Discovery of a novel form of Hedgehog that systemically circulates, and its signaling implications in Drosophila [Elektronische Ressource] / von Veena Kumari

Kumari

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2011
• Nombre de lectures: 180
• Langue: Deutsch
• Poids de l'ouvrage: 16 Mo

Extrait



  Discovery of a novel form of Hedgehog that systemically circulates, and its signaling implications inDrosophila.     DISSERTATION     zur Erlangung des akademischen Grades   Doctor rerum naturalium  (Dr. rer. nat.)     vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von    Veena Kumari  Masters of Sciences   geboren am 30. Marz1980 in Jehanabad, Bihar.     Dr. Suzanne Eaton, Max Planck Institute für Molekulare Zellbiologie und Genetik, Dresden 

           Date of Defense: 26th January 2011     Name of Reviewers: Prof. Kai Simons and Prof. Elisabeth Knust       Date of Submission: 21st Oct 2010



























Hiermit versichere ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Die Arbeit wurde bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Die vorliegende Dissertation wurde unter der Betreuung von Dr. Suzanne Eaton am Max-Planck-Institut für molekulare Zellbiologie und Genetik in Dresden im Zeitraum vom 1. Juli 2006 bis 15. Juli 2010 verfasst. Meine Person betreffend erkläre ich hiermit, dass keine früheren erfolglosen Promotionsverfahren stattgefunden haben. Ich erkenne die Promotionsordnung der Fakultät für Mathematik und Naturwissenschaften, Technische Universität Dresden an.  

           I herewith declare that I have produced this paper without the prohibited assistance of third parties and without making use of aids other than those specified; notions taken over directly or indirectly from other sources have been identified as such. This paper has not previously been presented in identical or similar form to any other German or foreign examination board.    The thesis work was conducted from 15.07.2006  to  30.07.2010  under the supervision of Dr.Suzanne Eaton at Max Planck Institute of Molecular Cell Biology and Genetecis, Dresden.   Dresden Signature 

Acknowledgement  Its a pleasure to thank those who made this thesis possible. I am grateful to my supervisor Suzanne to have given me an opportunity to work in he her lab as a PhD student. Her guidance and support had been very motivating. I sincerely thank her for everything. I also thank my TAC members Kai Simons and Christoph Thiele for their inputs and suggestions. I thank Kai for his mentorship. The suggestions and discussions during the lab retreats and Joint Group Meeting were very fruitful, I thank all the associated members. I sincerely thank Kai and Eli for reviewing this thesis. Many thanks to Falko Riedel and Maria Carvalho for proofreading my thesis. It was a huge effort and not at all an easy job. I owe my deepest gratitude to Nagananda, Falko and Maria for maintaining my fly lines when I was away. I thank Falko for his constant support and suggestions. I thank Jens Roeper for helping me with the softwares, being supportive and encouraging. Eatons lab was a wonderful experience, I thank all the members for creating a conducive atmosphere to work. I thank Satyajit Mayor for his HhGFP flies. I thank LMF facility for maintaining such awonderful place to work. I also thank Sven, Stefan and Cornelia for maintaining such a nice fly facility and helping me impromptu. My heartfelt thanks to Photolab department help me with the thesis completion. I also thank them for helping me with my posters, one of which was an award winner. I would like to show my gratitude to all my friends here in Dresden who made this journey possible. I would also like to thank my parents who stood by me in my good and bad times till completion of this thesis. I thank everyone who is directly or indirectly related to the completion of this thesis. 

Summary:   Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. InDrosophila, (Lpp) Lipophorin particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development.  Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs inDrosophila. We wanted to understand the role of Hh in overall development ofDrosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3 rd  instar larva ofDrosophila. show that I imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3 rd  instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development ofDrosophila melanogasterthat includes tissue-based interaction.  

Abbreviations:  A Anterior ap Apterous Adh Alcoholdehydrogenase AP boundary Anterior Posterior Boundary Br Broad BrC Broad Complex Ci Cubitus Interruptus Ci 75  Ci Repressor Ci 155  Full length Ci CiR Ci repressor Col Collier Cos2 Costal-2 Crol Crooked Legs Cys Cysteine cDNA Complementary Deoxyribonucleic Acid. DILPsDrosophilaInsulin Like Proteins. DAG Diacylgycerols DNA Deoxyribonucleic acid DV Dorso-Ventaral DHH Desert Hedeghog Disp Dispatched Dpp Decapentaplegic en Engrailed EcR Ecdysone Receptor Fig Figure Fu Fused GFP Green fluorescent protein GSK3β Synthase Kinase Glycogen CD8GFP Cluster of Differentiation protein(transmembrane) tagged to GFP gDNA Genomic Deoxyribonucleic Acid Gly Glycine HDL High Density Particles Hh Hedgehog HhN Hedgehog N-terminal fragment HhC Hedgehog C-terminal fragment

