Guenther C et al. (1993) International C. elegans Meeting "egl-43 directs HSN migration."

Garriga G, Guenther C

[International C. elegans Meeting, 1993]

Guenther C et al. (1997) International C. elegans Meeting "HAM-1 and asymmetric cell division"

Hawkins NC, Guenther C, Garriga G

[International C. elegans Meeting, 1997]

We have characterized the gene ham-1 with the goal of understanding the molecular mechanisms responsible for establishing asymmetries during cell division. ham-1 mutations disrupt asymmetric cell divisions in five embryonic lineages, including the HSN/PHB neuroblast division. In wildtype, the HSN/PHB neuroblast divides asymmetrically to produce a smaller anterior daughter that dies and a larger posterior daughter, the HSN/PHB precursor. Direct observation of the dividing HSN/PHB neuroblasts in ham-1 embryos revealed that the anterior daughter is transformed into a second HSN/PHB precursor which can survive and divide to produce extra HSN and PHB neurons. HAM-1 encodes a novel protein that is asymmetrically localized in many dividing cells during embryogenesis. In the HSN/PHB neuroblast, HAM-1 is restricted to one side of the cell periphery and is predominately inherited by the posterior daughter cell upon division, the HSN/PHB precursor. This result, coupled with the observation that loss of ham-1 function affects the fate of the daughter cell that does not normally inherited HAM-1, has led us to propose a model in which ham-1 acts prior to cell division to segregate factors that confer specific fates to daughter cells. To identify determinants segregated by HAM-1, and proteins necessary for the asymmetric localization of HAM-1, we have conducted a yeast two hybrid screen. Two intereacting proteins of interest were identifed; a dishevelled (dsh) homolog on chromosome II and a protein with similarity to the Wiskott-Aldrich (WAS) protein on chromosome IV. In Drosophila, dsh has been shown to function in the wingless (wnt) signalling pathway, and in C.elegans, wnt signalling has been shown to orient asymmetric cell divisions. Thus, ham-1 might represent a novel member of this pathway. The human WAS gene encodes a multifunctional protein that can interact with both the plasma membrane and actin cytoskeleton. The role of these two interacting proteins is currently under further investigation.

Guenther C et al. (1996) West Coast Worm Meeting "ASYMMETRIC LOCALIZATION OF HAM-1 DISTRIBUTES DEVELOPMENTALPOTENTIAL IN SEVERAL DIVIDING NEUROBLASTS"

Garriga G, Guenther C

[West Coast Worm Meeting, 1996]

