2. Drosophila embryonic Dorsal-Ventral patterning petros.ligoxygakis@bioch.ox.ac.uk

3. èThe origin of D-V asymmetry èMaternal effect genes èDorsal/Ventral patterning èMultiple signalling pathways are involved in setting up D-V patterning. These pathways are used in different contexts during development and are highly conserved in different organisms. In this lecture

4. The egg production line AnteriorPosterior YoungOld

5. èAs the ovarioles develop, follicle cells migrate over the oocyte and the nurse cells pump their contents into the oocyte, leaving only a remnant of the nurse cells behind at the anterior end. èA Drosophila egg is 400 mm long, 160 mm in diameter.

6. Early development in Drosophila After thirteen divisions membranes ingress from the cortex to enclose each nucleus, to form the cellular blastoderm.

7. Dorso-ventral axis formation The egg is polarised on the DV axis (compare with animals where DV polarity is defined by the sperm entry point). The ovary is not polarised on the DV axis, so how is the asymmetry established?

8. Dorsal follicle cells Ventral follicle cells stage 8 è Step 1: Microtubule re-organisation. è Oocyte nucleus moves to an anterior position near the oocyte cell membrane. è grk mRNA is made in the nurse cells, transported to the anterior of the oocyte, then to the cortex overlying the oocyte nucleus, and anchored there. è Grk protein is synthesised, has limited diffusion, and signals to the follicle cells migrating overhead, which take on dorsal fates. grk mRNA Grk protein stage 10 gurken (a TGF  family protein) produced by the oocyte induces dorsal follicle cell fate, the first sign of D-V axis formation.

9. D/V patterning involves a Serine protease cascade è Share homology with trypsin-like family of extracellular serine proteases. è Typically secreted as inactive zymogen forms that are activated by proteolytic cleavage between N and C terminal domains. èPre-activated forms of Snake and Easter lacking N-terminal sequences have been used to order Gastrulation defective, Snake and Easter in a cascade è In vitro Easter can cleave Spatzle to create active form in embryos èSnake and Easter zymogens are freely diffusible, therefore local activation is critical

10. Problem: Dorsal protein is the transcription factor that interprets the DV information in the egg. Dorsal as well as Toll (the receptor of the pathway) and its ligand Spatzle are found throughout the syncytial blastoderm, not just the ventral or dorsal side! How can Dorsal act as a morphogen and Toll signalling generate its gradient only in the ventral side? Solution: The critical step is the generation of the ventral signal by the only ventral- specific gene: Pipe. Translocation of Dorsal from the cytoplasm to the nuclei of the ventral cells occurs during the 14th cycle of cell division. Nuclei that take up Dorsal express ventralising genes and repress dorsalising genes. Question: What is the asymmetrical cue that leads to translocation of Dorsal into the nuclei of only ventral cells (Pipe target)?

11. FOLLICULAR CELLS CHORION WindbeutelAndpipe Controlling the nuclear translocation of Dorsal Plasma membrane Tube Pelle Cactus Dorsal Toll Spaetzle EMBRYO Nuclear membrane PERIVITELLINE SPACE Nudel Gastrulation Defective Defective SnakeEaster Spn27AMyD88

12. Organisational similarities of proteolytic cascades in development, coagulation and immunity

13. The Toll receptor is central to D-V axis signalling

14. Generation of dorsal-ventral polarity In egg, after fertilisation. 7. Nudel and the Pipe target (factor x) interact to split the Gastrulation-deficient (Gd) protein. Nudel may determine the timing of this signal. 8. The activated Gd protein splits the Snake (Snk) protein, and activated Snk cleaves the Easter (Ea) protein. Gd, Snk and Ea are serine proteases 9. The activated Easter protein splits Spatzle; activated Spatzle binds to Toll receptor protein. 10. Toll activation activates Tube and Pelle, which phosphorylate the Cactus protein. Cactus is degraded, releasing it from Dorsal. 11. Dorsal protein enters the nucleus and ventralises the cell.

15. Searching for the target of Pipe Zhang et al (2009), Curr Biol 19, 1200-1205

16. Vitelline Membrane Like (VML) is a Pipe target and is localised at the anterior-lateral side of the oocyte

17. Genetic Interactions of vml with pipe Maternal Mutant Background Proportion of Embryos Exhibiting Denoted DV Phenotypes D0 D1 D2 D3 +/+; +/+; pipe7/p i pe2 ( n = 1681) 15.7% ± 0.9% 40.7% ± 1.2% 31.5% ± 1.1 12.1% ± 0.8% Vml/Vml; +/+; pipe7/p i pe2 ( n = 554) 89.5% ± 1.3% 10.3% ± 1.3% 0.2% ± 0.2% 0 Vml/+; +/+; pipe7/p i pe2 ( n = 327) 48.0% ± 2.8%39.1% ± 2.7% 11.6% ± 1.8% 1.2% ± 0.6% gdVM9 0/+; +/+; pipe7/p i pe2 ( n = 208) 78.8% ± 2.8% 20.2% ± 2.8% 1.0% ± 0.7% 0.8%

18. Toll signalling è Toll is a transmembrane protein found throughout the cell membrane of the egg that acts as a receptor for a localised external signal (Spatzle) è Toll membrane protein is activated by Spatzle on the ventral side of the embryo è In wild type embryos, amount of ligand is limited; in wild-type embryos Toll limits the diffusion of its own ligand by sequestering it as soon as it is produced. signal peptide (locates protein to membrane) transmembrane domain intra-cellular domain (26% amino-acid identity with human interleukin-1 receptor Domain structure of Toll

