Zen software version 3.1

Embryos were imaged using an Airyscan2 detector equipped with a Zeiss LSM 900 confocal microscope. Plan-Apochromat 63x/1.4 N.A. oil immersion objective was used. Images were acquired with the following settings: 944 × 944 pixels (33.8 × 33.8 μm), 16-bit depth, 41 z-slices separated by 0.2 μm. For the analysis of endogenous sna-MS2 (Supplementary Fig. 10), 780 × 780 pixels (33.3 × 33.3 μm) images were acquired. The fluorescence of GFP and mCherry was excited using 488- and 561-nm lasers, respectively. Excitation power was measured and calibrated using X-Cite XR2100/XP750 Optical Power Measurement System (EXCELITAS Technologies) to keep the same experimental setting throughout the experiments. Obtained images were processed using the “Airyscan processing” function of Zeiss ZEN software (version 3.1) in 3D with a value of 4.6 for all the channels. The temperature was kept between 22.0 to 23.0 °C during imaging.

Cross-sections (thickness: 10 µm) of the cryopreserved thoracic aortas from C57BL/6J mice were fixed in 4% paraformaldehyde for 15 min at room temperature, then stained with hematoxylin-eosin or Weigert solution to assess IMT or elastic fiber defects, respectively. The sections were examined by microscopy (Axioscan, Zeiss, Oberkocheņ, Germany) and analyzed with the ZEN software version 3.1 (Zeiss). For each animal, four non-contiguous sections separated by about 100 µm were blindly analyzed. For IMT, a mean value was obtained by averaging 32 measurements (eight different areas per section). For the assessment of the level of elastic fiber alterations, the number of interruptions of the circumferential elastic lamellae, the number of elastic lamellae and the thickness of the elastic lamellae (made of the arterial wall elastic fibers) were measured in the entire aorta wall for each cross-section, a mean value was obtained by averaging three to four measurements.

Fluorescence imaging was performed on a Zeiss LSM510 META confocal microscope, a Zeiss Axio Observer Z1 microscope with AxioCamMR3 camera, and a Leica DMI6000 with DFC365FX camera. The images were acquired and generated using Zen software version 3.1 (Carl Zeiss) and LAS AF Lite software version 4.0 (Leica), respectively. Hematoxylin and eosin whole slide images were acquired using a NanoZoomer- SQ digital slide scanner C13140-01 (Hamamatsu) and images were generated using NDP.view2 software version 2.9.29 (Hamamatsu).