Proteolytic restriction of Chordin range underlies BMP gradient formation

Published: 3 August 2020| Version 1 | DOI: 10.17632/xtk7yyxt5f.1
Francesca Tuazon


P-Smad5 quantitative immunofluorescence data from all figures in Tuazon et al., 2020. Segmented individual embryo and population (genotype) mean projections are available in their respective subfolders of each figure folder. If a dataset was used in multiple figures, it is available in all figure folders. Each genotype is available as a single .mat file and the formatting described in full in the ReadMe file. P-Smad5 immunostaining, imaging, and quantification were performed as previous described (Zinski et al., 2017), with the protocol and methodology thoroughly described in Zinski et al. (2019). Briefly, embryos were fixed in 4% paraformaldehyde at 4C, blocked in NCS-PBST, and probed overnight with a 1:100 dilution of anti-phosphoSmad1/5/8 antibody (Cell Signaling Technology Cat# 13820, RRID:AB_2493181), followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 647 (Molecular Probes Cat# A-21244, RRID:AB_141663) and a 1:2000 dilution of Sytox Green (ThermoFisher Scientific Cat# S7020). Embryos were cleared and mounted in BABB, a 1:2 ratio of benzyl alcohol (Sigma B-1042) and benzyl benzoate (Sigma B-6630). Mounted embryos were imaged on a Zeiss LSM710 or LSM880 confocal microscope with an LD LCI Plan-Achromat 25X/0.8 lmm Corr DIC M27 multi-immersion lens in the oil-immersion setting. A single bead from a calibration slide (ThermoFisher Scientific Cat#F369009, well A1) was imaged between embryos to account for any fluctuations in laser power. Post-acquisition P-Smad5 analysis utilized a custom MATLAB algorithm to identify individual nuclei center-points and extract P-Smad5 intensities in each nucleus (available in Zinski et al., 2017), which were normalized based on a standard calibration bead intensity (Zinski et al., 2019). Resulting embryos were aligned across the DV axis and conformed using Coherent Point Drift. Population means were generated after genotyping for in-tube/heterozygous sibling controls since all imaging and analysis was performed blinded. Mean profiles were generated by averaging P-Smad5 intensities of cells in a 30um band either at the margin or more animal positions. 3-D embryo-wide displays of mean P-Smad5 were generated by projecting all nuclei on a sphere divided into 4800 equilateral triangles and nuclei within each triangle averaged together. Nuclei density was similarly displayed, with a heatmap depicting nuclei number within each triangle relative to the total number of nuclei. The option to display either mean P-Smad5 intensity or relative nuclei density resides in the ‘icosdisplay’ function. Notable findings: 1) M-bmp1a -/- and +/- embryos displayed a significantly diminished P-Smad5 gradient in the early gastrula (5.7 hpf) that remarkably recovers by mid-gastrulation (7 hpf). 2) tolloid -/- and sizzled -/- embryos displayed normal P-Smad5 gradients at 5.7 hpf. 3) M-bmp1a-/-;tolloid-/- double mutants displayed an ablated P-Smad5 gradient at 5.7 and 7 hpf.


Steps to reproduce

See ReadMe and Zinski, et al. 2017


University of Pennsylvania, University of Pennsylvania Perelman School of Medicine


Quantitative Imaging