We have characterized the gene ham-1 with the goal of understanding the molecular mechanisms responsible for establishing asymmetries during cell division. ham-1 mutations disrupt five asymmetric cell divisions, including the HSN/PHB neuroblast (ABpl/rapppap) division. Based on our findings, we propose that HAM-1 distributes cell fate determinants during asymmetric cell divisions. HAM-1 is a novel protein. Therefore, to elucidate the role of HAM-1 in dividing neuroblasts, we generated a polyclonal antiserum against HAM-1. Staining fixed embryos with the HAM-1 antiserum has shown: a) HAM-1 is first expressed at the onset of gastrulation as crescents at the cell periphery in a subset of dividing cells. b) Later, HAM-1 appears as both crescents in dividing cells and rings in non-dividing cells. c) ham-1 mutants show altered HAM-1 staining. Almost no HAM-1 protein is visible in ham-1(n1438) embryos using the HAM-1 antiserum. ham-1(n1438) contains a small deletion that drastically reduces the level of ham-1 mRNA. HAM-1 is diffuse in the cytoplasm in ham-1(n1810) and ham-1(n1811) embryos. Both ham-1(n1810) and ham-1(n1811) contain the same missense mutation. All three alleles have similar affects on the five neuroblast divisions that require HAM-1. d) HAM-1 is asymmetrically expressed in the dividing HSN/PHB neuroblast. The HSN/PHB neuroblast divides asymmetrically to produce a smaller anterior daughter that dies and a larger posterior daughter, the HSN/PHB precursor, that subsequently divides to produce the HSN motor and PHB sensory neurons. HAM-1 is expressed as a crescent along the posterior of the dividing HSN/PHB neuroblast so that it will be inherited by the HSN/PHB precursor. The asymmetric localization of HAM-1 in the dividing HSN/PHB neuroblast suggests that it could act as a determinant to directly specify the HSN/PHB precursor fate. Alternatively, it could localize determinants to the HSN/PHB precursor. Our analysis of ham-1 mutant phenotypes suggests that HAM-1 tethers cell fate determinants during asymmetric cell divisions. Staining with HSN and PHB markers shows that these neurons are often duplicated in ham-1 mutants. Direct observation of the dividing HSN/PHB neuroblasts in wild-type and ham-1 embryos has shown that while the anterior daughter of the dividing neuroblast normally dies, it can occasionally survive and divide to produce extra HSN and PHB neurons in ham-1 mutants. Thus, the anterior daughter is transformed into a second HSN/PHB precursor by ham-1 mutations. The mutant phenotypes and localization of HAM-1 are consistent with HAM-1 tethering cell fate determinants in the dividing HSN/PHB neuroblast. Both daughters inherit cell fate determinants, in the absence of HAM-1, allowing them to adopt an HSN/PHB precursor fate. We are currently studying how HAM-1 is asymmetrically distributed in dividing neuroblasts.

Guenther C et al. (1995) International C. elegans Meeting "HAM-1 DISTRIBUTES DEVELOPMENTAL POTENTIAL DURING SEVERAL ASYMMETRIC CELL DIVISIONS"

Garriga G, Guenther C

[International C. elegans Meeting, 1995]

During C. elegans embryonic development, hundreds of asymmetric cell divisions generate cell diversity. To understand the molecular mechanisms responsible for establishing asymmetries during cell division, we have characterized mutations in the gene ham-1 that disrupt asymmetric cell divisions in five lineages: the RID, ADE/ADA, ADL, HSN/PHB and ALN/PLM lineages. Based on our findings, we propose a model in which ham-1 acts prior to cell division to segregate factors that confer specific fates to daughter cells. Immunocytochemistry has shown that the neurons generated by the affected lineages can be missing, present in the normal numbers or duplicated in ham-1 mutants. Since each affected lineage normally produces one cell that dies, we analyzed ham-1 ced-3 double mutants to determine if the missing cells in ham-1 were caused by cell death. All of the affected lineages including those that did not produce extra neurons in ced-3 mutants always produced extra neurons in ham-1 ced-3 mutants. Therefore, production of extra cells in ham-1 mutants is often masked by cell death.

Hawkins NC et al. (1998) West Coast Worm Meeting "DISHEVELLED INTERACTS WITH HAM-1"

Garriga G, Guenther C, Hawkins NC

[West Coast Worm Meeting, 1998]

