Theresa M. Grana et al. (2007) International Worm Meeting "Cell-cell adhesion molecules regulate gastrulation in C. elegans."

Theresa M. Grana, Elisabeth A. Cox, Jeff Hardin

[International Worm Meeting, 2007]

Morphogenesis requires dynamic regulation of cell adhesion and the cytoskeleton. The first major morphogenetic movement in development is gastrulation. C. elegans gastrulation begins at the 26-cell stage with the migration of the two endodermal precursors Ea/Ep from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on their specification and polarization, apical myosin accumulation and Wnt activated actin-myosin contraction that drives apical constriction and ingression (Nance et al., Wormbook, 2005; Lee et al., Current Biology, 2006). Here, we show that Ea/Ep ingression also requires cell-cell adhesion mediated by either HMR-1/cadherin or SAX-7/L1 CAM (L1 cell adhesion molecule). Either adhesion system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. In sax-7(eq1); hmr-1(RNAi) embryos we observe correct specification of Ea/Ep, based on timing of cell divisions and the presence of birefringent gut granules, and correct apical polarization of Ea/Ep, based on basolateral localization of PAR-2::GFP. Significantly, in sax-7(eq1); hmr-1(RNAi) embryos Ea/Ep fail to accumulate myosin (NMY-2::GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) alone, apical myosin accumulation is equivalent to wildtype. (In gastrulation stage embryos, HMR-1 (as indicated by HMP-2 staining) and SAX-7 are localized at all sites of cell-cell contact and do not appear on exposed cell surfaces.) SAX-7 and HMR-1 may function to localize myosin via signaling or by directing localization of myosin-binding molecules.

Fukuyama M et al. (1997) International C. elegans Meeting "SCREENS FOR MUTANTS DEFECTIVE IN SUBDIVISION OF THE AB-DERIVED ECTODERM INTO EPIDERMAL AND NERVOUS SYSTEM PRECURSORS"

Fukuyama M, Rothman JH, Braun D

[International C. elegans Meeting, 1997]

Among the six founder cells, AB generates the greatest diversity of differentiated cell types and is the major precursor of ectoderm, producing most of the epidermis and nervous system. Previous studies in several labs showed that in the absence of glp-1-dependent inductions by P1 descendants, AB gives rise to four apparently equivalent granddaughters. Each granddaughter then undergoes a developmentally asymmetric division to produce non-equivalent daughters: in the absence of glp-1-dependent induction, one daughter (an epidermal precursor, or EP) makes primarily epidermis while the other (a neuronal precursor, or NP) appears to make exclusively nervous system. To investigate the mechanism of this EP / NP subdivision of the AB-derived ectoderm, we are exploiting two reporter constructs that distinguish the two precursor types. While SCM::GFP, a marker of seam cells, is expressed exclusively by the EP descendants, a LAG-2::GFP reporter is expressed specifically in the NP descendants in the absence of inductions from P1 descendants. We are analyzing the expression patterns of these two markers in selected deficiency homozygotes and are performing a combined screen for maternal and zygotic embryonic lethal mutants in which expression of either or both of these markers is altered. Since our screens are not biased toward a particular terminal phenotype, and since mutants defective in the EP/NP segregation should be reflected directly in the number and distribution of cells expressing these markers, the screen may identify previously unknown genes required for the asymmetric cell division that subdivides the ectoderm into EPs and NPs.

Daniel J Marston et al. (2005) International Worm Meeting "Investigation of a role for cadherin-domain containing proteins in C. elegans gastrulation."

Daniel J Marston, Bob Goldstein

[International Worm Meeting, 2005]