19. Members of the Toll-like Receptor family TLRs are an evolutionary ancient and well conserved family of proteins Human TLR family consists of 10 members: TLR1-TLR10

20. Toll signalling in flies and IL1 signalling in mammals

21. Separation of dorsal and cactus proteins Cactus binding to Dorsal protein inhibits Dorsal’s nuclear entry sequence, and thus Cactus sequesters Dorsal in the cytoplasm. Dorsal is a transcription factor Cactus binds via ankyrin repeats Pelle protein kinase, probably through an intermediate, phosphorylates Cactus protein. Cactus degrades Dorsal protein can enter nucleus P

22. D-V patterning so far… è D-V patterning is initially set up during oogenesis, via asymmetric mRNA localisation. è Extracellular proteolysis provides ligand ventrally for the ubiquitous receptor encoded by Toll. è Cactus proteolysis, releasing the morphogen Dorsal into ventral embryonic nuclei. è D-V patterning depends on cell-cell signalling rather than on localised determinants in the egg. è The signals controlling Dorsal access to nuclei use exclusively maternal products until the receptor coded by Toll is activated. è Dorsal and cactus then have both maternal and zygotic contributions è Transcriptional targets of Dorsal are zygotic.

23. Dorsal-ventral polarity è D-V axis is initially set up by maternal genes and depends on cell-cell signalling rather than on localised determinants in the egg. The role of Dorsal: è dorsal, which encodes a transcription factor that can both activate and repress gene expression, is the morphogenetic agent for D-V polarity. è Loss-of-function mutations in dorsal give rise to dorsalised embryos (the product of dorsal is needed for differentiation of ventral cells). è Different concentrations of Dorsal specify different patterns of gene transcription and consequently different fates for cells.

24. Studying the Dorsal gradient

26. Differential nuclear localisation of dorsal protein regulates zygotic genes Dorsal activates specific genes to give the mesodermal phenotype (nervecord, muscles etc.) and proper gastrulation. –twist, snail and rhomboid Dorsal represses dorsalising genes –decapentaplegic (dpp) and zerknullt (zen)

27. How does Dorsal act as both a transcriptional activator and repressor? two types of complex - –activation –repression twist Dorsal (low affinity binding site) DSP1 (HMG-box binding protein) TATA binding protein zen Dorsal (high affinity binding site) groucho (transcriptional repressor)

28. (B) The interpretation of the Dorsal gradient by genes that demarcate the different dorsoventral territories; for simplicity, only two representative genes are shown. Subsequent processes will further subdivide these territories. The decapentaplegic (dpp) gene in particular codes for a secreted factor that will act as a local morphogen to control the detailed patterning of the ectoderm. The gradient of the Dorsal protein and its interpretation (A) The concentration gradient of Dorsal protein in the nuclei of the blastoderm, as revealed by an antibody.

29. (B) The interpretation of the Dorsal gradient by genes that demarcate the different dorsoventral territories; for simplicity, only two representative genes are shown. Subsequent processes will further subdivide these territories. The decapentaplegic (dpp) gene in particular codes for a secreted factor that will act as a local morphogen to control the detailed patterning of the ectoderm. The gradient of the Dorsal protein and its interpretation (A) The concentration gradient of Dorsal protein in the nuclei of the blastoderm, as revealed by an antibody.

30. A dorsal-ventral gradient in Dpp is produced by the antagonistic activity of the short gastrulation protein (Sog). The maternal gradient of dorsal protein in the nuclei represses dpp transcription ventrally but not dorsally. Sog is expresses in the ventral region of the embryo. Sog protein diffuses into the dorsal region and antagonizes the activity of Dpp protein, providing positional information in the dorsal region. Dpp Sog

31. Dorso-ventral patterning is controlled by the same genes in flies and frogs

32. Flies and frogs Dorsal neural tube of the vertebrate and ventral nerve cord of the fly appear to be generated by the same set of instructions. Tolloid Xolloid Sog Chordin Dpp BMP-4 fly frog

33. Can the fly sog gene rescue ventralised Xenopus embryos? sog and chordin can substitute for each other! Embryos completely ventralised by UV irradiation Ventralised embryos partially rescued by injection of sog mRNA Ventralised embryos completely rescued by injection of sog mRNA

34. Summary èSimilar signal transduction pathways in all multicellular organisms. èHomologous pathways form basic infrastructure, but targets may vary. èMolecular pathways are “tool-kits” comprising versatile ligands and receptors and molecular switches including proteolysis and reversible protein phosphorylation. èSignalling pathways can be recruited for different purposes. èDrosophila development shows significant similarities with vertebrate developmental systems.

35. Reading List Wolpert et al, Principles of Development Belvin MP and Anderson KV (1996) A conserved signalling pathway: the Drosophila Toll-dorsal pathway. Ann Rev Cell Dev Biol 12, 393-416 Moussian B and Roth S (2005) Dorsoventral axis formation in the Drosophila embryo-shaping and transducing a morphogen gradient. Curr Biol 15, R887- 899 Further reading: Zhang et al, 2009 Curr Biol 19, 1200- 1205