Ferguson, James et al. (2021) International Worm Meeting "Identifying the In Vivo Role of Non-centrosomal Microtubule Organizing Centers During Cell Migration"

Jacobs-Wagner, Christine, Papagiannakis, Alexandros, Feldman, Jessica, Ferguson, James

[International Worm Meeting, 2021]

The microtubule (MT) cytoskeleton plays key roles in cell migration via intracellular signaling and modulating cell adhesions and polarity. Within a cell, MTs emanate and grow from microtubule organizing centers (MTOCs), and the localization of an MTOC helps drive specific MT functions. In cultured migratory cells, the MTOC is canonically located at the centrosome with MTs growing towards the leading edge of the cell. in vivo cell migration studies, however, have identified variations in the MTOC site and MT organization, such as MTs radiating towards the trailing edge of the cell from the centrosome, or a broad MTOC at the trailing edge with MTs radiating towards the leading edge. The molecular factors governing MTOC site, and the impact of MT organization on the migratory functions, in vivo is unclear. During C. elegans development, the Sex Myoblasts (SM) are a pair of cells that undergo a long distance migration to the gonad. Utilizing the SM cells as an in vivo model, we are investigating the presence and function of non-centrosomal MTOCs in cell migration via live confocal imaging of intact animals. Through SM-specific, temporal and spatial MT degradation, we have found an essential role for MTs in SM cell migration. Upon MT degradation, the SM cells fail to fully migrate to the gonad. In addition, directional analysis of growing MTs has identified arrays of MTs running parallel to migration and a MTOC localized near the leading edge of the cell. In contrast to the canonical MT organization, this leading edge MTOC does not appear to be associated with the centrosome. These results highlight a MTOC and pattern of MT organization not typically associated with migratory cells. Further investigation into the significance of the MTs at the leading edge in association with cell adhesions and polarity proteins will provide insights into the mechanisms governing migration in vivo.

Melendez A et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON LIN-13. A ZINC-FINGER PROTEIN THAT ACTS IN VULVAL DEVELOPMENT"

Greenwald IS, Melendez A

[International C. elegans Meeting, 1997]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are sterile and display a Multivulva phenotype. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. Along with Gautam Kao, we rescued the Muv and sterility phenotypes of lin-13 with cosmids CO3B8 and KO4F4. We have narrowed the rescuing activity to a 12-kb (MluI-SalI) genomic fragment that encodes a predicted zinc finger protein, and a frameshift mutation introduced into this coding region abolishes rescuing activity. We have isolated cDNAs corresponding to this genomic region and have started characterizing them. The lin-13 transcript appears to be SL1-spliced and of approximately 7-kb in length, encoding a large protein of about 2,100 amino acids with 14 zinc-fingers of the C-X(2-4)-C-X(3)-F-X(5)-L-X(2)-H-X(3)-H class. lin-13 has been grouped with the synMuv genes, which define two functionally redundant pathways that negatively regulate vulval development (Ferguson et al. 1987; Ferguson and Horvitz, 1989). Studies of synMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We have begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development (lin-13 reporter constructs suggest that it is expressed in both hyp7 and the VPCs). We are also screening for lin-13 null alleles to understand further the role of lin-13 in development.

Melendez A et al. (1996) East Coast Worm Meeting "lin-13 encodes a zinc finger protein"

Melendez A, Greenwald IS

[East Coast Worm Meeting, 1996]

Two heat sensitive alleles of lin-13 were identified in screens for vulval mutants and genetically characterized (Ferguson and Horvitz, 1985; Ferguson et al. 1987). lin-13(n387) hermaphrodites raised at 25oC are Multivulva (Muv) and sterile. lin-13 homozygotes raised at 15oC are fertile and non-Muv but display a zygotically rescuable grandchildless phenotype. Deficiency studies suggest that the existing lin-13 alleles are hypomorphs, and that the lin-13 null phenotype is likely to be zygotic lethal. Therefore, lin-13 appears to have several roles in development. lin-13 maps very close and to the left of mab-5 (M. Costa and C. Kenyon, personal communication). Pools of cosmids for the implicated genomic region were tested in germline transformation experiments, and cosmids C03B8 and K04F4 can rescue both the sterility and Muv phenotype of lin-13 (G. Kao, A. Mele'ndez and I. Greenwald, unpublished observations). A 16-kb genomic (Mlu I) fragment rescued the Muv and sterility phenotypes of lin-13 mutants. The genomic DNA corresponding to the lin-13 rescuing Mlu I fragment contains at least two predicted open reading frames: a protein containing many zinc finger motifs and a protein with ankyrin repeats (A. Coulson, J. E. Sulston and R. H. Waterson, personal communication). We found that a 12-kb (BstE II-Mlu I) fragment, excluding the 5' end of the predicted zinc finger containing protein, no longer rescued. By contrast a 12-kb (Mlu I-Sal I) fragment that included the predicted zinc finger protein and eliminated most of the genomic region predicted to encode the ankyrin repeat protein still rescued. We have also introduced a frameshift mutation into the predicted zinc finger protein coding region of the 16-kb Mlu I genomic fragment and it no longer rescued. All of these results suggest that lin-13 encodes a zinc finger containing protein. lin-13 has been grouped with the SynMuv genes (Ferguson et al. 1987). Studies of SynMuv genes have suggested that some act in hyp7 and at least one acts in the VPCs (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995; J. Thomas and R. Horvitz, personal communication). We therefore plan to determine the cellular focus of lin-13 activity by using reporter genes and genetic mosaic analysis.

