Real-time monitoring of endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation

Abstract

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitored Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging platforms with single-molecule Fluorescence Correlation Spectroscopy (FCS), we demonstrate that Fgf8a, produced at the embryonic margin, propagates by free diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a mediated patterning. Manipulation of the Fgf8a input alters the signaling outcome, thereby establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, using diffusion-hindered versions of Fgf8a, we demonstrate that extracellular diffusion of the protein from the source is critical for it to achieve its morphogenic potential.

An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development

“…(F) Schematic of the Erk activity gradient, as read-out by modERK-KTR, and the extracellular levels of Fgf8a-GFP described in similarly staged embryos (~5.3 hpf) 40 .…”

Betaglycan promoter activity is differentially regulated during myogenesis in zebrafish embryo somites

“…43,44 The animal-vegetal axis gradient of FGF8a has been documented and is proposed to be created due to hindered-diffusion by heparan-sulfate containing proteoglycans. 45 The fact that Tgfbr3 is a heparan-sulfate proteoglycan that exists as an integral membrane protein of which the extracellular domains can be shed as a soluble receptor, invites to the speculation that it plays a role in hindering the diffusion of still unidentified, morphogens during development. Finally, a comparison of the abundance of betaglycan mRNA with the other genes coding for components of the canonical TGFβ signaling pathway (Figure 8C) reveals that the minimal set of components (ligands, receptors, and Smads) is present through all the embryonic stages, while the co-receptors endoglin (eng) and betaglycan (tgfbr3) need not to be present during early embryogenesis.…”

“…However, free diffusion of morphogens may be “hindered” by interactions with molecules in the extracellular matrix 43,44 . The animal‐vegetal axis gradient of FGF8a has been documented and is proposed to be created due to hindered‐diffusion by heparan‐sulfate containing proteoglycans 45 . The fact that Tgfbr3 is a heparan‐sulfate proteoglycan that exists as an integral membrane protein of which the extracellular domains can be shed as a soluble receptor, invites to the speculation that it plays a role in hindering the diffusion of still unidentified, morphogens during development.…”

Receptor binding and tissue architecture explain the morphogen local-to-global mobility transition

“…This slow-down has been experimentally shown, with local diffusion coefficients on the order of ∼ 60 𝜇m 2 /s being measured by Fluorescence Correlation Spectroscopy (FCS), and global diffusion coefficients being determined by Fluorescence Recovery After Photobleaching (FRAP) with values closer to ∼ 1 𝜇m 2 /s (43)(44)(45)(46). The reduction in diffusion on a global scale has been explained by multiple factors, including the binding of morphogens to receptors or extracellular binding molecules, as well as the hindrance of diffusion due to the complex structure of the developing tissue (23,43,(47)(48)(49). Further, use of timer reporters, where the average age of the morphogen molecules can be assessed at different spatial locations provides support for multiple effective dynamics modes (50,51).…”