Affi gel beads

The dissected palate of the E15.0 mutants was cultured on a Nuclepore filter (Whatman) in Trowell-type organ culture in chemically defined medium (BGJb, Gibco/Life Technologies). Affi-Gel beads (Bio-Rad) were incubated in TGFB3 (100 ng/μl, R&D Systems) and placed on the primary palate of the Cbfb mutant explants, as described previously (Sarper et al., 2018 (link)). BSA was used for the control beads. Fusion of the palatal process was evaluated histologically. The anterior portion of the palates was also dissected under the microscope and total RNA was extracted from these samples for analysis by qPCR.
To evaluate the possible rescue of cleft palate in Cbfb mutants by folic acid application, the palatal explants were cultured for 48 h in BGJb (Gibco) culture medium containing folic acid (N⁵-formyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid, Sigma-Aldrich) at a final concentration of 100 μg/ml. After culture, the in vitro explants were fixed at each stage in 4% paraformaldehyde overnight and then processed for histological observation.

Bilateral CN crush was performed and MPG tissues (n = 4) were isolated after two days, and were embedded in sterile culture plates containing 150 μl of reduced growth factor Matrigel (Corning Life Sciences). Affi-Gel beads (100–200 mesh, Bio Rad), incubated overnight at 4 °C with 1XPBS (n = 2) or SHH protein (25 μl of a 1 μg/μl solution, R&D Systems, n = 2), were embedded in the Matrigel near the MPG. Matrigel was gelled at 37 °C for 5 minutes prior to adding RPMI media (Sigma) and an antibiotic cocktail (100X Penicillin-Streptomycin-Glutamine, Thermo Fisher). Culture plates were placed in an atmosphere controlled incubator (5% CO₂) at 37 °C for three days.

Chick embryos were staged using the Hamburger-Hamilton embryo staging chart, and the clear vitelline membrane was removed over the caudal neural tube into which the tissue/bead was to be transplanted. Freshly dissected embryonic mouse spinal cord was sliced into 400-μm sections on a tissue chopper (McIlwain) and the slices placed into ice-cold L-15. Tissue to be transplanted was punched out with a pulled glass needle (1 mm × 0.78, Harvard Apparatus) and mouth pipette before being carefully placed into the most rostral part of the open neural tube. Sister punches were evaluated with anti-Nestin or anti-NKX6.1 antibodies to confirm that dmNes⁺RG or lateral/vVL cells could be isolated accurately and free from contaminating tissue (S6 Fig). Affi-gel beads (Bio-Rad) were soaked in protein or PBS control for 24 hours before transplantation into RFP electroporated/non-electroporated HH stage 10 chick embryos. Beads were carefully placed into the most rostral part of the open neural tube. The egg was sealed before incubation at 37°C for 24 hours. Embryos were then dissected out and fixed in ice-cold 4% PFA in for 2 hours prior to sectioning and immunohistochemistry. Operated regions were identified through the presence of the mouse tissue (α-M2 antibody).

Affigel beads (Biorad) were soaked in human Bmp2 protein (0.05 μg/μl¹ - R&D) dissolved in PBS/4 mM HCl or Noggin protein (0.05 μg/μl¹ - R&D) dissolved in PBS/4 mM HCl. Beads were soaked for 2 h and implanted into distal mesoderm using a sharp needle.

Protein loaded beads were prepared as follows: 5 Affi-gel beads (152–7302, Bio-Rad) were washed twice with PBS then incubated in 40 μl of BMP4 (5 μg/ml BMP4, 0.05% BSA, PBS), Gremlin (50 µg/ml, PBS), BSA (0.05%, PBS) or PBS for 1hr at room temperature, and rinsed in PBS. Gremlin beads were placed on embryonic kidneys that had been cultured for 24 hrs and beads were replaced daily until the end of the experiment. For experiments on natural kidneys, BMP4-soaked beads were also added to kidneys cultured for 24 hrs but replacement was found not to be necessary. BMP4-soaked beads were added to Grobstein kidneys (see above) once branching was observed, which was 24 hrs after being placed on polyester membranes. For Ganeva kidneys, BMP4-soaked beads were replaced daily for up to 4 days. In all those experiments involving beads, samples were cultured for 5 days after bead addition.

Affi-Gel beads (Bio-Rad) were incubated in TGFβ3 (100 ng/μl, R&D Systems), or bovine serum album for controls, and placed between the edges of dissected palatal shelves (Sarper et al., 2018 (link)).