Characterization and simulation of the mechanical forces that control the process of Dorsal Closure during Drosophila melanogaster embryogenesis

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(1)Characterization and simulation of the mechanical forces that control the process of Dorsal Closure during Drosophila melanogaster embryogenesis Maxime Dureau. To cite this version: Maxime Dureau. Characterization and simulation of the mechanical forces that control the process of Dorsal Closure during Drosophila melanogaster embryogenesis. Development Biology. Ecole normale supérieure de lyon - ENS LYON, 2015. English. �NNT : 2015ENSL0999�. �tel-01178037�. HAL Id: tel-01178037 https://tel.archives-ouvertes.fr/tel-01178037 Submitted on 17 Jul 2015. HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés..

(2) THESE ChatrhacteD rcoirznastroioplntihalanedm spreoilm uacensolagtaisfotnD oerefsm tahlberCym elogscuhnraensdiicuarlinfgorces en vue de l'obtention du grade de. Docteur de l'Università degli Studi di Genova En cotutelle avec l'Ecole Normale Supérieure de Lyon Disciplines: Physique & Sciences de la Vie Laboratoires: Dipartimento di Ingegneria Meccanica, Energetica, gestionale e dei trasporti & Laboratoire de Biologie Moléculaire de la Cellule. Ecoles Doctorales: Scienze e Tecnologie per l'Ingegneria, corso Ingegneria Matematica e Simulazione & Biologie Moléculaire Integrative et Cellulaire Présentée et soutenue publiquement le 29 juin 2015 Par Maxime Dureau. Directeurs de Thèse:. Patrizia Bagnerini, Stéphane Vincent.. Après l'avis de:. Jacques Demongeot, Professeur. Giorgio Battistelli, Professeur. Devant un jury composé de: Jacques Demongeot, Professeur Olivier Hamant, Directeur de Recherche Patrizia Bagnerini, Maître de Conférence. Giorgio Battistelli, Professeur Stefano Vignolo, Professeur Stéphane Vincent, Maître de Conférence.

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(4) PhDM anuscript ChatrhacteD rcoirznastroioplntihalanedm spreoilm uacensolagtaisfotnD oerefsm tahlberCym elogscuhnraensdiicuarlinfgorces submitted in fulllment of the requirements for the degree of. Doctor of the Università degli Studi di Genova Jointly with the Ecole Normale Supérieure de Lyon Disciplines: Physics & Life Sciences Laboratories: Dipartimento di Ingegneria Meccanica, Energetica, gestionale e dei trasporti & Laboratoire de Biologie Moléculaire de la Cellule. Doctoral Schools: Scienze e Tecnologie per l'Ingegneria, corso Ingegneria Matematica e Simulazione & Biologie Moléculaire Integrative et Cellulaire Presented & Defended publicly on June 29, 2015 By Maxime Dureau. Tutors:. Patrizia Bagnerini, Stéphane Vincent.. After an opinion from:. Jacques Demongeot, Professor. Giorgio Battistelli, Professor. In front of a jury composed of: Jacques Demongeot, Professor Olivier Hamant, Director of Research Patrizia Bagnerini, Lecturer. Giorgio Battistelli, Professor Stefano Vignolo, Professor Stéphane Vincent, Lecturer.

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(6) C1I1on.tnroTtdhueecntiotns model.......1133. 1 . 1 A b r i e f h i s t o r y o f i e s i n s c i e n c e . . . . . . 1 3 2 ' d e v l o p m e t . 9 1.2T h e a c t o r s o f D o r s a l C s u r . . . . . . . . 2 1 3 1 . 2 1 T h e Z i p i n g . . . A c t C a b l e . . . . . . . . 2 5 1 . 2 3 T h e m n i o s e r o s a . 7 i n g p r e v u m d e l s o f D o r s a l C l o s u r e . . . . 3 2 1.34P rU e s n t n d r s a d i n g t h e e c h a n i c b i o g i c a y s t e m s 4 0 11..44321A S isn gelseim lngfrocw o u lcn x p e r m e n t s . . . . . . . 1 a c t i n t w o r k s a n d c e l u l a r p r o p e r t i e s 4 e s p r e s i n s i d e t i s u . . . . 2 2 M a t e r i a l s a n d M e t h o d s 4 5 3I3m a g e t r e a t m e n t a n d S e g m e n t a i o n 4 7 tD m e n t . . . . . . . . . . . . . 4 7 .21S P r3.2gem -1tren aG iratop b y W a t e r s h e d 5 2 a c e a n d I m a g e p o s t r e a t e m e n t . h i c U s e r I n t r f s o r t i n g . . . . . . . . . . 6 1 d e l s 6 5 4A i4.1O m n s4.s1ceilraotsE o lorqyuaM a c e stiodnoeslsc.ila.tion m o .3.........67 Drosophila Melanogaster Drosophila. In Vitro.

(7) CONTENTS. 4. 4.2. 4.1.2. Optimization . . . . . . . . . . . . . . . . . . . . . .. 67. 4.1.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . .. 67. Mechanical Model . . . . . . . . . . . . . . . . . . . . . . . .. 69. 4.2.1. Cellular Mechanics . . . . . . . . . . . . . . . . . . .. 69. 4.2.2. Elastic Model . . . . . . . . . . . . . . . . . . . . . .. 69. 4.2.3. Visco-Elastic Model . . . . . . . . . . . . . . . . . . .. 72. 4.2.4. Non Linear Elastic component . . . . . . . . . . . . .. 74. Generalization of the Mechanical model . . . . . . . . . . . .. 76. 4.3.1. The Lagrangians . . . . . . . . . . . . . . . . . . . .. 76. 4.3.2. Optimization . . . . . . . . . . . . . . . . . . . . . .. 81. 4.3.3. Model Identication . . . . . . . . . . . . . . . . . .. 82. 5 A s t u d y o f L e a d i n g E d g e d y n a m i c s 8 9 6M od elin gA m n ioserosaC elD yn am ics 10 4.3. 6.1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101. 6.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 102. 6.3. Lagrangian modeling . . . . . . . . . . . . . . . . . . . . . . 103. 6.4. Model identication and validation . . . . . . . . . . . . . . 107. 6.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.

(8) Sum m aries Français Le travail de thèse présenté ici vise à caractériser et simuler les forces mécaniques impliquées dans le processus de fermeture dorsale chez l'organisme Drosophila melanogaster. Ce processus est embryonnaire: il est l'un des événements participant à la formation de la larve. En particulier, la fermeture dorsale participe à l'acquisition par l'embryon de sa forme nale par le biais de forces générées par de tissus cellulaires. Ainsi, l'objectif du travail présenté ici est d'approfondir nos connaissances sur la mécanique des tissus, ainsi que sur leur role dans l'embryogenèse. Les tissus impliqués dans la fermeture dorsale sont l'épiderme et l'amnioséreuse. A ce stade du développement, l'épiderme entoure quasiment tout l'embryon. Cependant, l'amnioséreuse couvre encore une zone située du côté dorsal, appelée trou dorsal. La fermeture dorsale consiste donc en la fermeture de ce trou dorsale, ainsi qu'en la jonction des parties latérales de l'épiderme, dans un processus similaire à la cicatrisation. An de réaliser la jonction de l'épiderme au niveau de la ligne dorsale, celuici doit être tracté. Cette traction est réalisée à la fois par l'amnioséreuse couvrant le trou dorsal, et par la rangée la plus dorsale de cellules de l'épiderme (Leading Edge cells). Celles ci participent à la fermeture de deux manières. D'une part, ces cellules forment un cable d'actine entourant l'ensemble du trou dorsal, qui, en se contractant, participe à réduire l'aire de l'amnioséreuse. D'autre part, les cellules du Leading Edge émettent des protrusions, qui s'arriment au Leading Edge opposé, et le tractent vers elles, 5.