In the C. elegans embryo gastrulation begins at the 26-cell stage with the migration of the two endoderm precursor cells, Ea and Ep, from the surface of the embryo to the embryonic interior. The cellular process driving this ingression of the Ea and Ep cells is an actin-myosin dependent constriction of the apical surface of these cells. Comparison with other systems would suggest that in order for this apical constriction to lead to ingression it is essential that cell-cell adhesion be maintained between the Ea and Ep cells, and/or the surrounding cells during the constriction. Supporting this is the observation that the surrounding cells are in close contact with the ingressing cells and they move round to cover the Ea and Ep cells as those cells move in to the embryo. There are more than 35 genes in the C. elegans genome thought to encode for adhesion proteins comprising several different classes, including cadherin proteins and IgCAMs. Currently there are no reported defects in gastrulation in strains carrying mutations in these genes. Furthermore when we used feeding RNAi to disrupt the function of those genes for which there is no mutant we detected no defects in gastrulation. One hypothesis is that there may be redundancy between multiple adhesion proteins. One way to overcome the challenge presented by redundancy is to inhibit the function of multiple adhesion proteins simultaneously. It is possible do this by microinjecting adult hermaphrodites with small pools of dsRNAs that code for different proteins. The adults then lay embryos with reduced expression of the proteins coded for by the pooled dsRNAs. Using this technique we have attempted to disrupt the function of multiple proteins that contain cadherin domains. We have identified a group of these proteins that are required for normal embryonic development. Furthermore we have been able to use 4D microscopy to follow cell migrations in embryos dissected out of the dsRNA treated adults to investigate the effects of reducing the function of these cadherin-domain containing proteins on cell movements during gastrulation.

Jen-Yi Lee et al. (2005) International Worm Meeting "Wnt/Frizzled signaling regulates actomyosin contraction during C. elegans gastrulation"

Daniel Marston, Jen-Yi Lee, Bob Goldstein

[International Worm Meeting, 2005]

Cell fate specification often plays a crucial role in determining cell behaviors, such as which cells will undergo morphogenetic movements. During C. elegans gastrulation, the two endodermal precursors, Ea and Ep, move into the center of the embryo. These cells exhibit an apical/basolateral polarized localization of the PAR proteins, which is required for the accumulation of myosin (Nance et al., 2003). Furthermore, Ea and Ep undergo an apical constriction that squeezes them into the center of the embryo (Lee and Goldstein, 2003). It is unclear what other genes regulate C. elegans gastrulation. Wnt-Fz signaling has been implicated in both cell fate specification and morphogenesis in other organisms. We have evidence that mom-2/wnt and mom-5/frizzled regulate C. elegans gastrulation, in addition to their roles in cell fate specification and cell cycle timing. MOM-2 acts in a pathway independent of the polarized distribution of PAR proteins, and the absence of mom-5 does not affect myosin accumulation, suggesting that Wnt-Fz signaling may directly affect apical constriction downstream of myosin accumulation, possibly to activate contraction. In support of this hypothesis, immunostaining embryos with antibodies recognizing a phosphorylated epitope on myosin light chain (MLC) shows an enrichment of phospho-MLC at the cortex of wild-type Ea and Ep during gastrulation, whereas no such enrichment of phospho-MLC is seen in mom-5 mutant embryos. Together, these data suggest that mom-2 and mom-5 act in a signaling pathway that regulate gastrulation through activation of the actin-myosin contractile machinery.

Van Auken K et al. (1997) International C. elegans Meeting "NOB-1 AND POSTERIOR EMBRYONIC PATTERNING"

Van Auken K, Edgar LG, Wood WB

[International C. elegans Meeting, 1997]

The nob-1 IIIR gene, identified in screens for larval lethal mutations that result in severe posterior deformities, is defined by two loss-of-function alleles, ct223 and ct351, which result in the Nob (no back end) phenotype, and one reduced-function allele, ct230, which results in viable animals with misshapen tails. To understand the basis for the Nob-1 phenotype, we analyzed embryonic lineages in animals homozygous for ct223 using a 4D microscope. We find no obvious alterations in lineages that give rise to posterior hypodermis, muscle and neurons. We do, however, observe variable defects in the Ep lineage that suggest Ep is adopting a fate similar to Ea. Consistent with this interpretation, we observe ectopic expression of a pal-1 lacZ reporter construct (normally expressed only in Eal/rpp) in two additional E derivatives, tentatively identified as Epl/rpp. These alterations in gut patterning do not fully explain the Nob-1 phenotype, however, as ablation of Ep, compatible with normal development in wild-type embryos, fails to rescue Nob embryos. To address what other defects may be responsible for Nob-1 morphology, we examined the expression of tissue-specific markers. Staining with MH27 antibody reveals defects in shape and position of posterior ventral hypodermis during enclosure. In addition, the posterior seam cells fail to elongate and fewer express a wIs1 seam cell-specific reporter. Preliminary analysis of expression of a ceh-13 GFP reporter during enclosure reveals potentially ectopic expression in AB-derived posterior hypodermis and neurons. Taken together with the ectopic posterior gut expression of pal-1, this suggests that nob-1 may regulate positional information in several posterior tissues during late embryogenesis. Mosaic analysis is in progress. Since nob-1 resides in a gap in the physical map, we are also identifying fosmid clones that may contain the gene, in preparation for cloning.

