Micro slide

SGBS cells were plated on eight-well ibidi micro slides (Ibidi GmbH, Germany) and differentiated as previously described. Immunofluorescence staining was carried out as described previously^{27 (link)}. Scanning was done by an iCys Research Imaging Cytometer (iCys, Thorlabs Imaging Systems, Sterling, VA, USA). The images were processed and analyzed (n = 3, 2000 cells per SGBS sample) by our high-throughput automatic cell-recognition protocol^{27 (link)} with some modifications using the iCys companion software (iNovator Application Development Toolkit version 7.0, CompuCyte Corporation, Westwood, MA, USA). As previuosly, cells were identified according to their Hoeschst 33342 nuclear staining. Then, the fluorescence signal intensity of the UCP1 immunostaining and the texture sum variance of the light scatter signal of lipid droplets were quantified in each cell within the 30-pixel immediate outward vicinity of the nucleus contour by the Cell Profiler software (The Broad Institute of MIT, MA, USA). Afterward, based on these fluorescence and light scatter signal of single cells, a semi-automated classification and enumeration of the differentiated white and brown adipocytes and undifferentiated preadipocytes was carried out applying the trained classification “Fast Gentle Boosting” of the Cell Profiler Analyst software (The Broad Institute of MIT, MA, USA).

For live imaging, frontonasal mass and lateral nasal prominence explants at stage 25 were transferred to Ibidi microslides (80821, Ibidi) and flipped so that the epithelial surface was contacting the glass. Matrigel (100%, 80 µl, Thermo Fisher) was added on top of the cultures and forceps were used to keep the explant in contact with the dish while the gel set. Media was added to each well (150 µl DMEM:F12 buffered with fresh 20 µM HEPES to equilibrate with room air). This method prevented drift while still allowing 3D morphogenesis to take place. To visualize the nuclei in live cultures, 1 μM Hoechst 33258 was added to the culture media 1 h prior to imaging.
Confocal time-lapse microscopy with 10× and 20× objectives (1.5 zoom on the 20× objective) was carried out (Fig. 4B) using a Leica SP5 inverted confocal microscope with an environmental chamber (37°C) and motorized stage. Image stacks were collected every 10 min for 4-6 h (Table 1). Images were captured at 1024×1024 or 2048×2048 resolution (for 10×), 400 Hz, UV 405 laser (11% power). We imaged ten independent cultures at 10× magnification and six of these were of high enough quality throughout the experiment to be used for manual tracking (Table 1). Another eight cultures were imaged at 30× (Table 1).

Tube formation was assessed using micro-slides (Ibidi, Martinsried, Germany). Per well 10 μl Matrigel was added and solidified for 30 min at 37°C, prior to seeding 1.0^∗10⁴ cells per well in 50 μl CECGM. Cells were imaged 6 h after seeding (Olympus CKX41).