(9) ✻. ❈❖◆❚❊◆❚❙. ❥✉sq✉✬à ❝❡ q✉❡ ❧❡s ❞❡✉① ♣❛rt✐❡s ❞❡ ❧✬é♣✐❞❡r♠❡ ❢✉s✐♦♥♥❡♥t✳ ❈❡s ♣r♦t✉s✐♦♥s ❛②❛♥t ✉♥❡ t❛✐❧❧❡ ❧✐♠✐té❡✱ ❧❛ tr❛❝t✐♦♥ ❡t ❧❛ ❢✉s✐♦♥ ✭❛♣♣❡❧é❡s ❛✉ss✐ ③✐♣♣✐♥❣✮ ♥❡ s♦♥t ♣♦ss✐❜❧❡s q✉✬❛✉① ❡①tré♠✐tés ❛♥tér✐❡✉r❡s ❡t ♣♦stér✐❡✉r❡s ❞✉ tr♦✉ ❞♦rs❛❧ ✭❛♣♣❡❧é❡s ❝❛♥t❤✐✮✱ ❧à ♦ù ❧❛ ❞✐st❛♥❝❡ ❡♥tr❡ ❧❡s ❞❡✉① ❧❡❛❞✐♥❣ ❡❞❣❡ ❡st s✉✣s❛♠✲ ♠❡♥t ♣❡t✐t❡✳ P❛r ❛✐❧❧❡✉rs✱ ❧✬❛♠♥✐♦sér❡✉s❡ tr❛❝t❡ ❡❧❧❡ ❛✉ss✐ ❧✬é♣✐❞❡r♠❡ ✈❡rs ❧❛ ❧✐❣♥❡ ❞♦rs❛❧❡✳ ❊❧❧❡ ♣r♦❞✉✐t ❞❡s ✜❧❛♠❡♥ts ❞✬❛❝t✐♥❡ ❡t ❞❡ ❧❛ ♠②♦s✐♥❡ ✭✉♥ ♠♦t❡✉r ♠♦❧é❝✉❧❛✐r❡✮✱ q✉✐ ❢♦r♠❡♥t ✉♥ rés❡❛✉ ❝♦♥tr❛❝t✐❧❡ ❛✉ s❡✐♥ ❞❡s ❝❡❧❧✉❧❡s ❧❡✉r ♣❡r♠❡tt❛♥t ❞❡ t✐r❡r s✉r ❧❡s ❝❡❧❧✉❧❡s ❞✉ ❧❡❛❞✐♥❣ ❡❞❣❡✳ P❛r ❛✐❧❧❡✉rs✱ ❧❡s ❝❡❧❧✉❧❡s ❞❡ ❧✬❛♠♥✐♦sér❡✉s❡ ✈♦✐❡♥t ❧✬❛✐r❡ ❞❡ ❧❡✉r ❝ôté s✉♣ér✐❡✉r ✭❝ôté ❛♣✐❝❛❧✮ ✈❛r✐❡r ❞❡ ♠❛♥✐èr❡ ♣ér✐♦❞✐q✉❡✳ ❇✐❡♥ q✉❡ ❝❡s ✈❛r✐❛t✐♦♥s ❞✬❛✐r❡ ❛✐❡♥t été ❧❛r❣❡♠❡♥t ❞é❝r✐t❡s✱ ❧❡✉r rô❧❡ ❞❛♥s ❧❛ ❢❡r♠❡t✉r❡ ❞♦rs❛❧❡ r❡st❡ ✐♥❝♦♥♥✉✳ ▲✬♦❜❥❡t ❞❡ ❝❡tt❡ t❤ès❡ ❡st ❞✬❛♠é❧✐♦r❡r ♥♦tr❡ ❝♦♠♣ré❤❡♥s✐♦♥ ❞❡s ❝♦♥❝❡♣ts ♠é✲ ❝❛♥✐q✉❡s ✐♠♣❧✐q✉és ❞❛♥s ❝❡s ♦s❝✐❧❧❛t✐♦♥s✱ ❡t ❞❡ ❝♦♥str✉✐r❡ ✉♥ ♠♦❞è❧❡ ♣❤②s✐q✉❡ r❡♣rés❡♥t❛♥t ❝❡s ♠♦✉✈❡♠❡♥ts✳ ▲❡ tr❛✈❛✐❧ ♣rés❡♥té ✐❝✐ ét✉❞✐❡ ❛✉ss✐ ❧❡s ♠♦✉✲ ✈❡♠❡♥ts ❞❡s ❝❡❧❧✉❧❡s ❞✉ ❧❡❛❞✐♥❣ ❡❞❣❡✱ ❞❛♥s ❧❡ ❜✉t ❞❡ ❝♦♠♣r❡♥❞r❡ ❧✬❡✛❡t ❞✉ ❝❛❜❧❡ ❞✬❛❝t✐♥❡ s✉r ❧❛ ❞②♥❛♠✐q✉❡ ❞❡ ❧❛ ❢❡r♠❡t✉r❡ ❞♦rs❛❧❡✳ ❆✜♥ ❞✬ét✉❞✐❡r ❧❡s ♠♦✉✈❡♠❡♥ts ❞❡s ❝❡❧❧✉❧❡s ❡t ❞❡s t✐ss✉s ✐♠♣❧✐q✉és ❞❛♥s ❧❛ ❢❡r♠❡t✉r❡ ❞♦rs❛❧❡✱ ✉♥ ❛❧❣♦r✐t❤♠❡ ♣❡r♠❡tt❛♥t ❞❡ ❞ét❡❝t❡r ❧❡s ❝♦♥t♦✉rs ❞❡s ❝❡❧❧✉❧❡s✱ ❧❡✉r ♣♦s✐t✐♦♥ ❛✐♥s✐ q✉❡ ❝❡❧❧❡ ❞❡ ❧❡✉rs ✈❡rt❡① ✭♣♦✐♥ts ❞❡ ❥♦♥❝t✐♦♥ ❡♥tr❡ tr♦✐s à q✉❛tr❡ ❝❡❧❧✉❧❡s✮ ❡t ❞❡ s✉✐✈r❡ ❧❡✉r ❞é♣❧❛❝❡♠❡♥ts ❛✉ ❝♦✉rs ❞✉ t❡♠♣s ❛ été ❞é✈❡❧♦♣♣é✳ ❯♥ ✐♥t❡r❢❛❝❡ ✉t✐❧✐s❛t❡✉r ❛ été ❝♦♥str✉✐t ♣♦✉r ❢❛❝✐❧✐t❡r ❧✬❛❥✉st❡♠❡♥t ❞❡s ♣❛r❛♠ètr❡s ♣❡r♠❡tt❛♥t ❝❡tt❡ ❞ét❡❝t✐♦♥✱ ❛✐♥s✐ q✉❡ ❧❛ ❝♦rr❡❝t✐♦♥ ❞✬❡rr❡✉rs é✈❡♥t✉❡❧❧❡s✳ ❉✐✛ér❡♥ts ♠♦❞è❧❡s ❞②♥❛♠✐q✉❡s ♦♥t ❡♥s✉✐t❡ été ❝♦♥str✉✐ts✱ ♣r❡♥❛♥t ❡♥ ❝♦♠♣t❡ ❧❡s ❞✐✛ér❡♥ts ❝♦♠♣♦rt❡♠❡♥ts ♠é❝❛♥✐q✉❡s q✉❡ ❧❡s ❝❡❧❧✉❧❡s ♣❡✉✈❡♥t ❛✈♦✐r✳ ❈❡s ♠♦❞è❧❡s ♦♥t été ❝♦♥str✉✐ts s❡❧♦♥ ❧✬❛♣♣r♦❝❤❡ ❧❛❣r❛♥❣✐❡♥♥❡✳ ▲❡s s②stè♠❡s ❞✬éq✉❛t✐♦♥s ❞ér✐✈❛♥t ❞❡s éq✉❛t✐♦♥s ❞✬❊✉❧❡r✲▲❛❣r❛♥❣❡ ♦♥t été rés♦❧✉❡s ♥✉♠ér✐q✉❡✲ ♠❡♥t✱ ❡t ❧❡✉rs ♣ré❞✐❝t✐♦♥s ♦♥t été ❝♦♠♣❛ré❡s ❛✉① ❞♦♥♥é❡s ✐ss✉❡s ❞❡ ❧✬❛❧❣♦r✐t❤♠❡ ❞❡ ❞ét❡❝t✐♦♥ s❡❧♦♥ ❧✬❛♣♣r♦❝❤❡ ❞❡s ♠♦✐♥❞r❡s ❝❛rrés✳ ▲❡s rés✉❧t❛ts ♦♥t été ✈❛❧✐❞és ❡♥ t❡st❛♥t ❧❡s rés✐❞✉s ❣râ❝❡ à ❧❛ ❢♦♥❝t✐♦♥ ❞✬❛✉t♦❝♦rré❧❛t✐♦♥✳ ❋✐♥❛❧❡♠❡♥t✱ ❧❛ ❞②♥❛♠✐q✉❡ ❞✉ ❧❡❛❞✐♥❣ ❡❞❣❡ ❛ été ét✉❞✐é❡ ❡♥ ❝❛r❛❝tér✐s❛♥t ❧❡s.

(10) ❈❖◆❚❊◆❚❙. ✼. ♠♦✉✈❡♠❡♥ts ❞❡s ❝❡❧❧✉❧❡s s✐t✉é❡s à ❧✬✐♥t❡r❢❛❝❡ ❡♥tr❡ ❧✬é♣✐❞❡r♠❡ ❡t ❧✬❛♠♥✐♦sér❡✉s❡✱ ❣râ❝❡ à ❧✬❛❧❣♦r✐t❤♠❡ ❞❡ ❞ét❡❝t✐♦♥✳ ▲❡s ❞②♥❛♠✐q✉❡s ❞✬❡♠❜r②♦♥s ♥♦♥ ♠✉tés ✭✇✐❧❞✲t②♣❡✮ ♦♥t été ❝♦♠♣❛ré❡s à ❝❡❧❧❡s ❞✬❡♠❜r②♦♥s ♣♦rt❛♥t ✉♥❡ ♠✉t❛t✐♦♥ ❛❢✲ ❢❡❝t❛♥t s♣é❝✐✜q✉❡♠❡♥t ❧❡ ❝❛❜❧❡ ❞✬❛❝t✐♥❡✳ ▲❡s rés✉❧t❛ts ♣rés❡♥tés ❞❛♥s ❝❡tt❡ t❤ès❡ ♥♦✉s ♣❡r♠❡tt❡♥t ❞❡ ♠✐❡✉① ❝♦♠♣r❡♥✲ ❞r❡ ❧❡s ♣r♦❝❡ss✉s ♠é❝❛♥✐q✉❡s ✐♠♣❧✐q✉és ❞❛♥s ❧❡s ♦s❝✐❧❧❛t✐♦♥s ❞❡s ❝❡❧❧✉❧❡s ❞❡ ❧✬❛♠♥✐♦sér❡✉s❡✳ ■❧s ♥♦✉s ❞♦♥♥❡♥t ❛✉ss✐ ❞❡s ✐♥❞✐❝❡s s✉r ❧❡✉rs ❝❛r❛❝tér✐st✐q✉❡s ❜✐♦❧♦❣✐q✉❡s✳ ■❧s ♥♦✉s ♣❡r♠❡tt❡♥t ❡♥✜♥ ❞❡ ♠✐❡✉① ❛♣♣ré❤❡♥❞❡r ❧❡ rô❧❡ ❞✉ ❝❛❜❧❡ ❞✬❛❝t✐♥❡ ❞❛♥s ❝❡ ♣r♦❝❡ss✉s ♣r♦❝❤❡ ❞❡ ❧❛ ❝✐❝❛tr✐s❛t✐♦♥✳.