We are studying the ham-1 gene to further understand the mechanisms involved in establishing asymmetries during cell division. HAM-1 is a 414 amino acid protein that is asymmetrically localized in a crescent at the cell peripehry in many dividing cells during embryogenesis. In particular, it is asymmetrically localized in the HSN neuroblast, which divides asymmetrically to generate a smaller anterior daughter cell that undergoes programmed cell death and a larger posterior daughter, the HSN/PHB precursor. The HSN/PHB precursor divides to produce an HSN neuron and a PHB neuron. HAM-1 is localized to the posterior periphery of the HSN/PHB neuroblast and is asymmetrically segregated to the HSN/PHB precursor upon division. In ham-1 mutants, the anterior daugher cell is often transformed into a second HSN/PHB precursor resulting in the duplication of the HSN and PHB neurons. Thus, it is the daughter cell that does not inherit HAM-1 whose fate is transformed. The mutant phenotype coupled with the protein localization had led to the model that HAM-1 acts to tether determinants to insure that they are asymmetrically segregated to the HSN/PHB precursor. In the absence of HAM-1, determinants are presumably distributed symmetrically throughout the HSN/PHB neuroblast and are subsequently inherited by both daughter cells. To identify determinants segregated by HAM-1, and proteins necessary for the asymmetric localization of HAM-1, we have conducted a yeast two hybrid screen. From this screen, a dishevelled (dsh) homolog, C34F11.9, was isolated. In Drosophila, dsh functions in the wingless (wnt) signalling pathway and is the first known component downstream of the putative wnt receptor. It is a cytoplasmic protein, that upon Wnt stimulation becomes hyperphosphorylated and membrane associated. In C. elegans,wnt signalling has been shown to orient asymmetric cell divisions. We hypothesize that DSH interacts with HAM-1 and may localize HAM-1 to the membrane via its PDZ domain, possibly in response to a wnt signal. Using RNA interference to determine the function of dsh in the asymmetric division of the HSN/PHB neuroblast we were unable to generate any phenotype. Since the lack of phenotype could be due to redundancy, a series of RNAi experiements were performed using two additional C.elegans dsh homologs in combination with C34F11.9. HAM-1 phenotypes were obtained from the RNAi experiments with the dsh homolog C27A2.6 alone. Experiments are in progress to determine if this dsh homolog also physically interacts with HAM-1 and further define its role in asymmetric cell division.

Teuliere, Jerome et al. (2013) International Worm Meeting "Cortical HAM-1 positions the cleavage furrow in myosin-dependent asymmetric neuroblast divisions that generates apoptotic cells."

Garriga, Gian, Hawkins, Nancy, Teuliere, Jerome

[International Worm Meeting, 2013]

While the mechanisms that execute apoptosis in C. elegans are understood, less is known about how somatic cells choose the apoptotic fate. Cell divisions that produce apoptotic cells are asymmetric, generating a larger cell that lives and a smaller cell that dies. Two examples are the anterior (Q.a) and posterior (Q.p) daughters of the Q cell. Distinct mechanisms, however, shift the position of the cleavage furrow in these two divisions to produce an anterior apoptotic cell (Q.aa) division and a posterior apoptotic cell (Q.pp) 3. Protrusion of the posterior membrane and anterior localization of myosin II produce an anteriorly-shifted Q.a cleavage furrow. Protrusion of the anterior membrane and posterior displacement of the mitotic spindle produce a posteriorly-shifted Q.p furrow. We find that the winged-helix protein HAM-1 specifically controls the Q.a furrow position. Q.a divided with a reversed polarity in ham-1 mutants to produce a larger anterior and a smaller posterior daughter, a Q.p division pattern. As in the wild-type Q.p division, the anterior Q.a membrane protrudes and the spindle is displaced posteriorly. The asymmetric distribution of myosin II remained asymmetric in QR.a, indicating that while myosin asymmetry is required for an anteriorly positioned Q.a cleavage furrow 3, it is not sufficient in QR.a. A functional GFP::HAM-1 transgene had a Q.a-specific expression, consistent with the ham-1 phenotype. Although HAM-1 antibodies only detected cortical HAM-1 in dividing cells 4, we detected HAM-1::GFP at the cortex and in the nucleus of both embryonic cells and in the Q lineage. Misexpression of HAM-1 in Q.p led to daughter size asymmetry defects. We observed this defect with either wild-type HAM-1 or a mutant that does not accumulate in the nucleus, suggesting that HAM-1 acts at the cortex to position the cleavage furrow. The spectrum of ham-1 mutant defects suggests that HAM-1 primarily regulates a subset of divisions that produce anterior cells fated to die. 3 Ou, G et al. Science 330, 677-680 (2010) 4 Guenther, C & Garriga, G. Development 122, 3509-3518 (1996).

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.