Supervisor: Milka Sarris
Guidance of cells by secreted chemoattractants, such as chemokines, is essential for immune responses. We and others previously showed that chemokines direct the migration of leukocytes as tissue-bound gradients, immobilized on extracellular glycans (heparan sulfate)1,2. It remains unclear how functional chemokine gradients are established and recognized by immune cells in situ3. In this project we will exploit the genetic tractability and imaging potential of the zebrafish model to address the following questions: i) What is the role of heparan sulfate in shaping interstitial chemokine gradients during infection in vivo? To address this question, we will exploit CRISPR-Cas9 gene targeting to generate knock-in transgenic zebrafish, whereby an endogenous chemokine (Cxcl8) that is normally produced at sites of infection1 is replaced by a fluorescently tagged, and thereby tractable, version of the same chemokine. Knock-in lines will be generated expressing either a wild-type Cxcl8-FP fusion (FP=fluorescent protein), a mutant Cxcl8-FP that cannot bind heparan sulfate and a membrane-tethered Cxcl8-FP that cannot form a gradient. Chemokine gradient formation and ability to guide neutrophils to sites of infection will be assessed through various advanced light microscopy techniques. ii) How does chemokine extracellular presentation affect neutrophil polarity and cytoskeletal dynamics? To address this question we will combine the generated knock-in lines with transgenic reporter lines available in our lab that allow live imaging of chemokine signalling and cytoskeletal dynamics in zebrafish neutrophils. iii) Is chemokine gradient formation dependent on the tissue setting or regulated by infection-induced factors? To ask these questions, we will exploit a new technology that we have developed, which allows light-mediated (optogenetic) control of chemokine secretion. We will generate transgenic zebrafish expressing a photo-releasable Cxcl8. We will illuminate different tissues to locally trigger chemokine secretion, in the presence or absence of infectious agents, and assess the potential of chemokines to establish functional gradients.
1. Sarris, M. et al. Inflammatory chemokines direct and restrict leukocyte migration within live tissues as glycan-bound gradients. Curr. Biol. CB 22, 2375–2382 (2012).
2. Weber, M. et al. Interstitial dendritic cell guidance by haptotactic chemokine gradients. Science 339, 328–332 (2013).
3. Sarris, M. & Sixt, M. Navigating in tissue mazes: chemoattractant interpretation in complex environments. Curr. Opin. Cell Biol. 36, 93–102 (2015).