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| WBPicture0000015146 | Description | Figure 1. Endogenously tagged LIN-12 alleles are tools to visualize and manipulate native LIN-12/Notch activity in vivo:A, schematic of the endogenous lin-12 locus in lin-12::mNG::3xFlag and lin-12::mNG::3xFlag::AID strains; The intracellular domain of LIN-12 is represented in dark gray with the transmembrane (TM) and PEST domains annotated. mNG is fused to the C-terminal end of the intracellular domain using a 9 amino acid flexible linker; B-D, nuclear localization of LIN-12::mNG::3xFlag in specific cells correlates with known Notch signaling events in C. elegans larval development. Arrowheads indicate cells with nuclear signal; B, LIN-12::mNG::3xFlag shows nuclear localization in ventral, but not dorsal, M lineage progenitors; C, LIN-12::mNG::3xFlag localization in VPCs is consistent with genetically determined functions in VPC specification. LIN-12::mNG::3xFlag exhibits clear nuclear localization in P5.p and P7.p cells where Notch signaling is known to be active, but localization is restricted to cell membrane and cytoplasmic domains in the neighboring P4.p, P6.p, and P8.p cells where Notch signaling is not active at this time; D, LIN-12::mNG::3xFlag nuclear localization in presumptive spermatheca (left) and central somatic gonad precursors (center); E, time-lapse imaging of LIN-12 intracellular domain localization in a dividing P7.p cell and its daughters reveals dynamic Notch activity in real time. Asterisk at 30 minutes indicates P7.p division. Nuclear LIN-12::mNG::3xFlag is apparent in P7.p prior to division and in both daughter cells immediately afterwards. Nuclear signal diminished over time in the posterior daughter P7.pp and was maintained in the anterior daughter P7.pa. Nuclei are outlined at key time points (0, 35, 85 minutes), and time elapsed is displayed as minutes:seconds; F-I, K-NAA treatment can be used to deplete endogenously tagged LIN-12::mNG::3xFlag::AID in vivo; F, nuclear LIN-12::mNG::3xFlag::AID fluorescence is visible in P5.p, P7.p, and several somatic gonad cells in a control animal imaged with moderate laser power; G, mCherry::HIS-11 fluorescence marks nuclei of cells that express TIR1; H, merged image showing LIN-12::mNG::3xFlag::AID, mCherry::HIS-11, and DIC. Arrowheads indicate cells with visible nuclear NICD signal; I, even with maximal laser power, LIN-12::mNG::3xFlag::AID fluorescence was barely visible in a representative animal treated with the synthetic auxin analog K-NAA. The bright circular punctae are due to autofluoresence in intestinal cells that is more apparent with higher excitation intensity; J, mCherry::HIS-11 marks nuclei of cells that express TIR1; K, merged image showing LIN-12::mNG::3xFlag::AID, mCherry::HIS-11, and DIC. Nuclear NICD signal was not observed in any cells. All images are oriented with anterior to left and dorsal to top. Scale bars = 10 um. | |||
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| Name | ffb41d710d2ef7832f5969096aed790f.jpg | ||||
| Depict | Expr_pattern | Expr16148 | |||
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| Anatomy (11) | |||||
| Cellular_component | GO:0005634 | ||||
| GO:0005768 | |||||
| GO:0005886 | |||||
| Acknowledgment | Template | Reprinted from <Journal_URL> | |||
| Publication_year | 2022 | ||||
| Article_URL | DOI | id | 10.17912/micropub.biology.000603 | ||
| Journal_URL | microPublicationBiology | ||||
| Publisher_URL | Micropublication | ||||
| Reference | WBPaper00064269 |