(11) ✽. ❈❖◆❚❊◆❚❙. ■t❛❧✐❛♥♦ ■❧ ❧❛✈♦r♦ ❞✐ t❡s✐ ♣r❡s❡♥t❛t♦ q✉✐ ❝❡r❝❛ ❞✐ ❝❛r❛tt❡r✐③③❛r❡ ❡ s✐♠✉❧❛r❡ ❧❡ ❢♦r③❡ ♠❡❝✲ ❝❛♥✐❝❤❡ ✐♠♣❧✐❝❛t❡ ♥❡❧ ♣r♦❝❡ss♦ ❞✐ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡ ❞❛❧❧✬♦r❣❛♥✐s♠♦ ❉r♦s♦♣❤✐❧❛ ♠❡❧❛♥♦❣❛st❡r✳ ◗✉❡st♦ ♣r♦❝❡ss♦ è ❡♠❜r✐♦♥❛❧❡✿ è ✉♥♦ ❞❡✐ ❛✈✈❡♥✐♠❡♥t✐ ❝❤❡ ♣❛rt❡❝✐♣❛♥♦ ❛❧❧❛ ❢♦r♠❛③✐♦♥❡ ❞❡❧❧❛ ❧❛r✈❛✳ ■♥ ♣❛rt✐❝♦❧❛r❡✱ ❧❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡ ♣❛rt❡❝✐♣❛ ❛❧❧✬❛❝q✉✐s✐③✐♦♥❡ ♣❡r ❧✬❡♠❜r✐♦♥❡ ❞❡❧❧❛ s✉❛ ❢♦r♠❛ ✜♥❛❧❡ ❛ttr❛✈❡rs♦ ❧❡ ❢♦r③❡ ❣❡♥❡r❛t❡ ❞❛ t❡ss✉t✐ ❝❡❧❧✉❧❛r✐✳ ■♥ t❛❧ ♠♦❞♦✱ ❧✬♦❜✐❡tt✐✈♦ ❞❡❧ ❧❛✈♦r♦ ♣r❡s❡♥✲ t❛t♦ q✉✐ è ❞✐ ❛♣♣r♦❢♦♥❞✐r❡ ✐ ♥♦str❡ ❝♦♥♦s❝❡♥③❡ s✉❧❧❛ ♠❡❝❝❛♥✐❝❛ ❞❡✐ t❡ss✉t✐✱ ❝♦sì ❝♦♠❡ s✉❧ ❧♦r♦ r✉♦❧♦ ♥❡❧❧✬❡♠❜r✐♦❣❡♥❡s✐✳ ■ t❡ss✉t✐ ✐♠♣❧✐❝❛t✐ ♥❡❧❧❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡ s♦♥♦ ❧✬❡♣✐❞❡r♠✐❞❡ ❡ ❧✬❛♠♥✐♦s✐❡r♦s❛✳ ❆ q✉❡st♦ st❛❞✐♦ ❞❡❧❧♦ s✈✐❧✉♣♣♦✱ ❧✬❡♣✐❞❡r♠✐❞❡ ❝✐♥❣❡ q✉❛s✐ t✉tt♦ ❧✬❡♠❜r✐♦♥❡✳ ▲✬❛♠♥✐♦s✐❡r♦s❛ ❝♦♣r❡ t✉tt❛✈✐❛ ❛♥❝♦r❛ ✉♥❛ ③♦♥❛ ❧♦❝❛❧✐③③❛t❛ ❞❡❧ ❧❛t♦ ❞♦r✲ s❛❧❡✱ ❝❤✐❛♠❛t♦ ❜✉❝♦ ❞♦rs❛❧❡✳ ▲❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡ ❝♦♥s✐st❡ ♥❡❧❧❛ ❝❤✐✉s✉r❛ ❞✐ q✉❡st♦ ❜✉❝♦ ❞♦rs❛❧❡✱ ❝♦sì ❝♦♠❡ ♥❡❧❧❛ ❝♦♥❣✐✉♥③✐♦♥❡ ❞❡❧❧❡ ♣❛rt✐ ❧❛t❡r❛❧✐ ❞❡❧❧✬❡♣✐❞❡r♠✐❞❡✱ ✐♥ ✉♥ ♣r♦❝❡ss♦ s✐♠✐❧❛r❡ ❛❧❧❛ ❝✐❝❛tr✐③③❛③✐♦♥❡✳ P❡r r❡❛❧✐③③❛r❡ ❧❛ ❝♦♥❣✐✉♥③✐♦♥❡ ❞❡❧❧✬❡♣✐❞❡r♠✐❞❡ ❛❧ ❧✐✈❡❧❧♦ ❞❡❧❧❛ ❧✐♥❡❛ ❞♦rs❛❧❡✱ q✉❡st♦ ❞❡✈❡ ❡ss❡r❡ tr❛✐♥❛t♦✳ ◗✉❡st❛ tr❛③✐♦♥❡ è r❡❛❧✐③③❛t❛ ❛❧ t❡♠♣♦ st❡ss♦ ❞❛❧❧✬❛♠♥✐♦s✐❡r♦s❛ ❝❤❡ ❝♦♣r❡ ✐❧ ❜✉❝♦ ❞♦rs❛❧❡✱ ❡ ♣❡r ❧❛ ✜❧❛ ♣✐ù ❞♦rs❛❧❡ ❞✐ ❝❡❧✲ ❧✉❧❡ ❞❡❧❧✬❡♣✐❞❡r♠✐❞❡✱ ❧❡ ▲❡❛❞✐♥❣ ❊❞❣❡ ❝❡❧❧s✳ ◗✉❡st❡ ♣❛rt❡❝✐♣❛♥♦ ❛❧❧❛ ❝❤✐✉s✉r❛ ❞✐ ❞✉❡ ♠♦❞✐✳ ❉❛ ✉♥❛ ♣❛rt❡✱ q✉❡st❡ ❝❡❧❧✉❧❡ ❢♦r♠❛♥♦ ✉♥ ❝❛✈♦ ❞✐ ❛❝t✐♥❡ ❝❤❡ ❝✐♥❣❡ ❧✬✐♥s✐❡♠❡ ❞❡❧ ❜✉❝♦ ❞♦rs❛❧❡ ❝❤❡✱ ❝♦♥tr❛rs✐s✐✱ ♣❛rt✐❝✐♣✐♦ ❛ r✐❞✉rr❡ ❧✬❛r❡❛ ❞❡❧❧✬❛♠♥✐♦s✐❡r♦s❛✳ ❉✬❛❧tr❛ ♣❛rt❡✱ ❧❡ ❝❡❧❧✉❧❡ ❞❡❧ ▲❡❛❞✐♥❣ ❊❞❣❡ ❡♠❡tt♦♥♦ ❞❡✐ ♣r♦tr✉s✐♦♥✐ ❝❤❡ s✐ st✐✈❛♥♦ ❛❧ ▲❡❛❞✐♥❣ ❊❞❣❡ ♦♣♣♦s✐t♦r❡✱ ❡❞ ❧✐ t✐r❛♥♦ ✈❡rs♦ ❡ss❡✱ ✜♥❝❤é ❧❡ ❞✉❡ ♣❛rt✐ ❞❡❧❧✬❡♣✐❞❡r♠✐❞❡ ❢♦♥❞♦♥♦✳ ◗✉❡st✐ ♣r♦tr✉s✐♦♥✐ ❤❛♥♥♦ ✉♥❛ t❛❣❧✐❛ ❧✐♠✐t❛t❛✳ ❈♦s✐✱ ❧❛ tr❛③✐♦♥❡ ❡ ❧❛ ❢✉s✐♦♥❡✱ ❝❤✐❛♠❛t❡ t❛♥t♦ ③✐♣♣✐♥❣✱ s♦♥♦ ♣♦ss✐❜✐❧✐ s♦❧❛♠❡♥t❡ ❛❧❧❡ ❡str❡♠✐tà ❛♥t❡r✐♦r✐ ❡ ♣♦st❡r✐♦r✐ ❞❡❧ ❜✉❝♦ ❞♦rs❛❧❡✱ ❝❤✐❛♠❛t❡ ❝❛♥t❤✐✱ ❧à ❞♦✈❡ ❧❛ ❞✐st❛♥③❛ tr❛ ✐ ❞✉❡ ❧❡❛❞✐♥❣ ❡❞❣❡ è s✉✣❝✐❡♥t❡♠❡♥t❡ ♣✐❝❝♦❧❛✳ P❡r❛❧tr♦✱ ❧✬❛♠♥✐♦s✐❡r♦s❛ t✐r❛ ❧❡✐ ❛♥❝❤❡ ❧✬❡♣✐❞❡r♠✐❞❡ ✈❡rs♦ ❧❛ ❧✐♥❡❛ ❞♦rs❛❧❡✳ Pr♦❞✉❝❡ ❞❡✐ ✜❧❛♠❡♥t✐ ❞✐ ❛❝t✐♥❡ ❡ ❞❡❧ ♠②♦s✐♥❡✱ ✉♥ ♠♦t♦r❡ ♠♦❧❡❝♦❧❛r❡ ❝❤❡ ❢♦r♠❛ ✉♥❛ r❡t❡ ❝♦♥tr❛tt✐❧❡ ✐♥ s❡♥♦ ❛❧❧❡ ❝❡❧❧✉❧❡✳ ◗✉❡st❛ r❡t❡ ♣❡r♠❡tt❡ ❧♦r♦.

