Regulation of Gastrulation in Drosophila. Prof. Dr. Maria Leptin

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1 Regulation of Gastrulation Development Genetics Unit University of Cologne, Germany 1 Introduction Transcription factors at the top of developmental hierarchies 2 Creation of three germ layers during gastrulation Creation of three germ layers during gastrulation Endoderm Ectoderm Mesoderm 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Cell shape changes during gastrulation 4 flymove.uni-muenster.de 5 The genetic cascade that regulates mesoderm morphogenesis Dorsal activates transcription of Twist and Snail on the ventral side Dorsal Twist Snail Twist: b-hlh, activator of gene expression in the mesoderm Snail: Zn-finger, repressor of gene destined to be expressed in the ectoderm and not in the mesoderm 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Twist is required for all aspects of the development of the mesoderm W.T twist Twist is required for all aspects of the development of the mesoderm 7 Maternal factors Adherens junctions Microtubules RhoGEF2 Concertina (heterotimeric G-protein) Postulated G-protein coupled receptor 8 Zygotic factors Twist Fog Activated concertina 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Known twist targets Early Twist target genes and their functions Dorsal Twist Snail Fog String Htl Dof N-cadh etc. TF secreted cdc25 FGF-R signal transd. 10 Wild type snail 11 Wt fog - Fog activates the G-alpha protein Concertina - Fog in the mesoderm is NOT ESSENTIAL 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Wild type string (cdc25) 13 Wild type heartless (FGF-R) or dof 14 Dorsal Twist Snail Fog String Htl Dof N-cadh etc. TF secreted cdc25 FGF-R signal transd. mut mut wt wt 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Identification of twist targets involved in ventral furrow formation 16 Twist targets twi, sna twi, sna target targets 17 New? Twist targets Gastrulation defects in embryos homozygous for chromosomal deficiencies Phenotype apparent for a short period of time Large genomic deletions T. Seher 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Genes uncovered by the deficiencies Df(3L)BK10 Df(3R)rdgC-co2 Df(2L)sc19-8 Df(3R)Tl P frühstart tribbles Traf1 et al. t48 Cell cycle regulation mystery - Expressed in mesoderm - Depends on Twist - Encodes KD kinase 19 Mitotic domains Victoria Foe (1989) Development 107, The pattern of cell divisions in the embryo is prefigured and determined by the expression of String/cdc25 Cell division 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 The first domain to express String is the prospective mesoderm 22 But it is only the tenth domain to divide But it is only the tenth domain to divide Anti phospho-histone3 23 Premature mitoses in homozygous mutant embryos Wild type Df(3L)ri79c or tribbles 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Q: Is the premature mitosis in the mutant the cause or the result of defects in cell shape changes? 25 A: Suppressing The cell premature mitoses cycle suppressor in homozygous Df(3L)ri79c genes have embryos* results a permissive in suppression function! of the gastrulation defects *(by deleting String) 26 tribbles mutants are VIABLE 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 New twist targets Genes uncovered by the deficiencies Df(3L)BK10 Df(3R)rdgC-co2 Df(2L)sc19-8 Df(3R)Tl P frühstart tribbles Traf1 t48 Cell cycle regulation mystery?? 28 T48 Wild type Df(3R)Tl-P 29 New twist targets Genes uncovered by the deficiencies Df(3L)BK10 Df(3R)rdgC-co2 Df(2L)sc19-8 Df(3R)Tl P frühstart tribbles Traf1 t48 Cell cycle regulation mystery?? - Expressed in mesoderm - Depends on Twist 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 T48 T48 structure: T48 is probably a transmembrane protein TM Gould, Brookmann, Strutt, White (1990) Nature 348, 308 Strutt and White (1994) Mech Dev 46, Localisation of T48 in the embryo Membrane localisation of T48 in the blastoderm epithelium Surface view Optical sections 32 T48 has a potential PDZ binding motif ITTEL 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 But for WHICH PDZ=domain? Computer-aided design of a PDZ domain to recognize new target sequences; Reina J, Lacroix E, Hobson SD, Fernandez-Ballester G, Rybin V, Schwab MS, Serrano L, Gonzalez C; Nat Struct Biol 2002 Aug 9(8): Potential interactors for t48: Scribble rhogef2 syntrophin-like2 Gregorio J. Fernandez, Ballester, Luis Serrano, Cayetano Gonzalez (EMBL) 35 Wild type rhogef2 (GLC) RhoGEF acts via RhoK; necessary for apical localisation of myosin Barrett et al. 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 T48 binds to RhoGEF2 (GST-pulldown, 35S labelled T48) Baz Sdt Dlg Rho GEF2 Scrb beads T48 intra Baz Sdt Dlg Rho GEF2 Scrb beads CycA-crb intra 37 S2 Schneider cells RhoGEF2-GFP T48HA Scribbled-GFP 38 T48 recruits RhoGEF2 to the plasma membrane T48HA + RhoGEF2-GFP T48DPDZ-HA RhoGEF2-GFP T48HA + Scribbled-GFP 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 An embryo just before gasrulation rhogef Armadillo/ beta-catenin 40 Armadillo/beta-catenin + rhogef A few minutes older embryo rhogef 41 Armadillo/beta-catenin + rhogef And a few minutes later rhogef 42 The screen versions of these slides have full details of copyright and acknowledgements 14

15 armadillo and rhogef in t48 mutants Younger Older rhogef armadillo and rhogef in t48 mutants arm 43 Wild type t48 BUT: fog/concertina pathway still active in t48 mutants t48 mutants have a mild phenotype and are viable 44 Double mutant (fog;t48) Younger Older 45 The screen versions of these slides have full details of copyright and acknowledgements 15

16 armadillo + rhogef in cta t48 double mutants 46 Synthesis How does it all fit together? - T48 and Fog are transcriptional targets of Twist - T48 can recruit rhogef to the membrane - T48 is essential only in the absence of Fog/Concertina signalling (and vice versa) 47 Microtubule plus end associated rhogef is displaced by activated concertina - + concertina Microtubules rhogef Rogers et al., (2004), Current Biology 14, The screen versions of these slides have full details of copyright and acknowledgements 16

17 Maternal components: Concertina (heterotimeric G-protein) Activated concertina Postulated G-protein coupled receptor Microtubules EB1 RhoGEF2 49 armadillo + rhogef in cta t48 double mutants Snail down regulates adherence junctions 50 RhoGEF and armadillo in snail mutants 51 The screen versions of these slides have full details of copyright and acknowledgements 17

18 Dorsal Twist Snail X? Fog T48 trbl frs??? RhoGEF Suppresion of cell division Myosin Diassembly of adherens junction Apical reassemby of adherens junctions Cytoskeletal constriction 52 Speculations Tribolium castaneum Twist (K. Handel and S. Roth) gastrulation Drosophila is a long germ insect Tribolium is a short germ insect It is possible that Drosophila accelerated the process Note: fog, frühstart, t48 are fast evolving genes 53 Institute of Genetics, Cologne Robert Wilson, Lisa Vogelsang,Thomas Seher, Verena Kölsch EMBL Luis Serrano Cayetano Gonzalez Gregorio Fernandez Ballester ZMBH Jörg Grosshans 54 The screen versions of these slides have full details of copyright and acknowledgements 18

19 55 The screen versions of these slides have full details of copyright and acknowledgements 19