HS HSPGs Hh-gal4 IHH JH IDGFs kDa LpI LpII Lp-Gal4 Lpp LDL P PKA PCR Ptc PTTH RNA RNAi RT-PCR SDS SDS-PAGE SacI secGFP SHH Smo SREBP SCAP Su (fu) Tub TOR UAS Wg WT ZPA ZNC 

                                   

                                   

Heparan sulfate Heparan sulfate proteoglycans Hedgehog-Gal4 (enhancer trap) Indian Hedgehog Juvenile Hormone Imaginal Disc Growth Factors Kilo Dalton ApolipophorinI ApolipophorinII Apolipophorin-Gal4 Lipophorin Particles Low Density Lipoprotein Posterior Protein Kinase A Polymerase Chain Reaction. Patched Prothoracicotropic Hormone Ribonucleic acid RNA interference Reverse Transcriptase Polymerase Chain Reaction. sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis Supressor of ActinI Secreted GFP Sonic hedgehog Smoothened Sterol Regulatory Element Binding Protein. SREBP Cleavage Activating Protein. Suppressor of fused Tubulin Target of Rapamycin Upstream Activator Sequence Wingless Wild Type Zone of Polarizing Activity Zone of Non-Proliferating Cells.

 Figures:  Figure 1.1: Morphogen gradient. 3 Figure 1.2: The life cycle of a fly with changing steroid hormone levels. 5 Figure 1.3: Systemic growth requires inter-organ communication. 6 Figure 1.4: An insight into the systemic growth pathways. 9 Figure 1.5: Drosophila wing imaginal disc from 3 rd  larvae showing instar hinge, blade, margin and notum. 12 Figure 1.6: Hh autoprocessing. 13 Figure 1.7:  Hh Signaling Pathway. 18 Figure 3.1: Hh circulates in the 3rd instar larval hemolymph. 21 Figure 3.2: Lpp and Hh cofractionate when subjected to density gradient centrifugation. 22 Figure 3.3: Hh levels are unaltered by knocking down Lpp levels in hemolymph. 23 Figure 3.4: Systemic Hh is not secreted from tissues covered by Hh-Gal4 driver. 24 Figure 3.5: Expression pattern of Hh-Gal4 in the fat body and in the wing disc. 25 Figure 3.6: Lp-Gal4 is not expressed in the imaginal wing disc. 26 Figure 3.7: Hh can travel from Wing Disc to Fat body and vice-versa. 27 Figure 3.8: Expression of Hh exclusively in fat body leads to activation of Hh pathway components in the wing disc. 29 Figure 3.9: Npc-Gal4 secretes much less Hh than compared to driver Lp- Gal4. 31 Figure 3.10: Systemic Hh can rescue the size of thehh ts /hh ts  wing imaginal disc. 32 Figure 3.11: Amount of Hh in the hemolymph and wing disc.   34 Figure 3.12: Dpp at the AP boundary is not rescued by Systemic Hh. 36

Figure 3.13: Systemic Hh cannot rescue short range Hh target gene expression. 37

Figure 3.14:

Figure 3.15:

Figure 4.1:

Figure 4.2:

Fat body as a target of Systemic Hh.







Hh accumulates in the fat body of late 3 rd instar larvae.

Hh as a systemic factor.

How systemic Hh could promote growth indirectly.





38

39

40

43

Table of Contents 1  Introduction. ............................................................................................. 3  1.1  Life Cycle ofDrosophila melanogaster........................................................................... 4  1.2  Growth and development ofD. melanogaster. ................................................................ 5  1.3   ...................................................................................................... 10Growth of wing disc.  1.4  Hh in the wing imaginal disc.......................................................................................... 11  1.5  Hh Processing, Secretion and Spread............................................................................. 12  1.6  Hh signaling. .................................................................................................................. 15  1.7  Target genes of Hh Signaling. ........................................................................................ 17 

2Scope of the Thesis. .............................................................................. 19

3Results. ................................................................................................... 20 3.1  The N-terminal active form of Hh systemically circulates in the 3rd instar larva ofD. melanogaster............................................................................................................................ 20  3.2  The systemically circulating form of Hh co-fractionates with Lpp particles................. 21  3.3  Systemic Hh levels do not depend on  Lpp  levels in the hemolymph............................. 22  3.4  The source of systemic Hh. ............................................................................................ 23  3.5   ...................................................... 24Systemic Hh can travel from one tissue to another..  3.6   28 .circulating Hh levels causes Fused Phosphorylation, and wing disc growth.Elevated  3.7  Hh can rescue wing disc growth............................................. 30Systemically circulating  3.8  target tissue of systemic Hh. ................................................................................... 37The 

4Discussion. ............................................................................................. 40 4.1  Hh systemically circulates.............................................................................................. 40  4.3  Target of systemic Hh. ................................................................................................... 41  4.4  Systemic Hh influences growth...................................................................................... 42  4.5  Future Implications......................................................................................................... 45 

5Material and Methods............................................................................. 46