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Imaging Morphogen Dynamics in vivo in 3D

Principal Supervisor: Dr Karuna Sampath - Warwick Medical School

Co-supervisor: Till Bretschneider

PhD project title: Imaging Morphogen Dynamics in vivo in 3D

University of Registration: University of Warwick

Project outline:

Background: Animals develop from a single cell to form multicellular organisms. This process requires signals called “morphogens” that instruct the cells how to behave during development and differentiation of progenitors. The morphogens move across cells and tissues to form gradients of the signal. Cells then respond in different ways depending on how much of the signal they receive. Thus, the correct synthesis and distribution of morphogens is crucial for normal development. The Nodal morphogen has many critical roles ranging from the directional coiling of snail shells and looping of the heart and visceral organs, to maintaining pluripotency (‘stemness’) of stem cells. Defects in Nodal factors lead to severe abnormalities including lethality. Although many studies have focussed on the functions and mechanisms of Nodal morphogens, it is still not known what are the dynamics of endogenous Nodal factors in developing embryos. The zebrafish embryo is transparent at early stages, and offers a unique advantage to image dynamics of molecules in vivo in 3D, and the current project will exploit this to examine the Nodal morphogen in live cells and embryos.

Aim: The project will use a combination of state-of-the-art in vivo microscopy methods and quantitative analysis of imaging data to address an important question: What are the kinetics of the Nodal morphogen in vivo ? The aim is to determine the dynamics of Nodal molecules in 3D as they are produced at their normal locations in living animals and diffuse across cells/tissues.

Key Approaches:

Genetics: Developmental genetics in zebrafish using CRiSPR/Cas9 genome editing to generate tagged Nodal reporter lines.

Imaging: Imaging fluorescent Nodal protein dynamics in live zebrafish embryos by confocal spinning disk microscopy and SPiM microscopy during early embryonic development.

Quantitative Approaches: Computational analysis of imaging data to determine the in vivo kinetics of Nodal proteins in embryonic progenitors.

References:

  1. Wang Y, Wang X, Wohland T, Sampath K. Extracellular interactions and ligand degradation shape the Nodal morphogen gradient. eLife 5: e13879. (2016). doi:10.7554/eLife.13879. PMID: 27101364
  2. Kumari, P., Gilligan, P.C., Lim, S., Winkler, S., Philp, R. and Sampath, K. An Essential Role for Maternal Control of Nodal Signaling. eLife 2:00683; (2013). DOI: 10.7554/eLife.00683 (2013).PMC3771576 Tian J, Andree’ B, Jones, CM, and Sampath K. The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signalling activities. Development 135: 2649-2658 (2008).
  3. Sampath K, Rubinstein AL, Cheng AHS, Liang JO, Fekany K, Solnica-Krezel L, Korzh V, Halpern ME, and Wright CVE. Induction of the zebrafish ventral brain and floor plate requires cyclops/nodal signalling. Nature 395, 185-9. (1998). Tyson RA, Zatulovskiy E, Kay RR, Bretschneider T. How blebs and pseudopods cooperate during chemotaxis. Proc Natl Acad Sci U S A. 2014 Aug 12;111(32):11703-8
  4. Bretschneider T, Diez S, Anderson K, Heuser J, Clarke M, Müller-Taubenberger A, Köhler J, Gerisch G. Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol. 2004 Jan 6;14(1):1-10 Bretschneider T, Siegert F, Weijer CJ. Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4387-91.

BBSRC Strategic Research Priority: Molecules, Cells and Systems - Molecular mechanisms

Techniques that will be undertaken during the project:

Molecular Genetics & Development: Molecular and Developmental genetics in zebrafish to identify tagged Nodal reporter lines generated by CRiSPR/Cas9 genome editing

Live imaging: Imaging fluorescent Nodal protein dynamics in live zebrafish embryos by confocal / spinning disk microscopy and SPiM microscopy during early development.

Quantitative approaches: Computational analysis of imaging data to determine the kinetics of Nodal proteins in embryonic progenitors.

Contact: Dr Karuna Sampath, University of Warwick