Jen-Yi Lee et al. (2001) International C. elegans Meeting "An in vitro System for Studying Cell Movements in Early Embryos"

Jen-Yi Lee, Bob Goldstein

[International C. elegans Meeting, 2001]

The proper migration of cells during embryogenesis is crucial for development. Gastrulation is the first major cell movement of the developing nematode C. elegans , yet surprisingly little is known about the mechanisms of this movement. We wished to use a blastomere culture system (1) to study gastrulation movements in vitro , since isolated cells can be used to address questions that cannot be addressed in intact embryos. As a first step, we asked whether or not the physical constraint of the eggshell and the underlying vitelline membrane were necessary for gastrulation. It was previously observed that gastrulation does not require the eggshell and that gastrulation-like movements may also occur in devitellinized embryos (1,2). We confirmed these observations by removing both the eggshell and the vitelline membrane and filming the embryos using a 4D microscope. Gastrulation begins with the ingression of the two E daughters, Ea and Ep, toward the center of the embryo, followed by MSpp, P 4 , MSpa, and D. We found that ingression of Ea and Ep occurs in vitro consistently (6/6 embryos), and at the time of normal gastrulation, suggesting that the in vitro system can be used to study normal gastrulation. Previous cell lysis experiments showed that an intact AB cell was not required for gastrulation (3). We wanted to further delimit the requirements for gastrulation by determining whether the progeny of an isolated, cultured P 1 cell could gastrulate. By isolating the P 1 blastomere and filming its development, we saw that gastrulation movements occurred even when only P 4 and MSpp contacted the E 2 cells. In addition, these results indicated that the normal geometry of the embryo was not essential for proper gastrulation. As with the devitellinized embryos, movements in the P 1 isolates occurred at the same time as in normal embryos. Taking advantage of the better optics resulting from reduced cell numbers, we wanted to begin to determine which cells were crawling by tracing and analyzing cell shapes in the P 1 isolates. Preliminary evidence showed that Ea and Ep remained roughly spherical, while P 4 and MSpp cells changed their shape during the time of gastrulation, making extensions towards each other, over Ea and Ep. These results suggest that the E cells do not crawl into the center of the embryo during gastrulation; rather, they appear to be pushed in by P 4 and MSpp. In vitro blastomere culture presents a useful system for studying cell migration in early development. We are currently using this system and gastrulation-defective mutants to test hypotheses relevant to how gastrulation is regulated. (1) L. Edgar, Methods in Cell Biology (1995) 48:303-321 (2) E. Schierenberg and B. Junkersdorf, Roux’s Arch Dev Biol (1992) 202:10-16 (3) J. Laufer et al., Cell (1980) 19:569-577

Valentini S et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Absence of Effects of Sir2 Over-expression on Lifespan in C. elegans or Drosophila"

Leko V, Valentini S, Piper M, Houdinott M, Burnett C, Gems D, Bedalov T, Sutphin G, Partridge L, McElwee J, Kaeberlein M, Goss M, Cabreiro F, Howard K

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Sirtuins are NAD-dependent histone deacetylases (HDACs) reported to increase lifespan in yeast, C. elegansand Drosophila, and to mediate effects of dietary restriction (DR) on aging. The role of Sir2 orthologs in animal aging has been tested by over-expression using transgenes, which was found to increase lifespan in C. elegans1,2 and Drosophila3. Gene effects on lifespan can be confounded by genetic background variation4. We verified the impact of Sir2 overexpression on aging, carefully controlling for genetic background and performing tests in multiple laboratories, but saw no effect on lifespan in either organism. InC. elegans, backcrossing to wild type of one long-lived line (LG100) with high level over-expression of sir-2.1 (ref. 1) abrogated longevity without reducing transgene expression. RNAi of sir-2.1 reduced transgene-induced over-expression but not longevity. LG100 also exhibited a dye-filling (Dyf) defect which, unlike the transgene, co-segregated with longevity. Backcrossing of a different long-lived line (NL3909) with low level over-expression of sir-2.1 (ref. 2) also abrogated longevity without reducing transgene-induced over-expression. In Drosophila, over-expression of dSir2 (EP-UAS-dSir2 with the ubiquitously-expressed tubulin-GAL4 driver) extended lifespan relative to wild-type flies, as reported3. However, flies bearing both EP-UAS-dSir2 and tub-GAL4 were not longer lived than genetic controls containing either EP-UAS-dSir2 or tub-GAL4 alone, i.e. increased longevity is not attributable to dSir2 over-expression. Moreover, DR-induced longevity was not dSir2 dependent. Additionally, the HDAC activity of recombinant dSir2 was not activated by resveratrol. These results illustrate the importance of excluding genetic background effects in studies of the impact of individual genes on lifespan. They also suggest that Sir2 over-expression does not increase lifespan in C. elegans or Drosophila, though it remains possible that Sir2 over-expression might increase worm or fly lifespan under other conditions. Nonetheless, these findings raise the possibility that the capacity of Sir2 over-expression to increase lifespan may be restricted to yeast. 1 Nature 410, 227 (2001). 2 Dev Cell 9, 605 (2005).3 PNAS 101, 15998 (2004). 4 Nature 450, 165 (2007).

