1. Animal Development Part 1: Using Drosophila to study pattern formation petros.ligoxygakis@bioch.ox.ac.uk Petros Ligoxygakis Associate Professor of Genetics

2. How does a complex animal develop from a single cell - the fertilised egg? Central question in developmental biology:

3. First problem How is the initial symmetry of the egg broken?

4. In mammals breaking symmetry is different

5. Second problem All cells produced from the first cell have the same developmental potential (same DNA / genes) So how are all the required cell types (nerve cells, muscle cells, epithelial cells etc.) produced? By differentiation. What is the mechanism of differentiation?

6. Molecular Biology of the Cell, 4th Edition Cellular diversity

7. Car analogy: Biochemical vs. Genetic Approaches http://review.ucsc.edu/spring04/bio-debate.html

8. Biochemistry vs. Genetics Using biochemistry alone: Can find out the chemical composition and properties of each component. But it lack biological context and function. Using genetics alone: Can find out what happens when you remove one or more components. But it is difficult to isolate direct from indirect consequences. Solution: Use both methods and also combine high throughput approaches (genomics, transcriptomics, proteomics) with advance microscopy methods in living cells. http://review.ucsc.edu/spring04/bio-debate.html

9. Drosophila melanogaster

10. Drosophila melanogaster as a model organism 1.Rapid life cycle and easy to culture in the lab 2.Genetically tractable -100 years of work. Genetic toolbox - for example easy to make transgenes. Easy to do biochemistry and image living cells and tissues 3.Medical significance: 71% human disease genes - similar in flies. Great model for human brain, behaviour and evolution 4.Nobel Prizes: Role of chromosomes in heredity 1933 Analysis of the genetic basis of pattern formation 1995 Discovery of innate immunity - similar in humans 2011

11. The life cycle of Drosophila

12. Pattern formation genes

13. First problem How is the initial symmetry of the egg broken?

14. Victoria K. Jenkins, Allison K. Timmons, Kimberly McCall, Diversity of cell death pathways: insight from the fly ovary Trends in Cell Biology, Volume 23, Issue 11, 2013, 567 - 574 Drosophila female ovaries are a “virtual egg production line” Weil, Parton, Davis, Jove (2012)

15. A P D V gurken (TGF-α) mRNA Breaking the symmetry of Drosophila oocyte oskar mRN A bicoid mRNA How are mRNAs sorted to different destinations? How are they translationally regulated?

16. Establishment of AP axis in oogenesis and bicoid localisation by Gurken signalling. 1.In early stage egg chambers MTOC is in the oocyte, and gurken mRNA is localised at posterior. 2.Translation and limited diffusion means signal sent to overlying posterior follicle cells (received via torpedo receptor). 3.A signal is sent back which activates protein kinase A in the egg 4.oocyte cytoskeleton is re-organised and directs the localisation of bicoid and oskar mRNA, defining the A-P axis.

17. Oocyte nucleus migration through microtubule pushing

18. oskar and pole cell determination osk mRNA localises posteriorly Localises to plus-ends of MTs; requires kinesin motor Oskar protein recruits osk mRNA (positive feedback) to nucleate formation of "pole plasm" at the posterior of the oocyte. Pole plasm inhibits expression of "anterior" genes. Blastoderm cells that inherit the pole plasm develop as germ cells osk

19. Bicoid specifies anterior structures bcd mRNA localises at anterior of oocyte Like grk, transported by dynein along MTs mRNA is translated after fertilisation; encodes a transcription factor. Diffuses in syncytium –> gradient. Activates different target genes in different regions

20. oskar mRNA and protein A P bicoid and oskar mRNA localisation in the Drosophila blastoderm embryo bicoid mRNA Bicoid protein

21. Bicoid is a morphogen Six doses of bicoid One dose of bicoid

22. mRNA localization in the blastoderm embryo Apical Basal A P David Ish-Horowicz Ilan Davis Simon Bullock

23. Delanoue et al, 2007 (collaboration with Catherine Rabouille, Ubrecht) RNA transport by molecular motors along the cytoskeleton Cryo immuno-EM

24. Microtubule motor dependent movement of RNA - + Dynein Dynactin bcd or grk RNA osk RNA Microtubule Kinesin Ash1 RNA Actin Myosin - + (In yeast) Anterior Posterior

25. Further reading Textbook Web resources http://flybase.org/ Reviews St Johnston D. (2002). The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet. 3:176-88. Parton, R., Davidson, Al, and Davis, I. and Weil, T. (2014). Subcellular mRNA localisation at a glance. http://jcs.biologists.org/content/127/10/2127.long http://jcs.biologists.org/content/127/10/2127/F1.poster.jpg Primary papers Zhao, T., Graham, O.S., Raposo, A., St Johnston, D. (2012). Growing microtubules push the oocyte nucleus to polarize the Drosophila dorsal-ventral axis. Science 336: 999–1003. http://flybase.org/reports/FBrf0218447.htmlhttp://flybase.org/reports/FBrf0218447.html Wilkie, G.S., Davis, I. (2001). Drosophila wingless and pair-rule transcripts localize apically by dynein-mediated transport of RNA particles. Cell 105: 209-219. http://flybase.org/reports/FBrf0135682.htmlhttp://flybase.org/reports/FBrf0135682.html Ephrussi A, Lehmann R. (1992) Induction of germ cell formation by oskar. Nature 358(6385):387-392. http://www.ncbi.nlm.nih.gov/pubmed/11972155 Alberts et al. (2015) Molecular Biology of the Cell, 6th Edition. Chapter 6: pages 362-366: The RNA World Chapter 16, page 889: The cytoskeleton and molecular motors Chapter 21, page 1145: Development Chapter 22, page 1217: Stem cells