Wen C-h et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON TWO GENES THAT INTERACT WITH LIN-12: SEL-9 AND SUP-17"

Wen C-h, Greenwald IS

[International C. elegans Meeting, 1997]

Two sel-9 mutations were originally isolated as suppressors of a lin-12 hypomorphic allele (Sundaram and Greenwald, 1993), and we have isolated 6 additional alleles of sel-9 by a noncomplementation screen. We have examined the genetic interactions between sel-9 and lin-12 alleles. None of the alleles of sel-9 behave like sDf47, a deficiency of the locus(Hunter and Wood, 1992). These results indicate either that none of the sel-9 alleles are null alleles, or that the deficiency does not behave like a null allele (perhaps because it reduces the activity of another interacting gene). The complex genetics have made sel-9 difficult to clone, and we will present the status of our efforts at the meeting. sup-17 was identified by suppressors of lin-12(d) alleles (Ferguson and Horvitz, 1985; F. Tax, J. Thomas, E. Ferguson and H.R. Horvitz, 1997). We have been investigating interactions of sup-17 with lin-12 and glp-1 hypomorphs, and with other genes that interact with lin-12. We have also begun to try to isolate sup-17, beginning with a 180kb YAC clone that Mark Metzstein (personal communication) used to rescue sup-17 in the process of correlating the genetic and physical maps in the ces-1 region. We have used in vivo recombination in yeast to truncate this YAC clone, and have identified a 60 kb region that is able to rescue sup-17. We hope to have made additional progress towards cloning sup-17 by the meeting.

A Melendez et al. (1999) International C. elegans Meeting "LIN-13, a zinc-finger protein that acts in vulval development"

A Melendez, IS Greenwald

[International C. elegans Meeting, 1999]

The synthetic Multivulva (synMuv) genes comprise two classes of genes that act in the determination of vulval precursor cell (VPC) fates (1,2). Under certain conditions, alleles of lin-13 display some of the genetic properties of a Class B SynMuv gene (2; our observations, which we will present). We have found that lin-13 encodes a large nuclear protein of 2,249 amino acids with 14 zinc-fingers of the C2-H2 class. Both of the existing lin-13 alleles are nonsense mutations. lin-13(n387) is a TCA to TGA (S525stop) change that disturbs the first zinc-finger and would encode a predicted protein of 524 amino acids. lin-13(n388) is a CGA to TGA (R857stop) change right after the second zinc finger and would encode a predicted protein of 856 amino acids. Thus, both nonsense mutations would result in greatly truncated LIN-13 protein products. The molecular nature of the lin-13 alleles was surprising, since Ferguson and Horvitz (1) showed that lin-13 (n387)/nDf16 is lethal which suggested that lin-13(n387) was hypomorphic, and the lin-13 null phenotype is zygotic lethality. To explore the issue of the lin-13 null phenotype, we used RNAi (3). We assayed the phenotype of the progeny of N2 hermaphrodites injected with dsRNA derived from lin-13 cDNA genomic clones. Injected mothers were incubated at 15degC or at 25degC. Progeny raised at 25degC were Muv and sterile, whereas progeny raised at 15degC were sterile but not Muv. Thus, the phenotypes caused by RNAi resemble those caused by the existing lin-13 alleles, and raise the distinct possibility that the null phenotype of lin-13 is the heat sensitive phenotype displayed by the two existing alleles. We have also begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development ( lin-13 reporter constructs suggest that it is expressed mainly in hyp7 and possibly in the VPCs). We will report on our progress and we will also speculate about the connection between lin-13 and the class B synMuv gene lin-35Rb . 1. Ferguson, E.L. and Horvitz, H.R. (1985) Genetics 110, 17-72. 2. Ferguson, E.L. and Horvitz, H.R. (1989) Genetics 123, 109-121. 3. Fire, A., et al. (1998) Nature 391, 806-811.

Doyle TG et al. (1998) East Coast Worm Meeting "PROGRESS REPORT ON THE CHARACTERIZATION OF SEL-8"

Greenwald IS, Doyle TG

[East Coast Worm Meeting, 1998]

Lin-12/Notch proteins mediate cell-cell interactions that specify cell fate. The isolation of extragenic suppressors and enhancers of lin-12 is an approach that can identify factors that influence lin-12-mediated signalling. The single sel-8 allele was identified as a suppressor of lin-12(d) mutations (F.Tax, J. Thomas, E. Ferguson and H.R. Horvitz, 1997). We have been performing a detailed genetic analysis of sel-8. Interactions between sel-8 and other alleles of lin-12 as well as with other mutants have been found. Three-factor and deficiency mapping have narrowed the locus of sel-8 to a small region of LG III near unc-93. We hope to have progress on the cloning of sel-8 by complementation rescue by the meeting.