Vancoppenolle B et al. (1996) European Worm Meeting "Comparison of the early embryonic cell lineage of Caenorhabdi- tis elegans and Pellioditis marina, two rhabditid nematodes."

Vancoppenolle B, Coomans AV, Schnabel R, Borgonie G

[European Worm Meeting, 1996]

The early embryonic cell lineage of Pellioditis marina, a marine rhabditid with relatively short developing time was traced using a 4D-microscope. Although the general pattern of cell divisions is congruent with the lineage described for Caenorhabditis elegans by Sulston and coworkers, striking differences can be observed concerning migrations, timing of divisions and cell deaths. The AB, MS and C lineage of P. marina differ from those of C. elegans both in the occurence of additional cell deaths as wel as in the abscence of certain cell deaths. Additionaly, Caap does not divide in accordance with the characteristic period of the rest of the C lineage. In contrast with C. elegans, the E founder cell in P. marina undergoes a migration before gastrulation and devides into Ea and Ep only after E has entered the interior of the embryo. D and P4 divide in a similar way as in C. elegans.

Vancoppenolle B et al. (1997) International C. elegans Meeting "COMPARISON OF THE EMBRYONIC CELL LINEAGE OF CAENORHABDITIS ELEGANS AND PELLIODITIS MARINA"

Borgonie G, Vancoppenolle B, Schnabel R, Coomans AV

[International C. elegans Meeting, 1997]

The early embryonic cell lineage of Pellioditis marina, a marine rhabditid with relatively short developing time (9hrs at 25!C) was traced using a 4D-microscope. Although the general pattern of cell division is congruent with the lineage described for Caenorhabditis elegans by Sulston and Co-workers, striking differences can be observed concerning migrations, timing of divisions and cell deaths. The AB, MS and C lineage of Pellioditis marina differ from those of Caenorhabditis elegans both in the occurence of additional cell deaths as well as in the abscence of certain cell deaths. Additionaly, Caap does not divide in accordance with the characteristic period for the rest of the C-lineage. In contrast with Caenorhabditis elegans, the E founder cell in Pellioditis marina undergoes a migration before gastrulation and divides into Ea and Ep only after E has entered the interior of the embryo. D and P4 divide in a similar way as in Caenorhabditis elegans.

Wittmann C et al. (1995) International C. elegans Meeting "CEH-13 MIGHT PLAY A ROLE IN BOTH CELLULAR AND EMBRYONIC ANTEROPOSTERIOR ASYMMETRY"

Buerglin TR, Wittmann C, Brunschwig K, Mueller F, Tobler H

[International C. elegans Meeting, 1995]

Located between lin-39 and mab-5, ceh-13 is a member of the homeobox cluster of chromosome III. Parsimony analysis has revealed that it is the labial orthologue. In comparison with the other genes from the HOM-C and Hox clusters, labial-type genes from both D. melanogaster and vertebrates fulfil the most anterior function in colinearity with their position in their respective cluster. In D. melanogaster, labial has at least one additional function during the formation of the gut. Using a b-galactosidase reporter construct and a purified anti-CEH-13 specific antibody, we have followed the expression pattern of ceh-13. Nuclear CEH-13 is first detected in Ep. In 28-cell stage embryos, additional staining appears in the posterior sisters of the last AB division. Expression in later stages is compatible with the interpretation that ceh-13 function is restricted to the anterior domain of late embryos and may also play a role in the differentiation of ventral nerve cord cells.