Knock, knock, knocking in fluorophores – visualization of Fgf8a’s activity by knock-in of fluorescent protein at the endogenous locus
Summary by Will Anderson: Harish, R. K., M. Gupta, D. Zoller, H. Hartmann, A. Gheisari, A. Machate, S. Hans and M. Brand (2023). "Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation." Development 150(19).
Image credit: Midjourney
An intriguing phenomenon in biology is the transformation of a group of undifferentiated cells into a complex, multicellular organism with a variety of cell types and tissues. One mechanism proposed to facilitate this differentiation is the use of proteins called morphogens. The “ideal” morphogen is a ligand that: 1) is secreted from a discrete source, 2) diffuses through surrounding tissue to form a gradient, and 3) directs differential gene expression and cell fates based on its concentration at points along said gradient [1,2]. Fgf8, a highly conserved ligand, has been proposed to be one such molecule [3]. While previous work on Fgf8’s zebrafish ortholog, Fgf8a, has characterized ectopic fluorophore-tagged Fgf8a [4], the investigation of the ligand’s activity has been constrained by the lack of a method to visualize endogenous Fgf8a’s behavior. Here, Harish et al. have utilized recent advances in the CRISPR/Cas9 system to successfully knock in EGFP at the endogenous zebrafish Fgf8a locus, and for the first time have facilitated the observation of the ligand’s distribution in its natural context [5].
After verifying that Fgf8a’s behavior and distribution is not disrupted by the insertion of EGFP in transgenic embryos, the authors analyzed the signal intensity of the tagged molecule at different stages of development and discovered that it spreads in the two ways characteristic of morphogens. First, it is secreted from a defined source (the embryonic margin), and second, it forms gradients along both the animal-vegetal and dorsal-ventral axes. Next, seeking to understand how these gradients are formed, the authors used fluorescence correlation spectroscopy and found that the movement of tagged Fgf8a protein through extracellular space fits a “two-component model” of random diffusion. Individual molecules fell into one of two categories – the majority were characterized as “fast moving,” and the remaining as “slow moving.” Based on previous studies [5,6], they inferred that the slow-moving portion of molecules were rendered less mobile by their interaction with extracellular matrix (ECM) components such as heparan sulfate proteoglycans (HSPGs).
To further explore this relationship with HSPGs, as well as Fgf8a’s ability to diffuse, the authors injected heparinase I, which cleaves a side chain of HSPGs, into Fgf8a-EGFP embryos and observed a decrease of the slow-moving portion of Fgf8a molecules and an increased ability for the ligand to distribute across the embryo. This also resulted in broadened expression domains of multiple Fgf8a target genes. These data indicate that Fgf8a binding with ECM components such as HSPGs indeed affects the ligand’s ability to diffuse through the embryo. The authors also restricted Fgf8a’s capacity to diffuse by tethering it to the cellular membrane and observed a reduction in the expression range of the Fgf8a target gene spry4, further indicating that changes in the ligand’s distribution affect gene expression.
All told, this work demonstrates an exciting new frontier in the zebrafish field - exploring protein distributions using endogenous knock-ins of fluorescent proteins - and provides compelling evidence for Fgf8a’s function as a morphogen in zebrafish.
References
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2. Rogers, K. W. and A. F. Schier (2011). "Morphogen gradients: from generation to interpretation." Annu Rev Cell Dev Biol 27: 377-407.
Toyoda, R., S. Assimacopoulos, J. Wilcoxon, A. Taylor, P. Feldman, A. Suzuki-Hirano, T. Shimogori and E. A. Grove (2010). "FGF8 acts as a classic diffusible morphogen to pattern the neocortex." Development 137(20): 3439-3448.
Yu, S. R., M. Burkhardt, M. Nowak, J. Ries, Z. Petrasek, S. Scholpp, P. Schwille and M. Brand (2009). "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules." Nature 461(7263): 533-536.
Harish, R. K., M. Gupta, D. Zoller, H. Hartmann, A. Gheisari, A. Machate, S. Hans and M. Brand (2023). "Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation." Development 150(19).
Yan, D. and X. Lin (2009). "Shaping morphogen gradients by proteoglycans." Cold Spring Harb Perspect Biol 1(3): a002493.