(12) ❈❖◆❚❊◆❚❙. ✾. ❞✐ t✐r❛r❡ s✉❧❧❡ ❝❡❧❧✉❧❡ ❞❡❧ ❧❡❛❞✐♥❣ ❡❞❣❡✳ P❡r❛❧tr♦✱ ❧❡ ❝❡❧❧✉❧❡ ❞❡❧❧✬❛♠♥✐♦s✐❡r♦s❛ ✈❡❞♦♥♦ ❧✬❛r❡❛ ❞❡❧ ❧♦r♦ ❧❛t♦ s✉♣❡r✐♦r❡✱ ❧❛t♦ ❛♣✐❝❛❧❡✱ ✈❛r✐❛r❡ ✐♥ ♠♦❞♦ ♣❡r✐♦❞✐❝♦✳ ❙❡❜❜❡♥❡ q✉❡st❡ ✈❛r✐❛③✐♦♥✐ ❞✐ ❛r❡❛ s✐❛♥♦ st❛t❡ ❞❡s❝r✐tt❡ ❧❛r❣❛♠❡♥t❡✱ ✐❧ ❧♦r♦ r✉♦❧♦ ♥❡❧❧❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡ r❡st❛ s❝♦♥♦s❝✐✉t♦✳ ▲✬♦❣❣❡tt♦ ❞✐ q✉❡st❛ t❡s✐ è ❞✐ ♠✐❣❧✐♦r❛r❡ ❧❛ ♥♦str❛ ❝♦♠♣r❡♥s✐♦♥❡ ❞❡✐ ❝♦♥❝❡tt✐ ♠❡❝❝❛♥✐❝✐ ✐♠♣❧✐❝❛t✐ ✐♥ q✉❡st❡ ♦s❝✐❧❧❛③✐♦♥✐✱ ❡ ❞✐ ❝♦str✉✐r❡ ✉♥ ♠♦❞❡❧❧♦ ✜s✐❝♦ r❛♣♣r❡s❡♥t❛♥t❡ q✉❡st✐ ♠♦✈✐♠❡♥t✐✳ ■❧ ❧❛✈♦r♦ ♣r❡s❡♥t❛t♦ q✉✐ st✉❞✐❛ ❛♥❝❤❡ ✐ ♠♦✈✐♠❡♥t✐ ❞❡❧❧❡ ❝❡❧❧✉❧❡ ❞❡❧ ❧❡❛❞✐♥❣ ❡❞❣❡✱ ♥❡❧❧♦ s❝♦♣♦ ❞✐ ❝♦♠♣r❡♥❞❡r❡ ❧✬❡✛❡tt♦ ❞❡❧ ❝❛✈♦ ❞✐ ❛❝t✐♥❡ s✉❧❧❛ ❞✐♥❛♠✐❝❛ ❞❡❧❧❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡✳ P❡r st✉❞✐❛r❡ ✐ ♠♦✈✐♠❡♥t✐ ❞❡❧❧❡ ❝❡❧❧✉❧❡ ❡ ❞❡✐ t❡ss✉t✐ ✐♠♣❧✐❝❛t❡ ♥❡❧❧❛ ❝❤✐✉s✉r❛ ❞♦rs❛❧❡✱ ✉♥ ❛❧❣♦r✐t♠♦ ❝❤❡ ♣❡r♠❡tt❡ ❞✐ s❝♦♣r✐r❡ ✐ ❝♦♥t♦r♥✐ ❞❡❧❧❡ ❝❡❧❧✉❧❡✱ ❧❛ ❧♦r♦ ♣♦s✐③✐♦♥❡ ❝♦sì ❝♦♠❡ q✉❡❧❧❛ ❞❡✐ ❧♦r♦ ✈❡rt✐❝✐ ✭♣✉♥t✐ ❞✐ ❝♦♥❣✐✉♥③✐♦♥❡ tr❛ tr❡ ❛ q✉❛ttr♦ ❝❡❧❧✉❧❡✮ ❡ ❞✐ s❡❣✉✐r❡ ✐ ❧♦r♦ s♣♦st❛♠❡♥t✐ ❞✉r❛♥t❡ ✐❧ t❡♠♣♦ è st❛t♦ s✈✐❧✉♣✲ ♣❛t♦✳ ❯♥ ✐♥t❡r❢❛❝❝✐❛ ✉t❡♥t❡ è st❛t♦ ❝♦str✉✐t♦ ♣❡r ❢❛❝✐❧✐t❛r❡ ❧✬❛❞❡❣✉❛♠❡♥t♦ ❞❡✐ ♣❛r❛♠❡tr✐ ❝❤❡ ♣❡r♠❡tt♦♥♦ q✉❡st❛ ❧♦❝❛❧✐③③❛③✐♦♥❡✱ ❝♦sì ❝♦♠❡ ❧❛ ❝♦rr❡③✐♦♥❡ ❞✐ ❡rr♦r✐ ❡✈❡♥t✉❛❧✐✳ ❉✐✛❡r❡♥t✐ ♠♦❞❡❧❧✐ ❞✐♥❛♠✐❝✐ s♦♥♦ st❛t✐ ❝♦str✉✐t✐ ♣♦✐✱ ♣r❡♥❞❡♥❞♦ ✐♥ ❝♦♥t♦ ✐ ❞✐✛❡r❡♥t✐ ❝♦♠♣♦rt❛♠❡♥t✐ ♠❡❝❝❛♥✐❝✐ ❝❤❡ ❧❡ ❝❡❧❧✉❧❡ ♣♦ss♦♥♦ ❛✈❡r❡✳ ◗✉❡st✐ ♠♦❞❡❧❧✐ s♦♥♦ st❛t✐ ❝♦str✉✐t✐ s❡❝♦♥❞♦ ❧✬❛♣♣r♦❝❝✐♦ ❞✐ ▲❛❣r❛♥❣✐❛✳ ■ s✐st❡♠✐ ❞✐ ❡q✉❛③✐♦♥✐ ❝❤❡ ❞❡r✐✈❛♥♦ ❞❡❧❧❡ ❡q✉❛③✐♦♥✐ ❞✐ ❊✉❧❡r✲▲❛❣r❛♥❣❡ s♦♥♦ st❛t✐ r✐s♦❧t✐ ♥✉♠❡r✐❝❛♠❡♥t❡✱ ❡ ❧❡ ❧♦r♦ ♣r❡❞✐③✐♦♥✐ s♦♥♦ st❛t❡ ♣❛r❛❣♦♥❛t❡ ❛✐ ❞❛t✐ ❣❡♥❡r❛t✐ ❞❡❧❧✬❛❧❣♦r✐t♠♦ ❞✐ ❧♦❝❛❧✐③③❛③✐♦♥❡ s❡❝♦♥❞♦ ❧✬❛♣♣r♦❝❝✐♦ ❞❡❣❧✐ ♠✐♥✐♠✐ q✉❛❞r❛t✐✳ ■ r✐s✉❧t❛t✐ s♦♥♦ st❛t✐ ❝♦♥✈❛❧✐❞❛t✐ ♣r♦✈❛♥❞♦ ✐ r❡st✐ ❣r❛③✐❡ ❛❧❧❛ ❢✉♥③✐♦♥❡ ❞✐ ❛✉t♦✲ ❝♦rr❡❧❛③✐♦♥❡✳ ❋✐♥❛❧♠❡♥t❡✱ ❧❛ ❞✐♥❛♠✐❝❛ ❞❡❧ ❧❡❛❞✐♥❣ ❡❞❣❡ è st❛t❛ st✉❞✐❛t❛ ❝❛r❛tt❡r✐③③❛♥❞♦ ✐ ♠♦✈✐♠❡♥t✐ ❞❡❧❧❡ ❝❡❧❧✉❧❡ ❧♦❝❛❧✐③③❛t❡ ❛❧❧✬✐♥t❡r❢❛❝❝✐❛ tr❛ ❧✬❡♣✐❞❡r♠✐❞❡ ❡ ❧✬❛♠♥✐♦s✐❡r♦s❛✱ ❣r❛③✐❡ ❛❧❧✬❛❧❣♦r✐t♠♦ ❞✐ ❧♦❝❛❧✐③③❛③✐♦♥❡✳ ▲❡ ❞✐♥❛♠✐❝❤❡ ❞✐ ❡♠❜r✐♦♥✐ ♥♦♥ ♠✉t❛♥t❡ ✭✇✐❧❞✲t②♣❡✮ s♦♥♦ st❛t❡ ♣❛r❛❣♦♥❛t❡ ❛ q✉❡st❡ ❞✐ ❡♠❜r✐♦♥✐ ❝❤❡ ♣♦rt❛♥♦ ✉♥❛ ♠✉✲ t❛③✐♦♥❡ ❝❤❡ ❛✛❡tt❛ s♣❡❝✐✜❝❛♠❡♥t❡ ✐❧ ❝❛✈♦ ❞✐ ❛❝t✐♥❡✳ ■ r✐s✉❧t❛t✐ ♣r❡s❡♥t❛t✐ ✐♥ q✉❡st❛ t❡s✐ ❝✐ ♣❡r♠❡tt♦♥♦ ❞✐ ❝♦♠♣r❡♥❞❡r❡ ♠❡❣❧✐♦ ✐ ♣r♦❝❡ss✐ ♠❡❝❝❛♥✐❝✐ ✐♠♣❧✐❝❛t✐ ♥❡❣❧✐ ♦s❝✐❧❧❛③✐♦♥✐ ❞❡❧❧❡ ❝❡❧❧✉❧❡ ❞❡❧❧✬❛♠♥✐♦sér❡✉s❡✳ ❈✐ ❞❛♥♥♦ ❛♥❝❤❡ ❞❡❣❧✐ ✐♥❞✐③✐ s✉❧❧❡ ❧♦r♦ ❝❛r❛tt❡r✐st✐❝❤❡ ❜✐♦❧♦❣✐❝❤❡✳ ❈✐ ♣❡r♠❡✲.

