niche cells, and how niche cell contact promotes stem cell survival. Previously, it was reported that ablation of niche precursors resulted in a loss of the primordial germ cells. We have repeated this experiment using fluorescent reporters to follow cells in the primordium. In animals where niche cells have been ablated, the germ cells are lost through an interesting mechanism in which the germ cells fragment and these pieces are consumed by the neighboring endodermal cells. We are currently investigating the mechanisms used by the niche cells to prevent germ cell death. Ultimately, we hope to identify how the niche cells are able to surround the stem cells, and determine the signals in the niche that protect the germ cells and enable their function in the animal.
-
[
International Worm Meeting,
2011]
Establishing epithelial cell polarity and cell-cell junctions are critical steps in tissue morphogenesis. In order to form mature junctions, epithelial cells must (1) respond to polarity cues and localize junction proteins to the site of future junction formation and (2) coalesce these proteins into fully-formed junctions. This process is essential during organogenesis, where the establishment and maintenance of junctions enables cells to segregate functional membrane domains. Our lab uses C. elegans to study the highly conserved process of epithelial polarization and junction formation. We have shown that the scaffolding protein PAR-3 is required to polarize the embryonic intestine but is dispensable for epidermal polarization. However, additional loss of apical membrane determinant EAT-20/Crb in PAR-3 mutant embryos compromises epidermal polarization. We are currently investigating how EAT-20 localizes and functions with PAR-3 in junction formation. Our findings will provide new insights as to how distinct cell types can employ unique methods to polarize. In other systems, PKC-3/aPKC regulates junction stability downstream of PAR-3. However, the molecular mechanism for this process has yet to be fully described. We have performed a genetic suppressor screen to identify genes functioning with
pkc-3 to regulate epithelial junctions. We are cloning two unknown
pkc-3 suppressors identified in this screen and characterizing how they regulate epithelial junctions. We have shown that these suppressors specifically rescue junction defects seen in
pkc-3ts mutants. Findings from this study will provide new insights into the basic mechanisms of junction formation.
-
[
International Worm Meeting,
2009]
Gastrulation in C. elegans begins when the endodermal precursor cells constrict their apical surfaces and ingress into the interior of the embryo. Subsequently, mesodermal cells and the two primordial germ cells (PGCs) ingress in a spatial and temporal sequence that is highly orchestrated. Proper ingression of endodermal precursor cells requires zygotic transcription and a PAR protein-mediated polarity that distinguishes the contacted and contact-free surfaces of the ingressing cells. By contrast, embryonic gene expression has not been observed in the PGCs at the time that they ingress, and these cells do not develop the contact/contact-free asymmetry of PAR proteins that polarizes somatic cells. Therefore somatic cells and the PGCs might utilize distinct mechanisms to ingress during gastrulation. We hypothesized that forces exerted by surrounding cells might internalize the PGCs. Using time-lapse videomicroscopy, we first identified ingressing somatic cells cells that make contact with ingressing PGCs. Ingressing descendants of the MS and D lineages contacted the PGCs on the surface of the embryo, and endodermal precursor cells, which ingressed earlier during gastrulation, contacted the PGCs from below. We used laser operation to determine which of these cells is important for PGC ingression. Our results suggest that PGC ingressions can occur when ingression of the flanking MS or D descendants is blocked by laser-killing of these cells, but PGCs do not ingress if the endodermal precursors are killed. In addition, the PGCs do not ingress in
end-1 end-3 double mutant embryos, in which endodermal cells are born in their normal position but do not ingress properly and do not form endoderm. We imaged embryos expressing transgenes that label PGCs and endodermal precursor cells and observed that these cells maintain a close association prior to, during, and after PGC ingression. Our experiments suggest that signals or forces from the endodermal precursor cells are critical for ingression of the PGCs.
-
[
International Worm Meeting,
2019]
Somatic niche cells are often critical regulators of germline precursor cells, controlling their survival, proliferation and differentiation. We are investigating how niche cells and germline precursor cells interact with the surrounding basement membrane (BM) in the C. elegans embryo. We find that BM performs two critical roles in development: first, BM helps to establish and maintain niche cell wrapping during embryogenesis; second, BM guarantees primordial germ cell (PGC) quiescence in the embryo. The gonad primordium in C. elegans is a simple, 4-cell structure formed during embryogenesis that will give rise to the entire germ line and somatic gonad in the adult worm. It is comprised of two primordial germ cells (PGC), each of which interacts with a single somatic gonadal precursor cell (SGP). Each SGP extends its cell membrane around the body of a PGC, so as to completely enwrap it. Wrapping establishes a morphology that is carried into larval development, and provides a protective niche for the PGCs in the embryo. The value of this niche is demonstrated by unwrapped PGCs, which die unexpectedly when they are cannibalized by neighboring endodermal cells. We show that the SGPs are required to recruit laminin to the outer surface of the gonad primordium. If the BM is disrupted, SGP wrapping is not maintained and PGCs escape quiescence late in embryogenesis, rather than in fed L1 larva. These failures in development of the gonad primordium lead to the disorganized larval gonad and tumorous, sterile germ line previously reported in BM mutants. Finally, we find that the BM receptor DGN-1/dystroglycan is expressed by the SGPs on surfaces contacting the BM, is required to maintain SGP wrapping, but does not affect PGC quiescence. We conclude that SGPs template a BM that assures the integrity of the niche throughout embryogenesis and that PGCs remain quiescent until after hatching. These results show how a developmentally programed structure, the BM surrounding an organ primordium, plays both structural and signaling roles in the embryonic PGC niche.
-
[
International Worm Meeting,
2021]
Organs are comprised of various tubes with distinct cellular compositions. The smallest of these tubes are made up of just a single cell and can form intracellularly, with a lumen extending through the center of the cytoplasm. Lumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain, but the molecular events that regulate this polarized vesicle delivery remain unknown. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. After acute protein depletion using the ZF1 degron, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.