Presentation on theme: "Animal Development Drosophila axis formation Part 2: D-V patterning"— Presentation transcript:
1 Animal Development Drosophila axis formation Part 2: D-V patterning Part I introduces Drosophila development, the life cycle, terminology and tissues. Oogenesis and early embryogenesis. The role of maternal and zygotic gene expression is discussed. There is a overview of the hierarchy of genes controlling anterior-posterior patterning and segmentation. This introduces the practical that follows in TT.The core part of this lecture is concerned with dorsal-ventral patterning, the different signals that establish asymmetry in the embryo and some of the mechanistic principles underlying these pathways. A recurring theme is how different signalling pathways may be recruited to different developmental processes both in Drosophila and in vertebrates.Synopsis: Review of Drosophila life cycle, terminology and tissues. Maternal and zygotic gene expression. Origin of the asymmetry and localised determinants in the egg. Subdivision and specification of the embryonic pattern. Transcriptional and translational regulation in embryogenesis. Where to study signal transduction pathways.
2 In this lecture The origin of D-V asymmetry Maternal effect genes Dorsal/Ventral patterningMultiple 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.
3 The egg production line Anterior PosteriorYoung Old
4 As 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.A Drosophila egg is 400 mm long, 160 mm in diameter.
5 Early development in Drosophila After thirteen divisions membranes ingress from the cortex to enclose each nucleus, to form the cellular blastoderm.
6 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?
7 gurken (an EGF family protein) produced by the oocyte induces dorsal follicle cell fate, the first sign of D-V axis formation.Dorsal follicle cellsgrk mRNAGrk proteinstage 10stage 8Ventral follicle cellsStep 1: Microtubule re-organisation.Oocyte nucleus moves to an anterior position near the oocyte cell membrane.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.Grk protein is synthesised, has limited diffusion, and signals to the follicle cells migrating overhead, which take on dorsal fates.
8 Mirr represses pipe expression in dorsal follicle cells. Mirr represses pipe expression in dorsal follicle cells. (A) The Iro complex. Horizontal lines indicate the regions deleted in three iroEGP deficiencies. iroEGP5 removes the first mirr exon and adjacent upstream sequences. (B-D′) Stage 10 mosaic egg chambers carrying iroEGP7 (B), iroEGP5 (C) and iroEGP6 (D) clones marked by absence of GFP (green, outlined); B′-D′ show pipe-lacZ expression (red). Dorsolateral (B,D) or lateral (C) views are shown. Arrowheads indicate ectopic pipe-lacZ expression. (E-G) Stage 10 egg chambers stained for mirr (E), ara (F) and caup (G) mRNA expression.Andreu M J et al. Development 2012;139:
9 Mirr binds to a conserved regulatory element in pipe. Mirr binds to a conserved regulatory element in pipe. (A) The pipe upstream region showing r1 sequences from D. melanogaster, D. virilis and D. grimshawi above; the r1mut sequence is also indicated. DNA fragments analyzed for enhancer activity are depicted beneath. (B-G) Lateral views of transgenic stage 10 egg chambers stained for lacZ reporter expression. Solid arrowheads indicate ectopic expression of constructs lacking the r1 element. The open arrowhead in E indicates loss of ventral expression in the fs(1)K101 background. In all cases, dorsal is up as confirmed by the dorsal position of the oocyte nucleus under Nomarski optics (not shown). (H,H′) Ventrolateral views of a stage 10 mosaic egg chamber carrying mirr overexpression clones marked by expression of GFP (H, green, outlined); H′ shows M2-lacZ expression (red). (I) EMSA analysis of in vitro synthesized Mirr protein binding to different oligonucleotide probes. Lanes labeled (–) contain TNT extract without mirr plasmid. The control probe (c) contains the ACAcgTGT sequence (Bilioni et al., 2005). The r1mut sequence is indicated in Fig. 2A; mut A-D probes contain the changes indicated in red.Andreu M J et al. Development 2012;139:
10 D/V patterning involves a Serine protease cascade Share homology with trypsin-like family of extracellular serine proteases.Typically secreted as inactive zymogen forms that are activated by proteolytic cleavage between N and C terminal domains.Pre-activated forms of Snake and Easter lacking N-terminal sequences have been used to order Gastrulation defective, Snake and Easter in a cascadeIn vitro Easter can cleave Spatzle to create active form in embryosSnake and Easter zymogens are freely diffusible, therefore local activation is critical
