M205c stereomicroscope

The anesthetized zebrafish embryos of the WT or KO groups, aligned side by side in groups of 5, were recorded for 15 s per day for 6 days in M205C stereomicroscope (Leica Microsystems; LAS V4.11 software) at 80x magnification for the heart rate evaluation by heartbeats count.

Anesthetized 0 hpf or one-cell stage embryos, mounted into an agarose-coated plate groove (#16500100, Invitrogen, Carlsbad, California, EU) were injected using M205C stereomicroscope (Leica Microsystems) using a microneedle (#5242952008 femtotips 930,000,043, 0.5–0.7 μm Eppendorf, Hamburg, DE) coupled to the Injectman® 4 pneumatic micromanipulator microinjector (Eppendorf, Hamburg, DE) pressurized with approximately 2–3 nL of CRISPR/Cas9 system into the cell, previously calibrated using micrometer-scale 1 mm in 0.01 mm divisions slide (#2280–13-1EA, Ted Pella). After injection, embryos were incubated in 0.5x E2 medium at 28 °C and analyzed after 24, 48, 72, 96, 120, and 144 hpf.

The model bioink consisting of plasma-alg-mc and cell culture medium was transferred into a 10 mL plotting cartridge (Nordson EFD, Oberhaching, Germany) either using the static mixer (experiment) or a spatula (control) and plotted by a pneumatic-driven extrusion plotter (Bioscaffolder 3.1, GeSiM mbH, Radeburg, Germany). The inks were extruded through dosing needles (d = 410 µm; Globaco, Rödermark, Germany) with 40–45 kPa air pressure, a plotting speed of 12 mm s⁻¹ and a layer height of 0.26 µm. Cuboidal scaffolds (base area 16 × 16 mm²) with a strand distance of 3 mm, four layers and a layer-to-layer orientation of 90° were fabricated. For the gellan gum-based bioink, ink and crosslinking medium were mixed using the “Tube” and “Screw-like v2” static mixer and then processed as described above. For the determination of shape fidelity, three scaffolds were plotted using ink directly from the tip of the cartridge (“Start”). Subsequently, ink was extruded for 30 s before three more scaffolds were plotted (“End”), all scaffolds, without crosslinking, were then imaged using a Leica M205 C stereo microscope equipped with a DFC295 camera (Leica Microsystems, Wetzlar, Germany). The strand width d was determined using the image processing software Fiji (Version 1.52p). For analysis, the ratio of d_End/d_Start was calculated.

Animals were anesthetized with Isoflurane and sacrificed by cervical dislocation. Ethical consent is documented and approved by the local authorities of the Regierungspräsidium Darmstadt. Embryos from timed-pregnant females of the SWISS strain were harvested at embryonic days (E) E10.5, E12.5, and 15.5 (Supplementary information: Supplementary Fig. 18). The respective developmental Theiler stages were determined as 18 (TS18), TS21, and TS23. From E10.5 embryos, the urogenital ridge was dissected under an M205C stereo microscope (Leica Microsystems, Germany) surgically isolated, and transferred into QIAzol®. Embryos were pooled for each time point. For E10.5 stage biopsies from three embryos were pooled biopsies to prepare RNA, for E12.5 and E15.5 stages two embryos were pooled for RNA preparation. From E12.5 (primitive bladder) and E15.5 (bladder) embryos, the distinct structures of the developing and distinct visible bladder were surgically isolated (Supplementary information: Supplementary Fig. 18), combined, and transferred into QIAzol®.

Anesthesia was performed by immersing larvae in 2 mL of 0.5x E2 medium containing 0.4% tricaine (ethyl-3-aminobenzoate, #MS-222, Sigma Chemical Co., St. Louis, MO, US) for 2 min at room temperature before analysis. At the end of the experiments, euthanasia was obtained by immersion in 4% tricaine diluted in 0.5x E2 medium. After exposure, larvae were checked in an M205C stereomicroscope (Leica Microsystems) to ensure that their hearts were not beating before being placed in a 10% bleach solution. When suitable, 24 hpf larvae were anesthetized and dechorionated by immersion in pronase (#P5147, Sigma) at 0.02 mg.mL^− 1 for 5 min.

The WT and KO embryos were anesthetized, aligned in a glass dish in the lateral position, and photographed under an M205C stereomicroscope (Leica Microsystems). The images obtained were used according to the methods previously reported by Bilder et al. [29 (link)]. For measurement of the body length, they were evaluated horizontally from the top of the head to the tip of the tail (μm); the head size evaluated by the antero-caudal measurement of the forebrain, midbrain, and hindbrain (μm); the yolk circumference area (μm²); the eye diameter (μm); the swim bladder circumference area (μm²); the angle of the head (in degree, °) evaluated by the opening of the head in relation to the yolk sac; and the pericardial area (μm²) using ImageJ v.1.8.0_172.