Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor.

The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking.

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.