Endogenous DVC1 was predominantly expressed during S and G2 phases of the cell cycle. Upon exit from mitosis, DVC1 expression was rapidly downregulated with kinetics similar to that of known APC-Cdh1 targets such as cyclin A, and reaccumulated as cells entered S phase.
To determine in which cell(s) tiar-2 functions, we constructed a GFP-TIAR-2 mini-gene translational reporter and observed broad expression in many cell types, including mechanosensory neurons.
HSP-4::GFP was exclusively localized in the PVD soma, colocalizing with a rough ER marker TRAM, HSP-4's endoplasmic reticulum localization pattern is consistent with the observation that its mammalian homolog BiP is localized in rough endoplasmic reticulum.
All five tagged alleles showed expression in two areas: the distal germline and spermatheca. Signal in the spermatheca was strongest at the membrane but also sometimes visible at a lower level in spermathecal nuclei. Expression in the distal germline was present in both sexes and strongest in the distal-most ~20 rows of germ cells, becoming weaker more proximally. Germ cells in the distal-most gonad showed membrane staining similar to that seen with antibodies to the extracellular domain of GLP-1 (e.g. Crittenden et al., 1994); in addition, staining in the nucleus was detected, but at a low level compared to membrane staining. Expression in L4 and adult animals was not observed outside the gonad or in the male somatic gonad (although we cannot exclude the possibility of expression below our threshold of detection). We also examined pre-gastrulation embryos and observed both membrane and nuclear expression of tagged GLP-1, in a pattern consistent with GLP-1 expression in the AB cell lineage, as reported previously (Evans et al., 1994; Priess, 2005).
All five tagged alleles showed expression in two areas: the distal germline and spermatheca. Signal in the spermatheca was strongest at the membrane but also sometimes visible at a lower level in spermathecal nuclei. Expression in the distal germline was present in both sexes and strongest in the distal-most ~20 rows of germ cells, becoming weaker more proximally. Germ cells in the distal-most gonad showed membrane staining similar to that seen with antibodies to the extracellular domain of GLP-1 (e.g. Crittenden et al., 1994); in addition, staining in the nucleus was detected, but at a low level compared to membrane staining. Expression in L4 and adult animals was not observed outside the gonad or in the male somatic gonad (although we cannot exclude the possibility of expression below our threshold of detection). We also examined pre-gastrulation embryos and observed both membrane and nuclear expression of tagged GLP-1, in a pattern consistent with GLP-1 expression in the AB cell lineage, as reported previously (Evans et al., 1994; Priess, 2005).
Time-lapse fluorescence microscopy was performed, including DIC for morphology. Gene expression patterns were summarized in 4 manners: Average over time, Average over time and at different positions along the anterior-posterior (AP) axis, a voxelized representation over time, and on individual cells overlaid from a reference coordinate dataset (https://doi.org/10.1016/j.ydbio.2009.06.014). The analysis was done with a pipeline based on the multi-purpose image analysis software Endrov (https://doi.org/10.1038/nmeth.2478), which further is needed to browse the raw recording data. Thumbnail movies were also generated, using maximum Z projection for the 3D fluorescence channel. Raw recordings available in the Endrov OST-file format are available at https://www.ebi.ac.uk/biostudies/studies/S-BIAD191?query=S-BIAD191