Vida Praitis et al. (2006) Development & Evolution Meeting "Characterizing a temperature-sensitive mutation required for tubule morphogenesis in C. elegans"

Vida Praitis, Juliette Mushi, Sarah Kniss, Michael Miller, Lensa Yohannes, Zelealem Yilma, Angela Schacht

[Development & Evolution Meeting, 2006]

During C. elegans morphogenesis, epithelial cells derived from the hypodermis, buccal cavity, and pharynx must attach to each other to form a continuous tube. While much of this process is understood at the cellular level, the molecules required to bring these epithelial sheets together are still being characterized. We are characterizing ru5, a temperature-sensitive embryonic lethal mutation that prevents buccal and pharyngeal epithelial cells from forming stable connections during morphogenesis, resulting in embryos with the Pun (pharynx unattached) phenotype. In ru5 mutants, the invagination that marks formation of the buccal cavity does not occur in ru5 embryos. Temperature shift experiments show the altered gene product is required midway through embryogenesis, when buccal and pharyngeal cells come together to form the mouth. To further characterize the ru5 phenotype, we examined the expression pattern of proteins found in the pharynx and head hypodermis during this stage of development. Our results indicate that developmental events, such as the establishment of the pharyngeal primordium, hypodermal enclosure, and reorientation of the anterior pharynx, appear normal. However, the localization of AJM-1 and SMA-1, two proteins that localize to apical domains in epithelial cells (Praitis, Ciccone, & Austin, 2005; Koppen, et al, 2001), is abnormal in the arcade cells and in the head hypodermis. These results suggest the failure to form a stable epithelium is due to defects in the organization, movement, or shape of the arcade cells or anterior hypodermis. We have mapped the ru5 locus and are currently working to identify the altered gene.

Vida Praitis et al. (2005) International Worm Meeting "A temperature-sensitive mutation that affects pharyngeal morphogenesis"

Angela Schacht, Zelealem Yilma, Lensa Yohannes, Michael Miller, Juliette Mushi, Vida Praitis, Sarah Kniss

[International Worm Meeting, 2005]

In C. elegans, pharyngeal morphogenesis is divided into several discreet steps including the creation of a pharyngeal primordium, reorientation of anterior pharyngeal cells, formation of stable epithelium between the buccal cells, pharynx, and intestine, and changes in cell shape (Portereiko & Mango, 2001). We have identified a temperature-sensitive embryonic lethal mutation which causes defects in pharyngeal morphogenesis, resulting in embryos with the pun (pharynx unattached) phenotype. Genetic experiments show the ru5 mutation is weakly semi-dominant and the phenotype can be maternally or zygotically rescued. ru5 embryos grown at the restrictive temperature undergo normal embryonic development until the bean stage when pharyngeal morphogenesis fails to occur properly. Temperature shift experiments show the gene product is required from 4 to 6 hours after the two-cell stage, during morphogenesis, but is otherwise not required for viability. To further characterize the ru5 phenotype, we examined the expression pattern of proteins found in the pharynx and head hypodermis during this stage of development. Our results indicate that early developmental events and the establishment of the pharyngeal primordium occur normally in ru5 embryos grown at the restrictive temperature. Reorientation of the anterior pharynx also appears normal, as the expression patterns of anterior markers such as AJM-1 and SMA-1 in ru5 embryos are indistinguishable from wild type. However, the localization of these proteins in the arcade cells and in the head hypodermis are abnormal and the invagination associated with the buccal cavity does not occur in ru5 embryos. These results suggest the failure to form a stable epithelium is due to defects in the organization, movement, or shape of the arcade cells or anterior hypodermis. Using conventional and snp-snp mapping strategies, we mapped the ru5 mutation to a small interval on LG I. We are currently working on cosmid rescue experiments to identify the altered gene product.

Vida Praitis et al. (2007) International Worm Meeting "Characterizing a temperature sensitive mutation that results in late gastrulation defects."

Lensa Yohannes, Dae Gon Ha, Juliet Mushi, Vida Praitis, Michael Miller, Angela Schacht, Adityarup Chakravorty, Sarah Kniss

[International Worm Meeting, 2007]

During C. elegans development, the mouth or buccal cavity is formed when epithelial cells of the anterior hypodermis connect to those of the pharynx. For buccal cavity formation to occurr normally, the hypodermis, arcade cells, and pharynx must be properly specified and migrate to their correct positions during gastrulation and morphogenesis. We are characterizing ru5, a weakly semi-dominant temperature-sensitive embryonic lethal mutation with variable expressivity that plays a critical role in buccal cavity formation. When grown at the restrictive temperature, 48% of ru5 embryos have defects that include an unattached pharynx (PUN) and body morphogenesis defects (mild); 30% have head ruptures during enclosure (moderate); and 22% of ru5 embryos fail to enclose, due to defects in gastrulation (severe). Previous research has shown that mutations in genes important for cell migration or fate can produce the morphogenesis defects we observe (Nance, Lee, & Goldstein, Wormbook; Chisolm & Hardin, Wormbook). To determine whether the defects observed in ru5 embryos are due to changes in cell fate or position, we have been performing lineage analysis and examining the expression of cell fate markers. Lineage analysis has shown that early development and E-ingression are normal in all ru5 embryos examined. In a severe ru5 embryo, MS-, D-, and P4-derived blastomeres divide with normal timing, but a subset do not ingress, resulting in failure to close the ventral gastrulation cleft. These results are consistent with temperature shift experiments, which indicate that the ru5 gene product is required during a single period of embryogenesis, when the MS blastomeres are ingressing. Examination of cell fate markers shows that the phenotypes we observe in ru5 embryos are not due to major changes in cell fate. The number of pharyngeal and hypodermal cells is normal in all embryos examined and the cells of the hypodermis, nervous system, gut, muscles, and pharynx all differentiate. However, the organization of apical markers such as par-6, sma-1, and ajm-1 are defective in a subset of cells in the head. These results are consistent with the hypothesis that the ru5 gene plays a critical role in positioning MS-derived blastomeres during gastrulation. We have identified the ru5 locus using snp-snp mapping and found that a single cosmid in the region complements the ru5 phenotypes. We are currently subcloning the rescuing cosmid and sequencing candidate genes.

Vida Praitis et al. (2008) Development & Evolution Meeting "Characterization of a temperature-sensitive embryonic lethal mutation"

Lensa Yohannes, Zelealem Yilma, Sarah Kniss, Dae Gon Ha, Michael Miller, Walter Liszewski, Vida Praitis, Juliette Mushi, Adityarup Chakravorty, Angela Schacht

[Development & Evolution Meeting, 2008]

Proper development of organs in the C. elegans embryo requires an exquisitely coordinated sequenceof events.Cells must divide withspecific timing, become properly specified, and move in a coordinated fashionso that they are properly positioned in the embryo.We have been characterizing the ru5 allele, a temperature-sensitive embryonic lethalmutation, with defects in embryonic development. When embryos homozygous forthe mutation are grown at the restrictive temperature they show defects atdistinct stages of development: 22% of ru5 embryos fail to enclose, due to defects in gastrulation (severe); 30%have head ruptures during enclosure (moderate); and 48% of ru5 embryos have body morphogenesis defects that includean unattached pharynx (PUN) (mild).Temperature shift experiments show that the ru5 allele acts prior to these stages of development,during mid-gastrulation, a period marked by rapid cell division, migration, andcell-cell signaling (Nance, Lee, & Goldstein, Wormbook; Chisolm &Hardin, Wormbook).To determinewhether the defects observed in ru5embryos are due to changes in cell number, fate or position, we performedlineage analysis and examined expression of cell fate markers.This analysis has shown thatdevelopmental events occurring prior to mid-gastrulation appear normal. When ru5 embryos are examined at later stages, the number andposition of pharyngeal and hypodermal cells is normal and the cells of thehypodermis, nervous system, gut, muscles, and pharynx all differentiateproperly.However, in someembryos, a subset of MS-, D-, and P4-derived blastomeres do not ingressproperly, and cells in the head and tail regions appear to be mis-positioned orimproperly oriented.These resultsare consistent with the hypothesis that the ru5 allele interferes with proper fate and/or positionof subset of MS-derived blastomeres during mid-gastrulation.We have identified the ru5 locus using conventional and snp-snp mapping.We tested cosmids from this region andfound that all failed to rescue the mutant phenotype.This suggests the gene of interest lies in a cosmid gap.Using complementation analysis and RNAi of known genes in the region, we haveidentified a candidate gene, which we are currently sequencing.

Materi WP et al. (1998) West Coast Worm Meeting "THE ROLE OF UNC-119 IN AXONOGENESIS IN C. ELEGANS"

Pilgrim DB, Maduro MF, Materi WP

[West Coast Worm Meeting, 1998]

UNC-119 is the first example of a novel class of protein found in both the developing and adult nervous systems of the nematode, the fly, the zebrafish (see poster by Angela Manning) and in mammals. While UNC-119 is found throughout the C. elegans nervous system, beginning around the 60-cell stage, the human and rat homologs (HRG4 and RRG4, respectively) have only been found in retinal tissue, beginning shortly after birth. The fly homolog (DmUnc-119) is expressed throughout the larval central nervous system. In the worm, four unc-119 null alleles have been characterized in addition to the wild type. Apart from the canonical nearly paralyzed, uncoordinated phenotype, these mutants also exhibit constitutive feeding behavior even in the absence of food and are unable to enter into the dauer larva stage when subjected to overcrowding and starvation. Both the behavioral and morphological phenotypes suggest that UNC-119 plays a role in growth cone guidance in the developing nervous system. An UNC-119::GFP fusion protein is found in the cytoplasm of all cells of the nervous system. In an unc-119 mutant the ventral nerve cord (VNC) is defasciculated and the lateral commisures, which normally project from the VNC dorsolaterally to join the dorsal nerve cord have an abnormal, highly branched structure. We have integrated GFP constructs, driven by promoters specific to subsets of neurons, into both wild-type and unc-119 mutant strains and compared neural structures at high magnification using confocal microscopy. The projections of many neurons are unaffected by the unc-119 mutation while abnormally long axons, abnormally branched axons and defasciculated axons are associated with neurons affected by the mutation. We have also conducted a yeast 2-hybrid screen using an unc-119 bait construct and have found several positive interactions that are selective for UNC-119 versus a SNF-4 bait. These preliminary results are presently being confirmed using the human homolog, HRG4, as bait in a 2-hybrid experiment. Co-immunoprecipitation experiments are also underway.