Volume 41, Issue 2, Pages 195-203.e3 (April 2017) PCP and SAX-3/Robo Pathways Cooperate to Regulate Convergent Extension-Based Nerve Cord Assembly in C. elegans  Pavak K. Shah, Matthew R. Tanner, Ismar Kovacevic, Aysha Rankin, Teagan E. Marshall, Nathaniel Noblett, Nhan Nguyen Tran, Tony Roenspies, Jeffrey Hung, Zheqian Chen, Cristina Slatculescu, Theodore J. Perkins, Zhirong Bao, Antonio Colavita  Developmental Cell  Volume 41, Issue 2, Pages 195-203.e3 (April 2017) DOI: 10.1016/j.devcel.2017.03.024 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 41, 195-203. e3DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Convergent Extension of the Ventral Nerve Cord (A) Maximum projections of C. elegans embryos expressing cnd-1p::PH::RFP. Red contours outline the embryo. White dashed box marks the VNC. Scale bar, 10 μm. (B) Single slices showing three distinct rosettes observed during CE in the VNC. White arrow points to the vertex of the most anterior rosette in which only three cells are labeled. Red dashed boundary outlines the rosettes. Accompanying schematics and color key identify the specific neurons in each rosette as well as those which arise from the right (R) and left (L) lineages. Asterisk marks RIGL. The color scheme is used in the rosette schematics and 3D reconstructions. Scale bar, 5 μm. (C) Example assembly and resolution dynamics of the central rosette identified in (B) imaged in a different embryo. Dashed circle outlines the rosette. In the corresponding schematics magenta lines mark shrinking cell-cell contacts and cyan marks newly formed cell contacts. Colored circles identify each cell according to the legend provided in (B). Scale bar, 5 μm. (D) 3D reconstruction of DA and DD neurons (same color scheme as in panel B) during rosette resolution (0′–6′) and subsequent single-cell intercalations beginning with DD3 (9′–19.5′). White arrowheads point to intercalating cells. (E) 3D reconstruction showing final linear configuration of DA and DD neurons in the VNC prior to the onset of twitching; color scheme as in (B). Developmental Cell 2017 41, 195-203.e3DOI: (10.1016/j.devcel.2017.03.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 PCP Genes and sax-3 Act in Parallel Genetic Pathways to Regulate Neuron Position in the Ventral Nerve Cord (A) Schematic of DD, DA, and DB neuron positions in the ventral nerve cord (VNC), retrovesicular ganglion (RVG), and preanal ganglion (PAG). (B–E) Representative images of L1 stage DD1–6 (arrowheads) in wt and mutant backgrounds labeled with a ynIs37[flp-13p::gfp] reporter. (F) Quantification of DD2–4 positions relative to DD1 in L1 stage wt and mutant worms. Neurons (color coded as indicated along the top) are plotted along the anterior-posterior (AP) axis where DD1 marks the 0% AP position and the anus is the 100% AP position. Means and 95% confidence intervals are shown for each DD neuron with DD2–6 neurons in mutants compared with the corresponding wt neuron (n = 40–53 worms). ∗∗p ≤ 0.001 using the two-tailed t test. (G–J) Representative images of L4 stage DD (green arrowheads) and DA/DB (red arrowheads) neurons in wt and mutant backgrounds visualized with a zyIs27[flp-13p::gfp unc-129p::mCherry] reporter. (K and L) Representative images of L1 stage DD (green arrowheads) and DA/DB (red arrowheads) neurons in wt and sax-3(zy5); prkl-1(ok3182) double mutant visualized with zyIs27. (M and N) Representative fluorescent and Nomarski images of L1 stage nervous system in wt and vang-1(ok1142) sax-3(zy5) double mutant visualized with the pan-neuronal otIs173[rgef-1p::mCherry] reporter. Bracket indicates region in double mutant devoid of neuronal cell bodies. All scale bars, 50 μm. Developmental Cell 2017 41, 195-203.e3DOI: (10.1016/j.devcel.2017.03.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Localization of PCP Components, SAX-3/ROBO, and NMY-2 during Rosette Assembly (A–C) Neuronal but not epidermal-specific expression of (A) vang-1, (B) prkl-1, and (C) sax-3 rescue the anterior displacement of DD neurons in the corresponding mutant backgrounds. DD neuron quantification and color scheme is as described in Figure 2F. Each DD2–6 neuron in transgenic worms (TG+) are compared with their corresponding neuron in non-transgenic (TG−) siblings. Means and 95% confidence intervals are shown for each DD neuron. ∗∗p ≤ 0.001, using the two-tailed t test (n = 44–62 worms). (D–G) Partial maximum projections of volumes acquired of embryos expressing the cnd-1 membrane label and functional GFP fusions with (D) PRKL-1 (zyIs33), (E) VANG-1 (zy60), (F) NMY-2 (cp13), and (G) SAX-3 (zyIs43) during rosette formation. Dashed ovals in early time point mark a contracting cell contact; dashed circles at later time point mark the resulting rosette vertex. (H) Enrichment of VANG-1::GFP and NMY-2::GFP at the contracting cell-cell contact during rosette assembly relative to mean GFP levels at non-contracting cell contacts (n = 3). (I) Enrichment of VANG-1::GFP and NMY-2::GFP at the rosette center after rosette formation relative to mean GFP levels at the pairwise cell contacts between all cells in the rosette (n = 3). Error bars show SE. Scale bars, 5 μm (D) and 2 μm (E–G). Developmental Cell 2017 41, 195-203.e3DOI: (10.1016/j.devcel.2017.03.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Phenotypes of vang-1, prkl-1, and sax-3/Robo Mutants in Rosette Resolution and Orienting Cell Intercalation (A) Maximum projections and 3D reconstructions showing the embryonic VNC in representative wt and mutant embryos expressing cnd-1p::PH::RFP at the 1.5-fold stage. White arrowheads highlight ectopic cell contacts not observed in the wt at this stage. Color key used is the same as in Figure 2. Scale bar, 5 μm. (B) Schematic highlighting the location of expanded views (dashed box) showing a stereotyped neighbor exchange between posterior DA and DD neurons in wt and mutant embryos expressing cnd-1p::PH::RFP. Red dot marks DA5, blue dot marks DD4, and purple dot marks DD6. Red arrows point to the contraction of the posterior edge of DD4 and white arrows point to the contraction of the anterior edge of DD4. Schematics illustrate the corresponding wt and mutant behaviors. (C) Frequency with which the anterior edge of DD3/4 was observed to contract in wt and mutants. (D) Relative enrichment of VANG-1::GFP at the posterior edge of DD3/4 versus the anterior edge of DD3/4 in embryos immediately prior to contraction as in (B) in wt (n = 5) and prkl-1(ok3182) (n = 4) embryos. Error bars show SE. (E) Examples of varied rosette resolution dynamics observed in wt, vang-1(ok1142), prkl-1(ok3182), sax-3(zy5), and sax-3(zy5);prkl-1(ok3182) embryos expressing cnd-1p::PH::RFP. Red dashed line outlines the cells participating in the rosette. (F) Box-and-whisker plots showing the distribution of observed rosette lifetimes in wt (n = 7), vang-1(ok1142) (n = 9), prkl-1(ok3182) (n = 10), sax-3(zy5) (n = 5), and sax-3(zy5);prkl-1(ok3182) (n = 4) embryos. Red line marks the median, and whiskers encompass 0–100% of range. Developmental Cell 2017 41, 195-203.e3DOI: (10.1016/j.devcel.2017.03.024) Copyright © 2017 Elsevier Inc. Terms and Conditions