(13) English. CONTENTS. 10. ttono inne di temere meglio il ruolo del cavo di actine in questo processo vicino alla cicatrizzazione.. The work presented here aims at characterizing and simulating the mechanical forces involved in the process of Dorsal Closure in the organism. Drosophila melanogaster.. This process is embryonic: it is an event partici-. pating in the formation of the larvae. In particular, Dorsal Closure participates in the acquisition of the nal form of the embryo. Therefore, the work presented here aims at fathoming our knowledge on tissues mechanics, as well as their role in embryogenesis. The tissues involved in Dorsal Closure are the epidermis and the amnioserosa. At this stage of development, the epidermis surrounds almost all the embryo. Nevertheless, the amnioserosa still covers a large area of the dorsal side called dorsal hole. Hence, Dorsal Closure aims at shutting this hole and joining the lateral sides of the epidermis, in a process similar to wound healing. In order to fuse the two sides of the epidermis on the dorsal line, the epidermis must be drawn dorsalward. This movement is driven by the amnioserosa on the one hand, and by the dorsalmost row of the epidermis (called Leading Edge cells) on the other hand. The latter participate in Dorsal Closure in two ways. First, the Leading Edge cells form atranscellular Actin Cable around the dorsal hole. The cable, contracting, will reduce the area of the dorsal hole covered by te amnioserosa. Second, the Leading Edge cells emit protrusions that will attach to the opposite Leading Edge and drag it toward themselves, untill the two sides of the epidermis fuse. These protrusions have a limited range, hence the dragging and fusion only take place at the anterior and posterior ends of the dorsal hole (called canthi), where the distance between the two Leading Edges is small enough. The Amnioserosa also drags the epidermis toward the dorsal line. Its cells produce actin laments and myosin (a molecular motor), that will form a contractile network within the cells. Interstingly, Amnioserosa cells see the.

(14) 1 av rT e a o f t h e i r t o p s i d e ( a p i c a l s i d e ) v a r y i n a p e r i o d i c w a y . A l t h o u g h t h e s iv t i n s a v e b e n w d e y t u i d , t h e r o l n D o r s l C o s u r e r e m a i n u n k n o w . iL h s P h D a i m s a t i m p r o v i n g o u r k n o w l e d g e o f t h e m e c h a n i c a l c o n c e p t s i n i n t h e o c l a t i n s , a d t o b u i d a p h y s i c a l o d l r e p r e s t i g h e s ltm o v e d m e s . T h e w o r k p r e n e d h e r l s o t u d i e s t h e m o v m e n s o f t iiIn e a d n g E d g e c l s , i n o d t o u n s t a n d t h e c o f t h e A c t i C a b l e o n s u r e . h y a m i c s o f D o r s a l C l o o r d e r t o t u d y t h e c e m o v m e n t s a n d t h e r o l e o f t h e t i s u e s i n v o l v e d o p d , a w i n g o d e t c t h e c l s D s a l C l s r e , a n a l g o r i t h w a s d e v l ew d g e , t h e i r p o i t o , s w e l a s t h o f t h e i r v e r t c e s ( m u l i p l e j u n t i o n n e r f u r c e l ) n d t r a c k m o i m . A s r i t e r f a c e b tV w e m e n t o f t h e p a m t r s a s a l s o d v l o p e d , i n o r d e r t o f a c i l t e t h e a d j u s t lb o w i n g t h e d e t c t i o a s w l a s t h e c o r c i o n o f p o s i b l e r o r s . e r n b u i l t , t a k i n g i n t o a c u n t h e p o s i a r u s d y n a m i a l m d e l n g lL e m e c h a i c l b e h v i o r s c e l s m a y h a v e . T h e s m o d e l s w e r b i l f o l w tftoh lalgtrh a g r n g a n a p r o a c h . T h y s t e m s o f q u a t i o n s r i v n g f r o m t h e E u l e r v e d , n d t h e i p e d i c t i n s c o m p a d n e q u t i o n s w e r n u m e r i c a l y l e b i o l g i c a l d a t x t r a c t d t h n k s t o t h e a l g o r t h m p r e s t e d e r l i e r , w i n g t h e e s s q u a e p r o a c . T h e m o d v a l i d a t i o n w a p r f o m d tF h a n k s t o a u t o c r l t i o n f u n t i o n t s t . ,roilw y h e L e d i n g E d g e d y a m c s w a u d i e d c h a r c t e r i s n g t h e c e l u isW llah i s n d h e a m n i o s r o s a . m o v e m n t s a t h e i n t r f a c e b e t w e n t h e p r m d t y p e m b r y o s d y a m i s w r c o m p a r d t o t h o e o f m u t e d e m b y t h e A c t i n C a b l e f o r m a i n . ip n g s c i c d e f c t s i n T e r e u l t p r e s n t d i n t h i s m a n u s c r i p t w b e t e r u n d e r s t a n d i n g o f t h e lisp igriocaelscn v d A m n o s e r o a e l s o s c i l a i o n s . T h y l s o v e c l u s rcan ob ctlh sein srtb ih n v o h rsim a ciltaeriotiw co.u F id n ah ,ealin y th eg.yseteroefthatin CONTENTS.

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(16) CInhtaropdteurct1ion hnom T isednoactwiraltm heasinacoim snagtifm provihnygsiocu.rTnhdersytanedm igchofsbeniolfgrictahlepfhoel-. le1om w.1binrygwT oenrkhiesD oD rfthrseaolm osdspulhroe,iglaandisem C vM loepm elanntaolgevansttoecrurm in.godduerilngthe oocinarglapnhrdtseinm A m lcuom rlasgereadnti,iosnvm ihleuwsbm aoarngnstot.orrgieaexnstifsom erdvxhetrnasidcveenlcytouvsrteyur.diseitsoauhnwdeelor-skgtnaonwdsnm bio,loangeid-l SnA cbgarletirteihcndotysui,fsgrufhodirtbCeshxgiaepatnhel,rtiasm onW urdsnst.fetaeW , a l s o c a l e d f r u i t y , o r v i n e lrxopspuugrdripwm oessretinhagilnsts1htc3uhardediteihresostndteophcebcdietshcfouthlrdsbtouesxtdcnefosirtvugerly-. Drosophila Melanogaster. Drosophila Melanogaster. 1.1.1 A brief history of ies in science. First uses of the fruit y. Drosophila Melanogaster. X X th. Drosophila Melanogaster.

(17) 1 4 sM a a m o d e l o r g a n i s m b e g a n i n 1 9 0 u n d e r t h e d i r e c t i o n o f T h o m a s H u n t orT gh n a t C l u m b a U n i v e r s t y . o m a s M o r g a n ' s e a r l y w o r k o n d r o s p h i l a f o c u s e d o n s e x d e t r m i n g e t h e f a c t o r s [ 1 ] , [ 2 ] . A t h i t i m e , t h e S u t n B o v e r t h e r y , r c h r o m o s ocoh rb y fstoem iry n h e r i t a n c e , d e v l o p d b y T e o d o r i a n d W a l t e S u t n i n c e n t u r , w a s l w l y s e t l g . C h r o m a i n w a s r s t e a r s o f h o f v d b y W a l t h e r F r i d m a n i n 1 8 7 9 , h o a e r d i s c o v e d t h e p i o s m e s . M i o s i w a s d e s c r b e d d u r i n g t h f o l o w i n g e c a d s , m e i sd ih ietsram d cin reay v iteioan d n 1 9 0 5 b y J . B F a m r a n d J . E S . M r e , a n d X Y t h s x sr[3y].tem w as rstsu gestedb yB W ilsonN etieSevn s H . M o r g a n g a v e a p r o f o f t h i s e x d e t r m i n a t i o n s y t e m t h a n k s t o t h e .d T e w i h r e d e y f e m a l ri o s p h i l a m d e l . H c s e d w e y m a l e f r u ion ev ,fd a n d t h e n t h e r e y d p r o g e n y t o g e t h e r , i n t e s t s t c r o s i l M e n d l ' s l a w s p r d i c e d a g e n d e r p e r f o r m d w i t h . W h e n d e n t r e p a r i o n o f r e d a n d w t e y i e , M o r g a n g o t v e r y l s w i t h r e d e y s , a n e q u a l r e p a r t i o n f e d a n d w h i t e y e s t h f m a e r s t o d c o n icsn lh m a e s , h o n i n F i g u r e 1 . T h s t i o s c n l y b u d eorm in tosm g h aets"p erop yoescod lb cyW h airlsocn teran idtS raen ivn m ts.edth rou ghth"acesry L e t u s c o n s i d e r t h a t m a l e s c a r y o n l y o n e X c h r o m o s m e , a n d a n o t h e r c r , w h i l f e m a l e s c a r y t w o X c h r m o o n e c a l e d Y o t a c o u n i n g f o r e y sw m s . L t h e w h i t e y e a l e b r e s i v e . H n c , c r o i n g r e d e y f e a l e s i(b trosth ihg)t.eh w h e y m a l s a s h o w n i n F i g u r 1 2 a , o e x p e c t s v r i n t h . F m a l p r o g e n y w l c a r y n r a t i o n o f t h e t e t c r s t o h a v e e d e y s ) a n t h e w h i t e y e X c h r o m o s m e e d e y X c h r o m o m e ( T h w h i t e y e a l e b e i n g r e c s i v e , y l h a v r d e y s . M a l s , e n b y t h f a t h e r , n o t o n t h e o t e r a n d , w i c a r y t h e Y h r o m o s m e g i v d erm ingyecolr,an dth eX crom sefrothm oth er:.A th is CHAPTER 1. INTRODUCTION. X X th. 100%. Drosophilas. 100%. X+. XW. X+.

