Wen, Chentao et al. (2021) International Worm Meeting "3DeeCellTracker, a deep learning-based pipeline for segmenting and tracking cells in 3D time lapse images"

Wen, Chentao, ,, Ishihara, Takeshi, Miura, Takuya, Kimura, Kotaro, Nemoto, Tomomi, Hillman, Elizabeth

[International Worm Meeting, 2021]

Optical monitoring of cell movements and activities in three-dimensional space over time (3D + T imaging) has become substantially easier due to recent advances in microscopy technology. However, the development of software for segregating cell regions from the background and for tracking their dynamic positions remains a bottleneck in the field. Individual laboratories still need to develop their own software to extract important features from 3D + T images obtained using different optical systems and/or imaging conditions. Moreover, even when identical optical systems are used, optimization of many parameters is often required for different datasets. We developed a software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for the first time for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker on a desktop PC with GPU successfully segmented and tracked 100-200 head neurons in both semi-immobilized and "straightened" freely moving worms, ~100 cells in a naturally beating zebrafish heart, and ~1,000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, such as spinning confocal, SCAPE, and 2-photon microscopes, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results, and we extracted complex patterns of calcium dynamics in the worm brain neurons and the rhythmic patterns in synchronization with heart chamber movement in the zebrafish heart cells. Thus, 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have not been analyzed previously.

Harris TW et al. (1996) West Coast Worm Meeting "CHARACTERIZATION OF SYNAPTIC FUNCTION MUTANTS"

Harris TW, Jorgensen EM

[West Coast Worm Meeting, 1996]

Sustained neurotransmission requires the recycling of synaptic vesicle components from the plasma membrane via endocytosis to restore the pool of synaptic vesicles in the presynaptic nerve terminal. We have taken two approaches to study synaptic vesicle endocytosis in C. elegans. First, we are cloning synaptic function mutants and characterizing their phenotypes. Second, we are identifying worm homologs of vertebrate endocytic proteins, then looking for exisiting mutations or screening for new mutations in these genes. Currently, we are cloning ric-1 and unc-70, and we have identified two worm homologs of synaptojanin, an inositol-5-phosphatase. Two observations suggest that ric-1 is likely to be a presynaptic defect: 1) ric-1 homozygotes are resistant to inhibitors of cholinesterase; and 2) homozygotes exhibit the same jerky, uncoordinated, coiler phenotypes as animals lacking acetylcholine. We have mapped ric-1 to a cosmid gap within the mec-14 - lin-39 interval on LGIII. We are now cloning transposon insertions associated with spontaneous alleles (kindly provided by Jim Rand) isolated from the strain TR638 which displays high levels of transpogenesis. Dominant alleles of unc-70 have a curly posture and are hypersensitive to inhibitors of cholinesterase, suggesting that such mutants release elevated levels of acetylcholine. Recessive alleles of unc-70 are lethal, but escapers are resistant to inhibitors of acetylcholinesterase indicating that these mutants reduce the levels of secreted acetylcholine. Thus, the level of UNC-70 activity seems to regulate the level of neurotransmitter released. We have rescued unc-70 null alleles by microinjection and these rescued animals have a curly phenotype that resembles dominant alleles of unc-70. Increasing evidence suggests a link between phosphoinositide metabolism and endocytosis, including the requirement of PI-3 kinase for efficient growth receptor internalization and downregulation, via a dynamin/clathrin mediated-process. Recently, a neuronal-specific inositol-5-phosphatase, dubbed synaptojanin, has been identified in rats. Synaptojanin interacts with amphyphysin and the adaptor protein GRB2. The only other protein currently known to interact with amphyphysin is dynamin. Synaptojanin colocalizes with dynamin at the nerve terminal, suggesting a role for synaptojanin in endocytosis. In conjunction with our collaborators, Pietro deCamilli and Yasuo Nemoto, we have identified two loci homologous to rat synaptojanin. We are presently mapping the physical location of these genes.

Cornils, Astrid et al. (2009) International Worm Meeting "Specific insulin-like peptides translate distinct sensory information to regulate C. elegans development."

Gloeck, Mario, Alcedo, Joy, Cornils, Astrid

[International Worm Meeting, 2009]

Insulin-like peptides (ILPs) can convey environmental cues to regulate diverse biological processes. C. elegans has 40 ILPs (1, 2) that might act as ligands of DAF-2, an insulin/IGF-1 receptor homolog in the worm (3). Although it is unclear how these ILPs regulate worm physiology in response to the environment, downregulation of DAF-2 signaling due to harsh environmental conditions prevents the development of reproductive adults and induces the formation of developmentally arrested dauer larvae (reviewed by refs. 4, 5). Since many ILPs are expressed in sensory neurons and interneurons (1), it is possible that these ILPs translate different sensory information to promote either reproductive development or dauer formation, a process regulated by specific chemosensory neurons (6, 7). We are focusing on the ILPs DAF-28 and INS-6, which are predicted to act as DAF-2 agonists (2), and the ILP INS-1, which is predicted to act as a DAF-2 antagonist (1). Our genetic analyses of deletion mutants showed that daf-28 and ins-6 act together to inhibit dauer entry and to promote dauer exit. At the same time, we also found that the relative importance of daf-28 and ins-6 on dauer entry vs. dauer exit are reversed. Together our data suggest that both daf-28 and ins-6 ensure reproductive development under good environmental conditions. In contrast, we observed that ins-1 promotes dauer entry and inhibits dauer exit, which suggests that ins-1 is required to ensure dauer formation under harsh environmental conditions. Two of the dauer-regulating chemosensory neurons are ASI and ASJ (6, 7). ASI functions to inhibit dauer entry (6), while ASJ functions to promote both dauer entry and dauer exit (6, 7). daf-28 is expressed in both the ASI and ASJ neurons and is downregulated in dauer larvae in these neurons (2). On the other hand, we found that ins-6 expression changes between the ASI neurons in well-fed worms and the ASJ neurons in dauer larvae, which is consistent with the observed functions of ins-6 in dauer entry vs. dauer exit. Thus, our data suggest that specific ILPs act coordinately and in different neurons to regulate different developmental programs in response to distinct sensory cues. References: (1) Pierce et al., 2001. Genes Dev. 15:672-686. (2) Li et al., 2003. Genes Dev. 17:844-858. (3) Kimura et al., 1997. Science 277:942-946. (4) Guarente and Kenyon, 2000. Nature 408: 255-262. (5) Nemoto and Finkel, 2004. Nature 429:149-152. (6) Bargmann and Horvitz, 1991. Science. 251:1243-1246 (7) Schackwitz et al., 1996. Neuron. 17:719-728.