Further Development 4.11: A Multitude of Ways to Shape FGF Secretion

Cell-to-Cell Communication: Mechanisms of Morphogenesis

FGF secretion represents a comprehensive example of the ways that HSPGs can influence paracrine factor diffusion. Cells secrete FGFs into the extracellular matrix, where the FGFs can interact with a diversity of HSPGs that function to both modulate the diffusion of FGF and influence FGF-FGFR binding (Balasubramanian and Zhang 2015). Like all proteoglycans, HSPGs possess side chains of sugar molecules—glycosaminoglycans—that vary in length and type, and different forms of HSPG-FGF interactions can differently shape the FGF gradient. Specifically, the morphogen gradient of Fgf8 is thought to be established through a source-sink model (also known as a “secretion-diffusion-clearance” mechanism; Yu et al. 2009). In this model, cells secreting Fgf8 are the source of the morphogen, and the receiving cells provide the sink through mechanisms of binding, internalization, or protein degradation for clearance of Fgf8 (Balasubramanian and Zhang 2015). Michael Brand’s lab tested this model in the zebrafish gastrula by microinjecting a cluster of cells with mRNA encoding Fgf8 fused to GFP. When these cells translated this mRNA, they produced and secreted Fgf8 complexed with green fluorescent protein. This allowed the researchers to quantify the amount of Fgf8 in the extracellular space at varying distances from the microinjected cells using fluorescence correlation spectroscopy (Figure 1A, B). Remarkably, the researchers were able to visualize an Fgf8-GFP gradient that differed under different circumstances (Figure 1C): free diffusion of the ligand achieved the greatest distance traveled; “directed diffusion” along HSPG fibers fostered rapid movement over several cell distances; “confined clustering” of Fgf8 on dense HSPG matrices significantly restricted diffusion; and endocytosis internalized the Fgf8-FGFR complex for lysosomal degradation in receiving cells (Yu et al. 2009; Bökel and Brand 2013). Thus, the target tissue is not passive. It can promote diffusion, retard diffusion, or degrade the paracrine factor.

Figure 1 The Fgf8 gradient. (A) Zebrafish blastulae were injected with mRNA encoding Fgf8-GFP (green stain) and mRFP-glycosyl phosphatidylinositol (GPI; red stain) to visualize, respectively, Fgf8 expression and the cell membrane. The confocal image is of a resulting zebrafish gastrula, showing Fgf8 protein being produced by and secreted away from isolated GFP-labeled cells (green). On the right is a schematic representation of select cells and the Fgf8 expression seen in the confocal image (compare α and β identifiers). Fgf8 is seen in a gradient in the extracellular matrix as well as being internalized in receiving cells. (B) Quantification of Fgf8 protein at different locations in (A), indicated by “X” marks in the schematic. Manipulation of endocytosis affecting the internalization of Fgf8 by receiving cells causes predictable changes in the range of Fgf8 secretion. Inhibition of endocytosis with the dominant negative GTPase dynamin causes a shallower Fgf8 gradient over a longer distance (green plot) (LOF, loss of function), whereas increased endocytosis with the overexpression of the endosomal protein Rab5c (GOF, gain of function) yields a steeper and shorter Fgf8 gradient (blue plot). (C) Five primary mechanisms for shaping the Fgf8 gradient. (1) The difference in the rate of fgf8 transcription and fgf8 mRNA decay can influence the amount of Fgf8 protein ultimately secreted from a producing cell. Once secreted, Fgf8 can (2) freely diffuse or (3) travel rapidly along HSPG fibers for directed diffusion. (4) In contrast, however, dense areas of HSPGs can also confine and restrict Fgf8 diffusion. (5) The Fgf8-FGFR complex can also be internalized by endocytosis and targeted for lysosomal degradation. Together, these different mechanisms result in the displayed gradient of Fgf8 and differential responses in cells that experience different concentrations of Fgf8 signaling (different-colored nuclei). (B after S. R. Yu et al. 2009. Nature 461: 533–536; C after C. Bökel and M. Brand. 2013. Curr Opin Genet Dev 23: 415–422 and R. Balasubramanian and X. Zhang. 2015. Semin Cell Dev Biol 53: 94–100.)

Literature Cited

Balasubramanian, R. and X. Zhang. 2016. Mechanisms of FGF gradient formation during embryogenesis. Semin. Cell Dev. Biol. 53: 94–100.
PubMed Link

Bökel, C. and M. Brand. 2013. Generation and interpretation of FGF morphogen gradients in vertebrates. Curr. Opin. Genet. Dev. 23: 415–422.
PubMed Link

Yu, S. R., M. Burkhardt, M. Nowak, J. Ries, Z. Petrásek, S. Scholpp, P. Schwille and M. Brand. 2009. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461: 533–536.
PubMed Link

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