(18) ✶✳✶✳. ❚❍❊. ❉❘❖❙❖P❍■▲❆ ▼❊▲❆◆❖●❆❙❚❊❘. ❋✐❣✉r❡ ✶✳✶✿. ▼❖❉❊▲. ✶✺. ❊①♣❡❝t❡❞ t❡st ❝r♦ss r❡s✉❧ts ❛❝❝♦r❞✐♥❣ t♦ ▼❡♥❞❡❧ ❧❛✇s ✈❡rs✉s. ▼♦r❣❛♥✬s r❡s✉❧ts✳ ▼♦r❣❛♥✬s r❡s✉❧ts s❤♦✇ ❛ ❣❡♥❞❡r✲r❡❧❛t❡❞ ❜✐❛s✳ ❬✹❪. ♣♦✐♥t✱ st❛t✐st✐❝s ❞♦ ♥♦t s❤♦✇ ❛♥② ❞✐✛❡r❡♥❝❡ ✇✐t❤ ▼❡♥❞❡❧ ❧❛✇✬s ♣r❡❞✐❝t✐♦♥s✳ ❉✐✛❡r❡♥❝❡s ❛r✐s❡ ✇✐t❤ t❤❡ ♥❡①t ❣❡♥❡r❛t✐♦♥ ✭❋✐❣✉r❡ ✶✳✷❜✮✿ ❝r♦ss✐♥❣ X + X W ❢❡♠❛❧❡s ✇✐t❤ X + Y ♠❛❧❡s✱ ▼♦r❣❛♥ ♦❜t❛✐♥❡❞ ❛ ♣r♦❣❡♥② ✇❤❡r❡ ❡✈❡r② ❢❡♠❛❧❡ ❤❛❞ r❡❞ ❡②❡s✿ t❤❡② ✐♥❤❡r✐t❡❞ t❤❡ X + ❝❤r♦♠♦s♦♠❡ ❢r♦♠ t❤❡✐r ❢❛t❤❡r✱ ❛♥❞ t❤✉s ❤❛❞ r❡❞ ❡②❡s✱ r❡❣❛r❞❧❡ss ♦❢ t❤❡ ❝❤r♦♠♦s♦♠❡ ❣✐✈❡♥ ❜② t❤❡ ♠♦t❤❡r✳ ▼❛❧❡s ✇♦✉❧❞ r❡❝❡✐✈❡ t❤❡ ❨ ❝❤r♦♠♦s♦♠❡ ❢r♦♠ t❤❡✐r ❢❛t❤❡r✱ ❛♥❞ ❤❡♥❝❡✱ t❤❡✐r ❡②❡ ❝♦❧♦r ✇♦✉❧❞ ❜❡ ❞❡t❡r♠✐♥❡❞ ❜② t❤❡ ❝❤r♦♠♦s♦♠❡ t❤❡② r❡❝❡✐✈❡❞ ❢r♦♠ t❤❡✐r ♠♦t❤❡r✿ ✺✵✪ ❝❤❛♥❝❡s t♦ r❡❝❡✐✈❡ t❤❡ X + ✱ ✺✵✪ t♦ r❡❝❡✐✈❡ t❤❡ X W ✳ ▼❡♥❞❡❧ ❧❛✇s ❝♦✉❧❞♥✬t ❡①♣❧❛✐♥ t❤❡s❡ r❡s✉❧ts✿ t❤❡② ❛r❡ ❜♦✉♥❞ t♦ s♦♠❛t✐❝ ✭♥♦♥ s❡①✲ ✉❛❧✮ ❝❤r♦♠♦s♦♠❡s✳ ❚❤❡s❡ st❛t✐st✐❝s ❝♦✉❧❞ ♦♥❧② ❛r✐s❡ ❢r♦♠ t❤❡ ✐❞❡❛ t❤❛t s❡① ✐s ❞❡t❡r♠✐♥❡❞ ❜② ✧❆❝❝❡ss♦r② ❝❤r♦♠♦s♦♠❡s✧✱ ❤❛✈✐♥❣ ❛ ❞✐✛❡r❡♥t ✐♥❤❡r✐t❛♥❝❡ ♣❛tt❡r♥✳ ❏✉st ❜② ✇❛t❝❤✐♥❣ ❉r♦s♦♣❤✐❧❛s ❡②❡ ❝♦❧♦r✱ ▼♦r❣❛♥ ✇❛s ❛❜❧❡ t♦ ❞❡✲ t❡r♠✐♥❡ t❤❡ ❡①✐st❡♥❝❡✱ ❛♥❞ t❤❡ ✐♠♣♦rt❛♥❝❡ ✐♥ ❣❡♥❞❡r ❞❡t❡r♠✐♥❛t✐♦♥✱ ♦❢ t❤❡s❡ s❡①✉❛❧ ❝❤r♦♠♦s♦♠❡s✳ ▲❛t❡r ♦♥✱ st✐❧❧ ✇♦r❦✐♥❣ ✇✐t❤ ❉r♦s♦♣❤✐❧❛✱ ▼♦r❣❛♥ st✉❞✐❡❞ r❡❝♦♠❜✐♥❛t✐♦♥ ❛♥❞ ❣❡♥❡t✐❝ ❧✐♥❦❛❣❡✱ ✐✳❡✳ t❤❡ t❡♥❞❡♥❝② ♦❢ ❝❤❛r❛❝t❡rs t♦ ❜❡ tr❛♥s♠✐tt❡❞ t♦✲ ❣❡t❤❡r✳ ●❛t❤❡r✐♥❣ ❞❛t❛ ♦♥ ❣❡♥❡t✐❝ ❧✐♥❦❛❣❡✱ ❛♥❞ ❝♦♥s✐❞❡r✐♥❣ t❤❛t t❤✐s ❧✐♥❦❛❣❡ ✇❛s ❞✉❡ t♦ ❛ ♣❤②s✐❝❛❧ ♣r♦①✐♠✐t② ♦♥ ❝❤r♦♠♦s♦♠❡s✱ ❤❡ ✇❛s ❛❜❧❡ t♦ ❜✉✐❧❞ t❤❡ ✜rst ❣❡♥❡t✐❝ ♠❛♣s r❡♣r❡s❡♥t✐♥❣ t❤❡ ♣♦s✐t✐♦♥ ♦❢ ❣❡♥❡s ♦♥ ❝❤r♦♠♦s♦♠❡s ❬✺❪✳ ❍❡ r❡❝❡✐✈❡❞ t❤❡ ✜rst ◆♦❜❡❧ Pr✐③❡ r❡✇❛r❞✐♥❣ ❛ ✇♦r❦ ♦♥ ❉r♦s♦♣❤✐❧❛ ✐♥ ✶✾✸✸✱ ✧❢♦r.

(19) ✶✻. ❈❍❆P❚❊❘ ✶✳. ✭❛✮. ✭❜✮. ❋✐❣✉r❡ ✶✳✷✿. ■◆❚❘❖❉❯❈❚■❖◆. ❈r♦ss t❛❜❧❡ ♦❢ ▼♦r❣❛♥✬s ✜rst ❝r♦ss✳. ❈r♦ss t❛❜❧❡ ♦❢ ▼♦r❣❛♥✬s s❡❝♦♥❞ ❝r♦ss✳. ▼♦r❣❛♥✬s ❝r♦ss❡s t❛❜❧❡s ❢♦r s❡①✉❛❧ ❝❤r♦♠♦s♦♠❡s✳. ❢♦r t❤❡ ❝❤r♦♠♦s♦♠❡ ❝❛rr②✐♥❣ t❤❡ r❡❝❡ss✐✈❡ ✇❤✐t❡✲❡②❡ ❛❧❧❡❧❡✱ ❛♥❞. XW. st❛♥❞s. X+. ❢♦r t❤❡. ❝❤r♦♠♦s♦♠❡ ❝❛rr②✐♥❣ t❤❡ ❞♦♠✐♥❛♥t ✇✐❧❞ t②♣❡ r❡❞✲❡②❡ ❛❧❧❡❧❡✳ ❬✹❪. ❤✐s ❞✐s❝♦✈❡r✐❡s ❝♦♥❝❡r♥✐♥❣ t❤❡ r♦❧❡ ♣❧❛②❡❞ ❜② t❤❡ ❝❤r♦♠♦s♦♠❡ ✐♥ ❤❡r❡❞✐t②✧✳ ❚❤❡s❡ st✉❞✐❡s ✇❡r❡ t❤❡ ✜rst st❡♣ ✐♥ t❤❡ ❜✉✐❧❞✐♥❣ ♦❢ t❤❡ ❦♥♦✇❧❡❞❣❡ ♦❢ ❜♦t❤ ❞r♦s♦♣❤✐❧❛✬s ❣❡♥❡t✐❝✱ ❛♥❞ t❤❡ ❣❡♥❡t✐❝ t♦♦❧❜♦① t❤❛t ♠❛❦❡s t❤❡ ❢r✉✐t ✢② s✉❝❤ ❛ ❤❛♥❞② ❧❛❜ ❛♥✐♠❛❧ ♥♦✇❛❞❛②s✳. ❖t❤❡r ♠❛❥♦r ❞✐s❝♦✈❡r✐❡s t❤r♦✉❣❤ ❢r✉✐t ✢✐❡s ❚✳❍✳ ▼♦r❣❛♥✬s ♦✉tst❛♥❞✐♥❣ r❡s✉❧ts ✇✐t❤ t❤❡ ❉r♦s♦♣❤✐❧❛ ♠♦❞❡❧ s✉❜s❡q✉❡♥t❧② ❡♥❝♦✉r❛❣❡❞ ♦t❤❡r s❝✐❡♥t✐sts t♦ ✉s❡ t❤✐s s♣❡❝✐❡s ❢♦r ❡①♣❡r✐♠❡♥t❛❧ ❜✐♦❧♦❣②✳ ❙✐♥❝❡ t❤❡♥✱ ♠❛♥② ❣r❡❛t s❝✐❡♥t✐✜❝ ❛❞✈❛♥❝❡s st❡♠ ❢r♦♠ ✇♦r❦s ♦♥ t❤❡ ❢r✉✐t ✢✐❡s✳ ❖♥❧② t❛❦✐♥❣ ✐♥t♦ ❛❝❝♦✉♥t ◆♦❜❡❧ Pr✐③❡s✱ ❢♦✉r ♦t❤❡r ♠❛❥♦r ❞✐s❝♦✈❡r✐❡s ❤❛✈❡ t♦ ❜❡ r❡♠❡♠❜❡r❡❞✳ ❍❡r♠❛♥♥ ❏♦s❡♣❤ ▼✉❧❧❡r ✭♦♥❡ ♦❢ ❚❤♦♠❛s ▼♦r❣❛♥✬s st✉❞❡♥ts✮ ✇❛s ❛✇❛r❞❡❞ t❤❡ ◆♦❜❡❧ Pr✐③❡ ♦❢ P❤②s✐♦❧♦❣② ❛♥❞ ▼❡❞✐❝✐♥❡ ✐♥ ✶✾✹✻ ✧❢♦r t❤❡ ❞✐s❝♦✈❡r② ♦❢ t❤❡ ♣r♦❞✉❝t✐♦♥ ♦❢ ♠✉t❛t✐♦♥s ❜② ♠❡❛♥s ♦❢ ❳✲r❛② ✐rr❛❞✐❛t✐♦♥✧✱ ✇♦r❦ ♣✉❜❧✐s❤❡❞ ✐♥ ✶✾✷✼✳ ■rr❛❞✐❛t✐♥❣ ✢✐❡s ✇✐t❤ ✈❛r✐♦✉s ❞♦s❡s ♦❢ ❳✲r❛②s✱ ❤❡ ❢♦✉♥❞ ❛ ❝♦rr❡❧❛t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ❛♠♦✉♥t ♦❢ r❛❞✐❛t✐♦♥ r❡❝❡✐✈❡❞ ❛♥❞ t❤❡ ♥✉♠❜❡r ♦❢ ❧❡t❤❛❧ ♠✉t❛✲.

(20) 1 7 tfm ioron sd a p e a r i n g i n i e s . H e l a t e r c o m u n i c a t e d o n t h e d a n g e r s o f r a d i a t i o n s h u m n s . L a t e r o n , h i s w o r k a l s a l o w e d s c i t i s t o g e a t e g e n c a l y iIn ead ip ,aperfoeliassetd e an cine1t980[7e],an th t o f m u a d n s [ 6 ] . d l a t e r w o r k s [ 8 ] , C h r i s t i a n e N u s l e i n lE V o h r d n d E i c F . W e s c h a u s u s e t h e f u l p t e n t i a l o f i n x rw i40th p e m e n t a l b i o l g y : t h y p r o d c d m u a t i o n s i n s D N A r a n d o m l y u s i n g e e c t s o f t h e s m u t i n s o n o v e r ldsico0h h y h a n e s u f o n a e , a n a n a l y z e d h i e s l i i j u s t h r e y e r s o f t i m . T h y w e r t h e n a b l e t i d e t i f y g n w a s m t a e d f o a c h e m b r y o n i c d e f c t h e y m o n i o r e . T h e v e r d t h t o f h 1 3 0 g e n s i n ' s g n o e , 5 0 w e r i m ru p o t a n t f o r e m b r y n i c d e v l o p m t , a d 1 4 0 w e r c r i t c a l . T h e y i d n t e d n i c s h a p e s b i s m n t , o o n l y n u m e r o u s g n s t h a t a r c r i t c a l f o r e m b r y o iifn , b u l s o i n a l ( a m l s ) . W i t h t h i s c r e n , t h e h a n i s m l o w i n g m u l t c e l u l a r o g a i s m s o igoerm n v l19tficr5od e lm a a r g e p a r t f t h e m e c o n s i n g l e c e l . F o r t i s w o r k , t h e y e r a w a r d t h e N o b e l P r i z e ton gertoh elorfw ieatrh E d w a d L e i s " f o r t h i d i s c o v e i e s c o n c e r n i g t h n t " . l y e m b r y o n i c d e v l p m e T w o t h e r s N o b e l P r i z e s a w a r d e n d i n g s o n s y t e m s w e r d u e c o v e r i s tlm o . I n 2 0 4 , R c h A x l a L i d a B u c k , " f o r h i r d i fim d o r a n t r e c p t o r s a n d t e o r g a n i z t i o n o f t h e o l f a t o r y s y t e m " a w o k n d 2 0 1 , J u e s H m a n , B r u c e B e u t lteh o s t y p e f o m e d n r s h u h . A rageiarn n d R a l p h S t i m a n f o r t e i r i s c o v e r i e s a r o n d i n t e a n d a d a p i v e . T e f o r e r w k i n g o n , n d t h e t w o l t e r o n m c , u i t y a l s w o r k u n r a v e l d a s h a r e d s y t e m t o p r o t e c t b o i n s e c t s a n d m a s t m i c o b s . T h e t w o f o m e r s , i n p a t i c u l a r , i d e n t d a f m i l y o f g e n icrobneactiosbothin raen sd poM lam b eaflsr.an ti-fu n galan dan t-m T h e s w o r k s m a d e t h e f r u i t i e s a n o r g a n i s m s o f a g r e a t i m p o r t a n c e h in on -xasveltof n dghw sh ow m u chcb inB iolgy.T 1.1. THE. DROSOPHILA MELANOGASTER MODEL. Drosophila. Drosophila. Drosophila. Metazoans. Metazoan. Drosophila. Drosophila. Drosophila.

