Volume 43, Issue 4, Pages 530-540.e4 (November 2017) An In Toto Approach to Dissecting Cellular Interactions in Complex Tissues  Pavak Kirit Shah, Anthony Santella, Adrian Jacobo, Kimberly Siletti, A.J. Hudspeth, Zhirong Bao  Developmental Cell  Volume 43, Issue 4, Pages 530-540.e4 (November 2017) DOI: 10.1016/j.devcel.2017.10.021 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 ShootingStar Platform (A) A schematic representation of data flow in the ShootingStar pipeline. (i) microscope control; (ii) tracking software and interfaces; and (iii) perturbation control. (B) Schematic illustration of the four primary steps of cell tracking in ShootingStar. Circles indicate cells detected at a particular time point. (C) Per volume processing times for images acquired of three species; C. elegans (blue), D. melanogaster (red), and D. rerio (black). MP, megapixels. (D) Cumulative accuracy of cell identities in tracking each of three C. elegans embryos (solid, dashed, and dotted lines. N = 3,256 cells each). Developmental Cell 2017 43, 530-540.e4DOI: (10.1016/j.devcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Systematic Monitoring of Automated Cell Ablations (A) Cell-cycle length of 127 remaining AB cell descendants in ablated C. elegans embryos (N = 2) plotted against the average cell-cycle length of the corresponding cell in wild-type embryos (N = 71). (i–iii) Max projections of mitosis in three cells from embryo 1 (dashed ovals). Red arrow indicates anaphase bridging (iii). Scale bar represents 2 μm. (B) Scatterplot of cell-cycle lengths shown in Figure 3 plotted in 3D against the lateral distance between every cell and the target cell and the axial distance between every cell and the target cell. Arrows (i–iii) highlight the same cells as in (A). (C) Time series showing the dynamics of cell death after laser ablation as visualized by a fluorescent histone reporter (white arrow) and membrane marker for a subset of neurons (cnd-1p::PH::GFP). Scale bar represents 10 μm. (D) Lineage tree showing ablations performed across three generations. Left is more anterior, right more posterior. (E) Max projection of a wild-type embryo at the comma stage. Scale bar represents 10 μm. (F) GFP channel from (E) showing the neurons named in (D) as left-right symmetric pairs (white ovals). (G) Representative embryo where ABprpapaa was ablated (N = 4). Dashed oval shows loss of neurons on ablated side. (H) Representative embryo where ABprpapaap was ablated (N = 4). Dashed oval shows surviving neuron. (I) Representative embryo where SIBVR was ablated (N = 4). Dashed oval shows two surviving neurons. Scale bars represent 5 μm in (F)–(I). See also Movie S1. Developmental Cell 2017 43, 530-540.e4DOI: (10.1016/j.devcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Automated Ablation of Hair Cells in Zebrafish Neuromasts (A) Max projection of a single neuromast in a 3 dpf D. rerio larva expressing cldnb::H2B::mCherry (magenta) and myo6b::β-actin::GFP (green). Inset shows expanded view of the GFP-labeled hair cell stereocilia. wt, wild-type. Scale bar represents 10 μm. (B) Time series of polarity establishment in wild-type hair cell visualized by β-actin::GFP. Red dot indicates no evident polarity, red arrow indicates polarity. Scale bar represents 0.5 μm. (C) Distribution of hair cell polarity in wild-type neuromasts (N = 26). (D) Example sequence from the automated ablation of a hair cell (white arrow) and the subsequent maturation of the surviving sister (dashed ovals). Scale bar represents 10 μm. (E) Distribution of hair cell polarity in mock-ablated neuromasts, where a neighboring mature hair cell was ablated instead of one of the two newly born hair cells (N = 24). (F) Distribution of polarity in hair cells whose siblings were ablated soon after birth (N = 9). (G) Time series of early polarization in a hair cell whose sibling was ablated soon after birth. Annotations as in (B). Scale bar represents 0.5 μm. Developmental Cell 2017 43, 530-540.e4DOI: (10.1016/j.devcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Ablation of Multiple Targets (A) 3D model of a lineaged C. elegans embryo showing the final positions of the six GLR cells derived from MSaaaaaa (red), MSapaaaa (blue), and MSppaaaa (magenta). (B) The mesodermal (MS) lineage showing the three mothers of the GLR cells (circled in red). (C) Max projection of representative embryo after ablation of GLR progenitors showing the location of the dead cells' nuclei (dashed circles). (D) Max projection of a wild-type embryo expressing a lim-4p::GFP reporter at the 1.5-fold stage showing normal nerve ring position and morphology. (E) Max projection of an embryo where all three GLR progenitors were ablated. Scale bars in (C–E) represent 10 μm. (F) Normalized nerve ring position in embryos at the 1.5-fold stage. 0 represents the position of the sensory dent, 1 represents the eggshell's posterior pole. (G) Image of a wild-type L1 showing the normal position of the nerve ring. Cell bodies were cropped from the green channel for clarity. Scale bars represent 10 μm. (H) Image of an L1 after GLR ablations. Red arrows point to defasciculated processes in the nerve ring. Scale bars represent 10 μm. (I) Distribution of cell-cycle lengths of 16 nearby cells (within 2 nuclear diameters of the targeted cell) in unablated embryos (N = 3) and embryos where all 3 GLR progenitors were ablated (N = 6). Each color denotes an embryo. (J) Image of an L1 after mock ablation where neighboring cells were ablated instead of the three GLR progenitors. Scale bars represent 10 μm. Developmental Cell 2017 43, 530-540.e4DOI: (10.1016/j.devcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Automated Single-Cell Photoconversion (A) Max projection of a C. elegans embryo expressing dyf-7p::Kaede. The white dashed box highlights the region shown in (B). The white arrow points to the cell body of a neural progenitor. Scale bar represents 10 μm. (B) Two-color imaging after photoconversion. The white arrow points to a cell process. The dashed outline shows tracing of a photoconverted cell outline superimposed on unconverted Kaede fluorescence. Accompanying 3D reconstructions illustrate the resolution of adjoining cells before (green) and after photoconversion (magenta). Scale bar represents 5 μm. (C) Terminal division of the mother of CEPV. The arrow points to a cell process, the arrowhead points to the posterior sister cell of CEPV. Scale bar represents 5 μm. (D) β-Tubulin localization in the mother of CEPV shortly after centrosome duplication. Yellow dashed lines trace the dendrite boundary. White asterisks mark tubulin accumulation at the centrosomes. Scale bar represents 1 μm. (E) β-Tubulin dynamics in the mother of CEPV before and during its division. Annotations are as in (D). Scale bar represents 1 μm. (F) Kymograph of β-tubulin dynamics. (i) The movement of the anterior centrosome into the proximal dendrite. (ii) Depletion of β-tubulin from the proximal dendrite. (iii) Cytokinesis. White dashed boundaries highlight each of the events described by (i)–(iii). Horizontal scale bar represents 2 min. Vertical scale bar represents 1 μm. Developmental Cell 2017 43, 530-540.e4DOI: (10.1016/j.devcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions