Imaging and Quantification of P-Smad1/5 in Zebrafish Blastula and Gastrula Embryos

Abstract

Spatiotemporal patterns of morphogen activity drive differential gene expression with a high degree of precision within a developing embryo and reproducibly between embryos. Understanding the formation and function of a morphogen gradient during development requires quantitative measurement of morphogen activity throughout an individual embryo and also between embryos within a population. Quantification of morphogen gradients in to presents unique challenges in imaging and image processing to minimize error and maximize the quality of the data so it may be used in computational models of development and in statistically testing hypotheses. Here we present methods for the preparation, immunostaining, imaging, and quantification of a bone morphogenetic protein (BMP) activity gradient in individual zebrafish embryos as well as methods for acquiring population-level statistics after embryo grouping and alignment. This quantitative approach can be extended to other morphogen systems, and the computational codes can be adapted to other imaging contexts in zebrafish and other organisms.

Fibrodysplasia ossificans progressiva mutant ACVR1 signals by multiple modalities in the developing zebrafish

“…During early embryonic development, a nuclear gradient of pSmad1/5 activity forms across the DV axis of the zebrafish embryo in response to BMP signaling ( Zinski et al, 2017 ; Zinski et al, 2019 ; Tucker et al, 2008 ; Figure 2a ). This gradient persists throughout gastrulation and specifies DV axial fates.…”

“…P-Smad1/5 immunostaining and imaging were performed as previously described ( Zinski et al, 2017 ; Zinski et al, 2019 ). For all immunostaining, embryos were fixed in 4% formaldehyde in PBST between shield stage and 60% epiboly (approximately 6–7 hpf), blocked with 10% FBS in PBST, and probed overnight at 4°C with a 1:200 dilution of anti-PSmad1/5/9 (Cell Signaling 13820).…”

“…High levels of BMP signaling specify ventral cell fates and intermediate signaling specifies lateral fates, while absence of signaling allows dorsal cell fate specification. The DV pattern is generated through a quantifiable pSmad1/5 signaling gradient within the gastrulating embryo that peaks ventrally and decreases dorsally ( Little and Mullins, 2006 ; Zinski et al, 2017 ; Zinski et al, 2019 ; Tucker et al, 2008 ; Mintzer et al, 2001 ). Perturbations to this BMP signaling gradient in the developing embryo result in distinct, dose-dependent patterning phenotypes ( Figure 1b ).…”

The BMP signaling gradient is interpreted through concentration thresholds in dorsal–ventral axial patterning

“…pSmad5 immunostaining, imaging, and quantification were performed as previously described [ 23 , 75 ]. Embryos were fixed in 4% paraformaldehyde at 4°C, blocked in NCS-PBST and probed overnight with a 1:100 dilution of anti-phosphoSmad1/5/9 antibody (Cell Signaling Technology, Cat# 13820, RRID:AB_2493181, Danvers, Massachusetts, USA), followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 647 (Molecular Probes, Cat# A-21244, RRID:AB_141663, Eugene, Oregon, USA) and a 1:1,000 dilution of Sytox Green (Thermo Fisher Scientific, Cat# S7020, Waltham, Massachusetts, USA).…”

“…Images were analyzed with a custom MATLAB algorithm to identify individual nuclei center points and extract pSmad5 intensities from within each nucleus [ 23 , 75 ], which were normalized based on the standard calibration bead intensity. Resulting embryos were aligned across the DV axis and conformed using Coherent Point Drift.…”