Fig. 2.

Distribution of POD-1 in migrating neuroblasts and generation of pod-1 conditional KO strain. (A) Schematic of Q neuroblast lineages. QL and QR neuroblasts divide three times and generate three neurons (QL: PQR, PVM, and SDQL; QR: AQR, AVM, and SDQR) and two apoptotic cells (X). (B) (Left) Representative fluorescence images of GFP-tagged POD-1 and mCherry tagged plasma membrane and histone in migrating QR.ap cell. The dashed lines indicate the cell periphery; (Right) the normalized GFP fluorescence distribution plot around the periphery of the representative image shown on Left. The trace starts from the posterior of QR.ap and moves counterclockwise along the cell periphery to the anterior and back to the rear. (Scale bar, 5 μm.) See SI Appendix, Fig. S2A for POD-1 localization in Q cell of GFP::POD-1 KI animals. (C) Quantification of POD-1::GFP fluorescence intensity ratio of the leading edge to the rear part of QR.ap cell. The corresponding mCherry-membrane intensity ratio was used as an internal control. Data are presented as mean ± SEM; **P < 0.01 by Student’s t tests. The number of examined animals indicated by N, n = 5. (D) (Left) Triple fluorescence images of AQR. (Right) The corresponding normalized fluorescence KI intensity distribution along the leading edge. Individual puncta of GFP::POD-1 or ARX-2::TagRFP are labeled and numbered in representative images and the corresponding plots. (Scale bar, 3 μm.) (E) Schematic of pod-1 gene model and single guide RNA (sgRNA) sequences (sgRNA1 and sgRNA2 target the second and third exon, respectively). (F) Representative gels of the T7 endonuclease I (T7EI) assay for pod-1 PCR products amplified from the genomic DNA of worms expressing Phsp::Cas9 and PU6::pod-1-sgRNA1 (Left) or PU6::pod-1-sgRNA2 (Right) with or without heat shock treatment. (G) Quantification of embryo survival rates in WT and pod-1 conditional knockouts; n = 50 to 100 from three different generations.