Koji Kyoda et al. (2008) Development & Evolution Meeting "Computer Analysis of Quantitative Cell Division Patterns of Early Embryos"

Shuichi Onami, Koji Kyoda, Eru Adachi, Mari Furukawa, Kumiko Shimada

[Development & Evolution Meeting, 2008]

We are computationally studying cell division pattern of early gene-knockdown embryos. Because conventional phenotype analysis was performed by manual inspection, the objectivity and sensitivity of phenotype detection were limited. Our computer system measures quantitative cell division pattern from 4D DIC microscope images; this pattern includes xyz positions and outlines of nuclear regions at 40-sec interval and their lineage. Here we report a phenotype analysis of RNAi embryos against all 97 genes on Chr. III known to give 100% Emb by RNAi. We obtained the cell division patterns of 31 wild type and 88 RNAi embryos against 77 genes from 1- to 8-cell stage; RNAi embryos against the other 20 genes did not show any cell division. By mathematically defining 281 phenotypic signatures and statistically comparing these signatures between wild type and RNAi embryos, we objectively detected 2583 phenotypes in the cell division patterns of RNAi embryos; the phenotypes consist of 2159 and 424 phenotypes for 257 spatial and 24 temporal signatures, respectively. Our computational analysis detected various novel spatiotemporal phenotypes, e.g., abnormal division axes of cells at 4-cell stage and the various types of abnormalities in cell division timing. We confirmed that our analysis detected all 23 phenotypes for 14 signatures on cell division pattern detected in previous large-scale manual analysis. Our analysis detected 178 previously undetected phenotypes for these signatures, indicating that the sensitivity of phenotype detection by computational analysis is higher than that by manual analysis. In addition, we developed a new correlation-based method to explore developmental mechanism using the quantitative cell division pattern data. We predicted developmental process map that consists of developmental signatures and their associations; the developmental signatures correspond to phenotypic signatures and the associations were inferred from correlations between phenotypic signatures in the data of wild type embryos. We predicted genes involved in these associations by finding the outliers on the correlation between phenotypic signatures in the data of RNAi embryos. Our computational analyses of quantitative cell division patterns provide useful information for understanding spatiotemporal developmental process.

Koji Kyoda et al. (2008) "Computational phenotype analysis of quantitative cell division patterns"

Mari Furukawa, Eru Adachi, Shuichi Onami, Koji Kyoda, Kumiko Shimada

[2008]

"We are performing phenotype analysis using quantitative cell division patterns of RNAi embryos. Because conventional phenotype analysis was performed by manual inspection, the detection of phenotypes has a limited objectivity. Our computer system measures quantitative cell division pattern from 4D DIC microscope images; this pattern includes xyz positions and outlines of nuclear regions at 40-sec interval and their lineage. Here we report a phenotype analysis of RNAi embryos against all 97 genes on Chr. III known to give 100% Emb by RNAi. We obtained the cell division patterns of 31 wild type and 88 RNAi embryos against 77 genes from 1- to 8-cell stage; RNAi embryos against the other 20 genes did not show any cell division. By mathematically defining 281 phenotypic signatures and statistically comparing these signatures between wild type and RNAi embryos, we detected 2583 phenotypes in the cell division patterns of RNAi embryos; the phenotypes consist of 2159 and 424 phenotypes for 257 spatial and 24 temporal signatures, respectively. Our computational analysis detected various novel spatiotemporal phenotypes, e.g., abnormal division axes of cells at 4-cell stage and the various types of abnormalities in cell division timing. We confirmed that our analysis detected all 23 phenotypes for 14 signatures on cell division pattern detected in previous large-scale manual analysis. Our analysis detected 178 previously undetected phenotypes for these signatures, indicating that the sensitivity of phenotype detection by computational analysis is higher than that by manual analysis. In addition, we developed a new correlation-based method to explore developmental mechanism using the results of phenotype analyses. We predicted developmental process map that consists of developmental signatures and their associations; the developmental signatures correspond to phenotypic signatures and the associations were inferred from correlations between phenotypic signatures. We predicted genes involved in these associations by finding the outliers on the correlation in the results of RNAi embryos. Our computational analyses of quantitative cell division patterns provide useful information for understanding spatiotemporal developmental process."

