Cyanine3 (cy3)

Appropriate horseradish peroxidase–, Alexa‐Fluor 488–, Alexa‐Fluor 647– and Cy3‐conjugated secondary antibodies were purchased from Dianova (Germany), GE‐Healthcare (United States), and Jackson Immunoresearch (Stratech, UK).

Whole mount in situ hybridization was performed essentially as described (36 (link)). Embryos underwent overnight fixation in 4% paraformaldehyde at 4°C. After dehydration, bleaching and rehydration, in situ hybridization was performed with DIG-labeled antisense riboprobes for Kit, Dct and MITF. All steps except probe hybridization and final colorimetric detection were performed on a Biolane HTI (Hölle & Hüttner AG, Tübingen, Germany).
Immunohistochemistry was performed on 10-μm-thick sections from the trunk of embryos after fixation, embedding, freezing and cryotome sectioning using the following primary antibodies: anti-Islet1 mouse monoclonal (1:500 dilution, Developmental Studies Hybridoma Bank), anti-MITF rabbit antiserum (1:1000 dilution, gift of H. Arnheiter, NIH, Bethesda, MD, USA), anti-FABP7 rabbit antiserum (1:300 dilution; Millipore), anti-SOX10 guinea pig antiserum [1:1000 dilution (37 (link))]. Secondary antibodies conjugated to Cy2, Cy3 (1:200 dilution, Dianova) or Alexa488 (1:500 dilution, Molecular Probes/Invitrogen) immunofluorescent dyes were used for detection. Nuclei were counterstained with 4΄,6-diamidin-2-phenylindole (DAPI).
β-Galactosidase (β-gal) staining was performed on whole embryos as previously described (32 (link)).

3rd instar larvae expressing handC driven Mon1::HA and Mon1^∆100::HA were dissected in PBS and fixed with 4% PFA in PBS. After washing, specimens were permeabilized with 1% Triton X-100 in PBS, followed by washing with BBT (0.1% BSA and 0.1% Tween-20 in PBS). Specimens were incubated for 30 min in blocking solution (1% BSA and 0.1% Tween-20 in PBS) followed by antibody staining (rabbit anti-Rab5, 1:250, Abcam Cat# ab31261, RRID:AB_882240, Cambridge, United Kingdom; mouse anti-Rab7, 1:10, Developmental Studies Hybridoma Bank Cat# Rab7, RRID:AB_2722471, University of Iowa, IA, USA) (52 (link)). Afterwards they were rinsed with BBT, blocked with blocking solution, and incubated with secondary antibodies (anti-rabbit Alexa Fluor 488, 1:200, Jackson ImmunoResearch Laboratories, Inc, Ely, United Kingdom, Code Number: 111-545-006); anti-mouse Cy3, 1:200, Dianova GmbH, Eching, Germany). Samples were embedded in Fluoromount-G mounting medium containing DAPI (Thermo Fisher, Waltham, MA, USA). Confocal images were captured with an LSM800 (Zeiss, Jena, Germany). For details, see SI Appendix.

Tissue models were fixed with 4% paraformaldehyde for 2 h at room temperature. The tissue models were then washed with phosphate-buffered saline (PBS), permeated using 1% saponin, blocked with 1% BSA in PBS and stained with primary antibodies overnight. The cells were stained with anti E-Cadherin (rabbit, 1:100, Proteintech, Illinois, USA), anti ZO-1 (rabbit, 1:100, Proteintech, Illinois, USA), anti-fibroblast (mouse, 1:100, Antikörper, Aachen, Germany), anti-neutrophil cytosolic factor 2 (rabbit, 1:100, Antikörper, Aachen, Germany), anti-VE cadherin (mouse, 1:100, Gibco/Thermo Fisher scientific, Massachusetts, USA), anti-CD31 (mouse, 1:100, Abcam, Cambridge, United Kingdom), and anti-MUC1 (rabbit, 1:100, Abcam, Cambridge, United Kingdom) antibodies. This was followed by decoration with fluorophore-coupled secondary antibodies (Cy5 and Cy3) (Dianova, Hamburg, Germany), Phalloidin (MoBiTec, Göttingen, Germany) and DAPI (Sigma Aldrich, Missouri, USA). Z-stacks of images were obtained through 25 μm from the top of the monolayer using Leica SP5 confocal system and processed by FIJI and FIJI Plugin 3D Viewer.^{35 (link),36} The confocal image of the whole 3D tissue model was surface rendered by IMARIS Version 8.4.1.