Abstract
Morphogenesis, the development of multicellular organisms, is controlled by morphogens, small signalling molecules, that form gradients across the embryo. While such gradients can emerge through several mechanisms, the establishment of stable gradients requires the reduction of morphogen diffusion by at least one order of magnitude, a process that has not been directly observed. Here, we develop Single-Plane Illumination Microscopy-based spatial Fluorescence Cross-Correlation Spectroscopy (SPIM- sFCCS) to directly measure the diffusion coefficient of the morphogen Squint in early zebrafish embryos as a function of topography and length scale. The diffusion coefficient of Squint is a function of its molecular size, the viscosity within the interstitial spaces, and possible crowding. Here, we show that beyond these general parameters, Squint’s diffusion coefficient changes on length scales that are commensurate with the diameter of the intercellular spaces in the embryo and that its slowdown is regulated by receptor binding, which is crucial for gradient formation and embryonic patterning.
Materials availability
Plasmids and maps can be provided on request.
Acknowledgements
We thank the NUS Centre for Bioimaging Sciences and SingaScope for providing microscope facility support and the Comparative Medicine (CM) and Department of Biological Sciences (DBS) fish facilities for providing zebrafish care. The PMT-mApple plasmid used was a gift from Le Yao/Christoph Winkler.
Funding
T.W. and T.J.C. acknowledge funding from the Singapore Ministry of Education (MOE2016-T3-1-005). K.S. acknowledges funding from UKRI-BBSRC and Leverhulme trust. A.V.S. and B.S. are supported by NUS Research Scholarships.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Nelanuthala, A.V.S., Saha, B., Sankaran, J. et al. Measuring morphogen transport over multiple spatial scales in live zebrafish embryos. Nat Commun (2026). https://doi.org/10.1038/s41467-026-73639-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-73639-3