Masumi Shimada et al. (2003) International Worm Meeting "Possible roles of MOE-1/OMA-1 CCCH-type zinc-finger family proteins in C. elegans oogenesis and embryogenesis"

Hiroyuki Kawahara, Masumi Shimada

[International Worm Meeting, 2003]

Oocyte maturation is an important prerequisite for the production of progeny. Although several germ-line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation had not been fully investigated. Using a multiple RNAi technique, we have identified three redundant CCCH-type zinc-finger genes, named by us moe-1, -2 and moe-3, as a group related by functions and nucleotide sequence1. Similar results were reported by Detwiler et al.(2001), and they gave the names MOE-1 and MOE-2 to OMA-1 and OMA-2, respectively2. We and Detwiler et al. have found that they have overlapping functions that are crucial for oocyte maturation; i.e. simultaneous removal of these proteins by RNAi induces arrest and expansion of growing oocytes. The results of our in situ hybridization have revealed that each of the moe/oma transcripts is expressed from the distal to proximal region of the gonad, while their corresponding proteins accumulate specifically in the cytoplasm of growing oocytes as well as on P granules. Thus, these gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH-type zinc-finger family proteins thus far discovered. Although MOE-2/OMA-2 protein is rapidly removed from P granules after fertilization, we found that MOE-2/OMA-2 associated with the centrosome-peripheral structure in dividing blastomeres. Our results suggest that moe/oma gene products are unique multifunctional proteins in terms of their redundancy and characteristic behavior during the course of oocyte maturation and subsequent embryogenesis. 1 Shimada, M., Kawahara, H., and Doi, H. (2002). Genes Cells 7, 933-947. 2 Detwiler, M.R., Reuben, M., Li, X., Rogers, E., and Lin, R. (2001). Dev. Cell 1, 187-199.

Yohei Sasagawa et al. (2007) International Worm Meeting "p97-UBXN complexes are essential for embryogenesis and gametogenesis in Caenorhabditis elegans."

Yohei Sasagawa, Teru Ogura, Kunitoshi Yamanaka

[International Worm Meeting, 2007]

An evolutionally conserved AAA chaperone p97 (also known as VCP/Cdc48p) plays an important role in a wide variety of diverse cellular processes, such as homotypic membrane fusion, alleviation of polyglutamine aggregation, cell cycle regulation, and ubiquitin/proteasome pathway, including endoplasmic reticulum (ER)-associated degradation (ERAD). In these processes, p97 is believed to convert chemical energy from ATP hydrolysis into mechanical force to disassemble or remodel protein complexes. The specific function of p97 is determined by its adaptor proteins, including Ufd1/Npl4 heterodimer, Ufd2, Ufd3, Otu1 and UBX (Ubiquitin regulatory X) proteins. The UBX protein family is highly conserved in eukaryotes. The UBX domain directly interacts with the N-terminal region of p97. However, the cellular function of UBX proteins has not been revealed. In this study, we will report the cellular function of Caenorhabditis elegans UBX proteins, UBXN-1 to UBXN-6.<div> We found C. elegans possesses six distinct UBX proteins, termed UBXN (UBX containing protein in Nematode). To study whether UBXN proteins interact with p97, we carried out pull down assay using purified GST-UBXN proteins and His-p97. All UBXN proteins interacted with C. elegans p97, suggesting that UBXN proteins function as p97 adaptors. To know their function, we knocked down each UBXN by RNAi method. RNAi depletion of UBXN-2 and UBXN-3 showed slight embryonic lethal phenotype (5-8%), while other UBXN depleted worms showed no obvious phenotype. Interestingly, when all UBXN proteins were simultaneously depleted by RNAi, severe embryonic lethal phenotype (50% of F1) was observed. In these dead embryos, defects in polarity and PAR-2 localization were observed. This result suggests UBXN proteins play redundant roles in the control of embryonic polarity. In addition, 50% of F1 escaper showed completely sterile phenotypes. No sperm was observed and oocytes were highly stacked in the sterile worms. This phenotype is similar to sex determination defects in fem-1 (hc17) and rpn-10 (tm1180) mutants. Shimada et al. reported TRA-2 ICD (intracellular domain) accumulated in rpn-10 (tm1180) mutant. To test whether UBXN proteins are also related to the degradation of TRA-2 ICD, we carried out immunostaining with their anti-TRA-2 ICD antibody. TRA-2 ICD was found to be highly accumulated in UBXN depleted worms as in the case of the rpn-10 mutant. These results suggest UBXN proteins are involved in gametogenesis. Taken together, we propose that p97-UBXN complexes play important roles in embryogenesis and gametogenesis.</div>.

