Intralineage adherens junctions are present but not absolutely required for individual encasing of NSC lineages.

(A) Representative confocal images of the expression of the Drosophila E-cadherin, Shg, at ALH0, ALH24, ALH48, and ALH72 at 25°C. n ≥ 7 VNCs for all time points. Shg is monitored through a shg::GFP fusion (magenta), CG membrane is visualised by cyp4g15-mtd::Tomato (green), NSCs are labelled with anti-Dpn (grey), and neurons are labelled with anti-ElaV (blue). Yellow stars indicate NSC position, dashed yellow circles indicate Shg::GFP signal between NSCs, and yellow arrowheads indicate Shg::GFP signal between NSC and CG. (B) Representative confocal images of the expression of the Drosophila β-catenin, Arm, at ALH72 at 25°C. n = 6 VNCs. Arm is detected with a specific antibody (magenta), CG membrane is visualised by Nrv2::GFP (green), NSCs are labelled with anti-Dpn (grey), and neurons are labelled with anti-ElaV (blue). Yellow stars indicate NSC position. (C) Representative confocal pictures of the thoracic VNC for control (wor >—(x w1118)) and shg knockdown by RNAi (wor > shg RNAi, line VDRC 27082) in NSC lineages (driver line Nrv2::GFP, wor-GAL4; tub-GAL80ts), at the NSC level, at the neuron level, and in orthogonal view. Larvae are dissected after 68 h at 29°C from ALH0. Control, wor >—(x w1118) (n ≥ 10 VNCs) and wor > shg RNAi (n ≥ 10 VNCs). CG membrane is visualised by Nrv2::GFP (green), and NSCs are labelled with anti-Dpn (grey). (D) Schematic of the respective NSC and CG patterns in shg knockdown in NSC lineages. (E) Representative confocal picture of the thoracic VNC for shg knockdown by RNAi (line VDRC 27082; wor > raeppli-NLS, shg RNAi) in NSC lineages marked with the multicolour lineage tracing Raeppli-NLS (blue, white, orange, and red). Top panel shows the whole thoracic VNC, and bottom panel a close-up of the yellow box. Raeppli-NLS is induced at ALH0 under the control of wor-GAL4 using hs-Flp. Larvae are dissected after 72 h at 29°C from ALH0. See S1 Table for detailed genetics, timing, and conditions of larval rearing. CG membrane is visualised with Nrv2::GFP (green). Two representative CG chambers displaying individual encasing of NSC lineages are delineated with a white dashed line. (F) Quantification of the percentage of NSC lineages non-individually encased from (E). Control, wor > raeppli-NLS (n = 7 VNCs) and wor > raeppli-NLS, shg RNAi (n = 9 VNCs). Data statistics: generalised linear model (Binomial regression with a Bernoulli distribution). Results are presented as box and whisker plots, where whiskers mark the minimum and maximum, the box includes the 25th–75th percentile, and the line in the box is the median. Individual values are superimposed. The data underlying this figure’s quantifications can be found in S1 Data. ALH, after larval hatching; Arm, Armadillo; CG, cortex glia; NSC, neural stem cell; Shg, Shotgun; VNC, ventral nerve cord.