Iskra Katic et al. (2003) International Worm Meeting "Characterization of sel-6, a suppressor of lin-12gain-of-function mutants"

Iskra Katic, Iva Greenwald

[International Worm Meeting, 2003]

LIN-12/Notch proteins mediate multiple cell-cell interactions that specify cell fate. Isolation of extragenic suppressors and enhancers of various lin-12alleles has been a powerful method for identifying factors that influence LIN-12 mediated signaling.In a screeen for suppressors of lin-12gain of function mutants, two sel-6alleles were identified (F.E. Tax, J.H. Thomas, E.L. Ferguson and H.R. Horvitz, 1997). We positionally cloned sel-6and found it to encode a protein that contains a potentially informative sequence motif and is conserved in higher eukaryotes. We are performing genetic and molecular analyses to address the role of SEL-6 in the LIN-12/GLP-1 signaling pathways, in the AC/VU as well as other cell fate decisions during C. elegansdevelopment. We will report on our progress at the meeting.

Campbell G et al. (1995) International C. elegans Meeting "SCREENING FOR CELL DEATH GENES AS SUPPRESSORS OF lin-24 and lin-33 MUTATIONS"

Horvitz HR, Campbell G

[International C. elegans Meeting, 1995]

The mutations n432 and n1057 in lin-24 and n1043 and n1044 in lin-33 cause variable abnormal morphologies and deaths of the Pn.p cells, as well as some vulval lineage defects (Ferguson and Horvitz, Genetics 110:17-72, 1985). These abnormalities lead to a vulvaless (Vul) phenotype, which can be suppressed by mutations in the cell death engulfment genes ced-2, ced-5, and ced-10; mutations in other programmed cell death genes (ced-3, ced-4, ced-9(gf), ced-1, ced-6, ced-7, ced-8) do not suppress the Vul phenotype (Saechin Kim, personal communication). These data suggest that a suppressor screen of the Vul phenotype of lin-24 and lin-33 mutants could allow us to identify additional alleles of ced-2, ced-5 and ced-10 and to define new genes involved in programmed cell death.

C Braendle et al. (2004) European Worm Meeting "FLEXIBILITY AND ROBUSTNESS OF VULVA DEVELOPMENT IN VARIABLE ENVIRONMENTS"

M Felix, C Braendle

[European Worm Meeting, 2004]

All organisms have to maintain great phenotypic stability (robustness) in the face of changes in their environment. To gain a better understanding of the mechanisms providing robustness, we study the developmental mechanisms underlying the expression of an invariant and reproducible phenotype: vulval cell fate specification in the nematode Caenorhabditis elegans. This cell patterning process is controlled by a network of tightly regulated and partially redundant signalling pathways (Ras/Notch/Wnt). The significance of these mechanisms is not fully understood, yet their properties, such as redundancy, may be important to maintain a correct vulva phenotype when development is perturbed. We have started to test this idea by studying vulva formation (of vulva mutants and wild type animals) in different environmental conditions (e.g. starvation, liquid culture, passing through the dauer stage). Such environmental conditions have previously been shown to affect the penetrance of several vulva mutations (Ferguson & Horvitz 1985, Battu et al. 2003, Moghal et al. 2003), indicating that vulval development is sensitive to environmental variation. We further aim to test whether pathway activation is differentially regulated in a context-dependent fashion, and to examine how such a differential regulation corresponds to changes in the precision of the vulval patterning process. If different environments alter the use of developmental mechanisms while maintaining precision of the vulva pattern, this would indicate that development exhibits flexibility, which serves to maintain phenotypic stability. This scenario would suggest that environmental variation plays a role in shaping and maintaining the mechanisms underlying vulva formation. We thus also aim to examine whether different C. elegans strains, potentially exhibiting different environmental histories, have evolved differences in the mechanisms controlling vulva development. References Ferguson & Horvitz (1985). Genetics: 110: 17-72.Battu et al.(2003). Development 130: 2567-2577. Moghal et al. (2003). Development 130: 4553-4566.

Doyle TG et al. (1997) International C. elegans Meeting "PROGRESS REPORT ON THE CHARACTERIZATION OF SEL-8"

Doyle TG, Greenwald IS

[International C. elegans Meeting, 1997]

Lin-12/Notch proteins mediate cell-cell interactions that specify cell fate. The isolation of extragenic suppressors and enhancers of lin-12 offers an approach to identifying factors that influence lin-12-mediated signalling. A single sel-8 allele was identified as a suppressor of lin-12(d) mutations (F. Tax, J. Thomas, E. Ferguson and H.R. Horvitz, 1997). We are performing a detailed genetic and molecular analysis of sel-8. The genetic analysis will involve characterizing the nature of the existing sel-8 allele, finding new alleles, and observing interactions between sel-8 and different alleles of lin-12 as well as other mutants. sel-8 maps to LG III; and we are presently using three-factor mapping to narrow down its location so it may cloned by complementation rescue. We hope to have progress on the cloning and the genetics of sel-8 by the meeting.