Fernando, Lourds et al. (2020) MicroPubl Biol

Nguyen, Victoria, Allen, Anna, Hansen, Tyler, Golden, Andy, Fernando, Lourds

[MicroPubl Biol, 2020]

The 26S proteasome is one of the major proteolytic machineries in cells and is composed of nearly 33 different subunits (Budenholzer et al., 2017; Chen et al., 2008; Papaevgeniou and Chondrogianni, 2014). The subunits are arranged as one or two 19S regulatory particle(s) capping a cylindrical catalytic 20S core particle (Budenholzer et al., 2017). Each subunit is highly conserved from yeast to mammals, and proper function of the proteasome is crucial for survival of organisms (Papaevgeniou and Chondrogianni, 2014). Spatiotemporal expression of specific subunits and their roles in regulating the proteasome activity is still not clearly understood. RPN-12 is a subunit of the 19S regulatory particle (RP) of the 26S proteasome in Caenorhabditis elegans (Boehringer et al., 2012; Takahashi et al., 2002). In C. elegans, loss or down regulation of a single proteasome subunit causes embryonic lethality, with the exception of three of the 19S RP subunits: RPN-10, RPN-12, and DSS-1 (Keith et al., 2016; Pispa et al., 2008; Shimada et al., 2006; Takahashi et al., 2002). While the roles of RPN-10 and DSS-1 were previously studied, more detailed investigation into the function of RPN-12 has not been performed. A heterozygous mutant containing a 952bp deletion of the coding region of rpn-12 was generated [rpn-12(av93)] using CRISPR/Cas9 genome editing technology. The heterozygous rpn-12 null mutant was then homozygosed and determined to be homozygous viable with apparent fertility issues. Only 36% of the rpn-12(av93) hermaphrodites produced progeny due to sperm production defects (this phenotype will be published elsewhere). Since efficient proteasome activity is important for the longevity of most organisms, we wanted to investigate whether rpn-12(av93) mutants may have an altered lifespan compared to wild type animals (Saez and Vilchez, 2014; Vilchez et al., 2012). To determine whether the lifespan of rpn-12(av93) mutant animals was affected, lifespan assays were conducted at 20C and 25C. The mean lifespan of rpn-12(av93) hermaphrodites (15.94 days) was not significantly different from wild type hermaphrodites (15.54 days) at 20C. Interestingly, at 25C the mean lifespan of rpn-12(av93) hermaphrodites (15.59 days) was increased compared to wild type animals (12.80 days). However, the maximum lifespan of both rpn-12(av93) and wild type hermaphrodites was not significantly different at 25C. The lifespan analysis at 20C was performed three times (cumulative wild type n=240 and mutant n=192) and at 25C was performed twice (cumulative wild type n=185 and mutant n=135). Our data suggests that RPN-12 is not essential for viability and lifespan of C. elegans under normal conditions (20C), but that absence of RPN-12 can result in a significant increase in mean lifespan under heat stress (25C).