11 Problem: Solution: Question: 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)?
12 Controlling the nuclear translocation of Dorsal Spn27ACHORIONNudelGastrulationDefectiveSnakeEasterPERIVITELLINE SPACESpaetzleEMBRYOWindbeutelAndpipeDorsalCactusTubeTollPelleMyD88Plasma membraneNuclear membraneFOLLICULAR CELLS
13 Organisational similarities of proteolytic cascades in development, coagulation and immunity
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 proteases9. 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,
16 Vitelline Membrane Like (VML) is a Pipe target and is localised at the anterior-lateral side of the oocyte
18 Toll signallingDomain structure of Tollsignal peptide(locates protein to membrane)intra-cellular domain (26% amino-acid identity with human interleukin-1 receptortransmembrane domainToll is a transmembrane protein found throughout the cell membrane of the egg that acts as a receptor for a localised external signal (Spatzle)Toll membrane protein is activated by Spatzle on the ventral side of the embryoIn 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.
19 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 factorPelle protein kinase, probably through an intermediate, phosphorylates Cactus protein.PCactus binds via ankyrin repeatsCactus degradesDorsal protein can enter nucleus
20 D-V patterning so far…D-V patterning is initially set up during oogenesis, via asymmetric mRNA localisation.Extracellular proteolysis provides ligand ventrally for the ubiquitous receptor encoded by Toll.Cactus proteolysis, releasing the morphogen Dorsal into ventral embryonic nuclei.D-V patterning depends on cell-cell signalling rather than on localised determinants in the egg.The signals controlling Dorsal access to nuclei use exclusively maternal products until the receptor coded by Toll is activated.Dorsal and cactus then have both maternal and zygotic contributionsTranscriptional targets of Dorsal are zygotic.
21 Members of the Toll-like Receptor family TLRs are an evolutionary ancient and well conserved family of proteinsHuman TLR family consists of 10 members: TLR1-TLR10
22 Conserved pathway for regulating transcription factors in both Drosophila and mammals SpatzleIL-1Toll and its ligand are also involved in the Drosophila immune reponse against fungal infections. Several toll-related genes have been discovered in humans, which may be involved in both the immune response and early development.TollIL-1RTubeMyD88PelleIRAK?TRAF-6CactusIKBDorsalNFKB
23 Conserved pathway for regulation of nuclear localisation Phosphorylated IKB is ubiquinated and subsequently degraded by the proteosome. Removal of IKB unmasks the nuclear localisation sites in both the p50 and p65 subunits of NFKB. NFKB enters the nucleus, binds to specific sequences in DNA and regulates transcription.IKB similar to CactusNFKB similar to DorsalDegradation of IKB allows NFKB to enter the nucleus
24 Dorsal-ventral polarity 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:dorsal, which encodes a transcription factor that can both activate and repress gene expression, is the morphogenetic agent for D-V polarity.Loss-of-function mutations in dorsal give rise to dorsalised embryos (the product of dorsal is needed for differentiation of ventral cells).Different concentrations of Dorsal specify different patterns of gene transcription and consequently different fates for cells.
27 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 rhomboidDorsal represses dorsalising genesdecapentaplegic (dpp) and zerknullt (zen)
28 How does Dorsal act as both a transcriptional activator and repressor? twist(low affinity binding site)DSP1(HMG-boxbinding protein)TATAbindingproteinDorsal(high affinity binding site)zengroucho(transcriptional repressor)two types of complex -activationrepression
29 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.(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.
30 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.(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.
31 A dorsal-ventral gradient in Dpp is produced by the antagonistic activity of the short gastrulation protein (Sog).DppThe 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.Sog
32 Dorso-ventral patterning is controlled by the same genes in flies and frogs
33 Flies and frogsDorsal neural tube of the vertebrate and ventral nerve cord of the fly appear to be generated by the same set of instructions.fly frogTolloid XolloidSog ChordinDpp BMP-4
34 Can the fly sog gene rescue ventralised Xenopus embryos? Ventralised embryos partially rescued by injection of sog mRNAEmbryos completely ventralised by UV irradiationVentralised embryos completely rescued by injection of sog mRNAsog and chordin can substitute for each other!
35 SummarySimilar signal transduction pathways in all multicellular organisms.Homologous pathways form basic infrastructure, but targets may vary.Molecular pathways are “tool-kits” comprising versatile ligands and receptors and molecular switches including proteolysis and reversible protein phosphorylation.Signalling pathways can be recruited for different purposes.Drosophila development shows significant similarities with vertebrate developmental systems.