Jeremy Nance et al. (2005) WormBook "Gastrulation in C. elegans."

Bob Goldstein, Jeremy Nance, Jen-Yi Lee

[WormBook, 2005]

Gastrulation is the process by which the germ layers become positioned in an embryo. C. elegans gastrulation serves as a model for studying the molecular mechanisms of diverse cellular and developmental phenomena, including morphogenesis, cell polarization, cell-cell signaling, actomyosin contraction and cell-cell adhesion. One distinct advantage of studying these phenomena in C. elegans is that genetic tools can be combined with high resolution live cell imaging and direct manipulations of the cells involved. Here we review what is known to date about the cellular and molecular mechanisms that function in C. elegans gastrulation.

Jeremy Nance et al. (2001) International C. elegans Meeting "Lineage control of cell behavior during gastrulation"

James R Priess, Jeremy Nance

[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.

Jeremy Nance et al. (2003) International Worm Meeting "PAR proteins and apical-basal polarity of early embryonic cells"

James R Priess, Jeremy Nance

[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.

Jeremy Nance et al. (2005) International Worm Meeting "Non-muscle myosin regulates apicobasal PAR-3 distribution and blastocoel size"

Ryan Cinalli, Jeremy Nance, James Priess, Kim Wachter, Kathryn Good

[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.

Jeremy Nance et al. (2002) West Coast Worm Meeting "PAR proteins and cell polarity after the one-cell stage"

Jeremy Nance, James R Priess

[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.

Achilleos, Annita et al. (2009) International Worm Meeting "Investigating the role of PAR-3 in C. elegans embryonic epithelial cells."

Nance, Jeremy, Achilleos, Annita

[International Worm Meeting, 2009]

Epithelial cells are required for animal development, barrier formation and nutrient uptake. These cells are highly polarized along their apical-basal axis, and form junctions near their apical surfaces that provide adhesion with neighboring epithelial cells. The PAR proteins PAR-6, PAR-3, and PKC-3 are required for junction formation and polarization of mammalian and fly epithelial cells. We showed previously that PAR-6 is required for maturation of junctions but not for polarization of C. elegans embryonic epithelial cells. Depleting PAR-3 by RNAi results in defects in the polarity of a subset of larval epithelial cells (Aono et al., 2004), suggesting that PAR-3 might have an earlier role than PAR-6 in polarizing epithelial cells. To determine if PAR-3 is required at the initial stages of epithelial cell polarization, we used a combination of transgenes and a putative null mutation in par-3 to remove both maternal and zygotic PAR-3 from the embryo before epithelial cells formed. Mutant embryos arrested during elongation and developed lesions that exposed internal cells. Intestinal and other internal epithelial cells that formed in the absence of PAR-3 had disrupted polarity, and junction proteins that normally accumulate at the apical surface (DLG-1, HMR-1) showed a dispersed and often clustered localization. Surprisingly, polarization and junction formation appeared superficially normal in epidermal epithelial cells lacking PAR-3. These findings highlight the different roles that PAR proteins play in regulating epithelial cell polarization and junction formation, and indicate that internal and external epithelial cells utilize at least partially different mechanisms to develop apico-basal polarity.

Chihara, Daisuke et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Morphogenetic mechanisms of stem cell niche assembly in the C. elegans embryo"

Nance, Jeremy, Chihara, Daisuke

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

We are using the C. elegans primordial gonad to understand how stem cells assemble into a niche during development. The C. elegans primordial gonad contains two somatic gonad precursor cells (SGPs) and two primordial germ cells (PGCs). The primordial gonad assembles during embryogenesis when PGCs and SGPs come together adjacent the intestine. As a first step in understanding niche assembly, we investigated how PGCs move to the site where the primordial gonad forms. PGCs and somatic cells move into the interior during gastrulation. Because somatic cells require transcription to ingress whereas PGCs are transcriptionally quiescent, we hypothesized that somatic cells might push or pull the PGCs into the embryo. We used videomicroscopy to identify cells that contact the PGCs, and used laser killing to determin e if the contacting cells are required for PGC ingression. The PGCs are surrounded by ingressing descendants of the MS and D lineages and interior endodermal cells. The only cells necessary for PGC ingression were the endodermal cells, which ingress into the embryo before the PGCs. Killing or altering the fate of the endodermal cells prevented PGC ingression but not ingression of other somatic cells. Using fluorescent membrane markers and live imaging, we showed that PGCs and endodermal cells maintain contact throughout gastrulation. PGCs express high levels of E-cadherin, and knocking down E-cadherin caused PGCs to detach from endodermal cells and remain on the surface of the embryo. We propose that PGCs upregulate E-cadherin to maintain tight adhesion with endodermal cells, which pull the PGCs into the embryo and position them at the site of primordial gonad assembly. Our results highlight the importance of germ cell gut interactions during development and of E-ca dherin-mediated adhesion in niche formation.

Abrams, Joshua et al. (2015) International Worm Meeting "The role of PAR proteins in vesicle recruitment to the apical membrane during lumen expansion in the C. elegans excretory canal."

Abrams, Joshua, Nance, Jeremy

[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.

Chihara, Daisuke et al. (2009) International Worm Meeting "Internalization of primordial germ cells during C. elegans gastrulation."

Chihara, Daisuke, Nance, Jeremy

[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.

McIntyre, Daniel et al. (2019) International Worm Meeting "Somatic gonad precursors assemble a basement membrane that guarantees primordial germ cell quiescence and gonad integrity during embryogenesis."

Nance, Jeremy, McIntyre, Daniel

[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.