(21) ✶✽. ❈❍❆P❚❊❘ ✶✳. ■◆❚❘❖❉❯❈❚■❖◆. ❧❡❛r♥❡❞ ❢r♦♠ t❤✐s ♦r❣❛♥✐s♠✳ ❯s❡❞ ❛t ✜rst ❜❡❝❛✉s❡ ♦❢ ✐ts ❜r❡❡❞✐♥❣ ❝♦♥✈❡✲ ♥✐❡♥❝②✱ ❞r♦s♦♣❤✐❧❛ ❤❛s ♥♦✇ ❜❡❝♦♠❡ ❛ ♠❛❥♦r ♠♦❞❡❧ ♦r❣❛♥✐s♠✳. ❆ ❝♦♥✈❡♥✐❡♥t ✢② ❚❤❡r❡ ❛r❡ s❡✈❡r❛❧ r❡❛s♦♥s ✇❤② t❤❡ ❢r✉✐t ✢✐❡s ❛r❡ ✉s❡❞ ♦♥ ❛ ❧❛r❣❡✲s❝❛❧❡ ✐♥ ❧❛❜♦r❛t♦r✐❡s✳ ❋✐rst ♦❢ ❛❧❧✱ ❉r♦s♦♣❤✐❧❛ ♠❡❧❛♥♦❣❛st❡r ✐s ✈❡r② ❝♦♥✈❡♥✐❡♥t ♦♥ t❤❡ ❝♦♥t❡①t ♦❢ ❛ ❧❛❜♦r❛t♦r②✳ ❚❤❡② ❛r❡ ❝❤❡❛♣ t♦ ❢❡❡❞✱ ❝❛♥ ❜❡ st♦❝❦❡❞ ✐♥ ❧❛r❣❡ ❛♠♦✉♥ts ✐♥ s♠❛❧❧ s♣❛❝❡s✱ ❛♥❞ ❛r❡ ✈❛st❧② ❜r❡❞✱ s✐♥❝❡ t❤❡✐r ❣❡♥❡r❛t✐♦♥ t✐♠❡ ✐s ♦♥❧② t✇♦ ✇❡❡❦✳ ❚❤✐s s❤♦rt ❣❡♥❡r❛t✐♦♥ t✐♠❡ ♦✛❡rs ❣r❡❛t ♣♦ss✐❜✐❧✐t✐❡s ✐♥ t❤❡ ❣❡♥❡t✐❝ ✜❡❧❞✳ ❚♦❣❡t❤❡r ✇✐t❤ t❤❡ ❢❛❝t t❤❛t ❢r✉✐t ✢✐❡s ❤❛✈❡ ♦♥❧② ❢♦✉r ♣❛✐rs ♦❢ ❝❤r♦♠♦s♦♠❡s ✭t❤r❡❡ ❛✉t♦s♦♠❛❧ ❛♥❞ ♦♥❡ s❡①✉❛❧✮✱ t❤❛t ❢❡♠❛❧❡ ❤❛✈❡ ❛ ✈❡r② ❤✐❣❤ ❢❡❝✉♥❞✐t② ✭r♦✉❣❤❧② ✶✵✵ ❡❣❣s ♣❡r ❞❛②✮✱ ✐t ❡♥❛❜❧❡s t❤❡ ❣❡♥❡t✐❝✐sts t♦ ❣❡♥❡r❛t❡ ❣r❡❛t ❛♠♦✉♥ts ♦❢ ❧✐♥❡s ✭♣r♦❞✉❝✐♥❣ ♠✉t❛t✐♦♥s ♦r ✐♥s❡rt✐♦♥s ❢♦r ✐♥st❛♥❝❡✮✱ ❛♥❞ t♦ st✉❞② t❤❡♠ ♦✈❡r s❡✈❡r❛❧ ❣❡♥❡r❛t✐♦♥s✱ ✐♥ ❛ r❡❛s♦♥❛❜❧❡ ❛♠♦✉♥t ♦❢ t✐♠❡✳ ■♥t❡r❡st✐♥❣❧②✱ ❛❧t❤♦✉❣❤ ❉r♦s♦♣❤✐❧❛ ✐s ❡✈♦❧✉t✐♦♥❛r✐❧② ♠♦r❡ ❞✐st✐♥❝t ❢r♦♠ ❤✉♠❛♥ t❤❛♥ ♦t❤❡r ♠♦❞❡❧ ♦r❣❛♥✐s♠s✱ s✉❝❤ ❛s ♠♦✉s❡ ♦r r❛t✱ ✐t st✐❧❧ s❤♦✇s ❛ ❛st♦♥✐s❤✐♥❣ ❣❡♥❡t✐❝ s✐♠✐❧❛r✐t② ✇✐t❤ ♠❛♠♠❛❧✐❛♥s ✐♥ ❣❡♥❡r❛❧✳ ■♥ ❢❛❝t✱ ✼✺✪ ♦❢ t❤❡ ❣❡♥❡s ✐♥✈♦❧✈❡❞ ✐♥ ❤✉♠❛♥ ❞✐s❡❛s❡ ❤❛✈❡ ❤♦♠♦❧♦❣s ✐♥ ❉r♦s♦♣❤✐❧❛✱ ❛♥❞ ✺✵✪ ♦❢ t❤❡ ♣r♦t❡✐♥s ♣r♦❞✉❝❡❞ ❜② t❤❡ ❢r✉✐t ✢✐❡s ❝❡❧❧s ♠❛t❝❤ ❛ ♣r♦t❡✐♥ ❢♦✉♥❞ ✐♥ ♠❛♠♠❛❧s✳ ■♥ ✈✐❡✇ ♦❢ t❤✐s✱ ❉r♦s♦♣❤✐❧❛ ❝♦♠❡s ✈❡r② ❤❛♥❞②✱ s✐♥❝❡ t❤❡② ❛r❡ ♠✉❝❤ ♠♦r❡ ❝♦♥✈❡♥✐❡♥t ❢♦r ❡①♣❡r✐♠❡♥t❛t✐♦♥s t❤❛♥ ♦t❤❡r s♣❡❝✐❡s ❝❧♦s❡r t♦ ❤✉✲ ♠❛♥s✱ ✇❤✐❧❡ t❤❡② st✐❧❧ s❤♦✇ ❛ s❛t✐s❢❛❝t♦r② ❣❡♥❡t✐❝ s✐♠✐❧❛r✐t② ✇✐t❤ ✉s✳.

(22) 1 9 1D .ros2philalifeandrep'rsodd luctionocp e v e m e n t y l e i s h o r t . U n d e r i d e a l c o n d i t o n s ) , t e r d v e l o p m n t i s a c h i e v d f r o m n e g e r a t i o n t o t h e e x i (d s e m b r y g e n s i i n a p 1T 0 d a y s . T h e g , u n d e r s u c h o n d t i o n s , a c h i v s i t p rfou ih o x m a t e l y 1 2 t o 1 5 h o u , a n d t h e n h a t e s . T h e r u l t i n g l a r v a d e v l o s n g 4 d a s , m l d i n g t w i c e i p r o c e s , o n a n d t w o d a y s f t e h a t c h i n g . o s l8ortsb e ra1ro2tu a v ih latn w t h e e c a p s u l a t e i n t h p u p a , u n e r g o m a m o r p foou rtsh .eforlitfh d a y s T h e d y w i l t h e n e m e r g e , a d i t w i l t k e a n t h e r y ecy flm le.estobecom a frtil. arehread IW n w h a t f o l o w s , w e w i l f o c u s o n p a r t i c u l a r f e a t u r e s o f e m b r y o . ib lryon d e s c r i b e b r y t h e f r u i t y ' s e g m a t i n . P i c t u r e s o f a f e w em i c t a g s a e p r e s n t d n F i g u r 1 . 3 y o g e n s i s d i v d e i n s e v n t e n s t a g e s r e g r o u p i n g e v r y e m b r eD v n t n e c s a r y t o g f r o m t h e u n c l u l a r t a r i g h f t f t l z a t i o n t o tgw h r e a d y t o h c h l a r v a [ 9 , 1 0 ] . d i v s i o n s , ich u n g t h e r s t f o u s t g e s t h e m b r y o u n d e r g o e s t h i r t e n u c l e a r ttiroeah o m m b a n e d i v i o n . T h e m b r y o b c m a h u g e e o n t a n g a e x p e r i e n c e s o m m r p h o l i c l n u b e r o f u c l e . T h e m b r y o a l s n g e s ( u d s a p e a r i n g a n d i s a p e r i n g o n t h m m b r a n e , n u c e i p o s e e r i h y ) . C e l w l s t a t a p e a r a t s t g 5 i a p r c e icvalgien a t h r i n f o r t h e r l t e i n d C e l u l a r i z a t o n . M i d v n t r a c e l s r e l s o p a t i o n . T h e s s t a g e s l a s l o g t h o u g h y t h r e h o u s . n s t a r t s a t s t a g e s i x : v e n t r a l c e l s e n t e r t h e M i d v e n t r a l c e l s i n v a g i n a t i o m gn asd treu ln tdid a oerm p roign ,aftoerm g a t i u e c l e d h v e n t r a l f u o w . M s o d fom h esls.A grosoapearsath ean 1.1. THE. DROSOPHILA MELANOGASTER MODEL. Drosophila. Drosophila. 's life cycle. 25 ◦ C. Drosophila. Drosophila. 's embryogenesis. Drosophila. Drosophila.

(23) ✷✵. ❈❍❆P❚❊❘ ✶✳. ■◆❚❘❖❉❯❈❚■❖◆. t❡r✐♦r ♣❛rt✱ ♠❛r❦✐♥❣ t❤❡ s❡♣❛r❛t✐♦♥ ✇✐t❤ t❤❡ ❢✉t✉r❡ ❤❡❛❞✳ ❆t st❛❣❡ s❡✈❡♥✱ ♣r❡❝✉rs♦r t✐ss✉❡s t♦ t❤❡ ❣✉t ❛❧s♦ ✐♥✈❛❣✐♥❛t❡✳ ❆t st❛❣❡ ✽ t♦ ✶✵✱ ♣r❡❝✉rs♦r ❝❡❧❧s t♦ t❤❡ ❞✐❣❡st✐✈❡ s②st❡♠ ❦❡❡♣ ♦♥ ✐♥✈❛❣✐♥❛t✐♥❣✱ ✇❤✐❧❡ t❤❡ ❣❡r♠ ❜❛♥❞ ✭❛♥ ❡♠❜r②♦♥✐❝ t❛✐❧✮ ✐s ❢♦r♠❡❞✳ ❚❤❡ ❣❡r♠ ❜❛♥❞ ✐s ❛ ✈❡♥tr❛❧ ❜❛♥❞ ♦❢ ❝❡❧❧s✱ t❤❛t ❡①♣❛♥❞s ❛♥❞ ❢♦❧❞s ❛t t❤❡ ♣♦st❡r✐♦r ❡♥❞ ♦❢ t❤❡ ❡♠❜r②♦✱ t♦ ❡✈❡♥t✉❛❧❧② ❝♦❧♦♥✐③❡ t❤❡ ❞♦rs❛❧ s✐❞❡✳ ❉r✐✈❡♥ ❜② ❛ ❝♦♥✈❡r❣❡♥❝❡ ❛♥❞ ❡①t❡♥s✐♦♥ ♣❤❡♥♦♠❡♥♦♥ ✭❝❡❧❧s ✐♥t❡r❝❛❧❛t❡ t♦ ✐♥❝r❡❛s❡ t❤❡ ♥✉♠❜❡r ♦❢ ❝❡❧❧ ✧❝♦❧✉♠♥s✧✱ ❛♥❞ t❤❡♥ ❡①♣❛♥❞ ❛❧♦♥❣ t❤❡ ❛♥t❡r✐♦✲♣♦st❡r✐♦r ❛①✐s✮✱ ❣❡r♠ ❜❛♥❞ ❡✈❡♥t✉❛❧❧② ❜❡❝♦♠❡s ❛ ✉✲s❤❛♣❡❞ str✉❝✲ t✉r❡ s✉rr♦✉♥❞✐♥❣ t❤❡ ❆♠♥✐♦s❡r♦s❛✳ ■♥ t❤❡ ❜❡❣✐♥♥✐♥❣ ♦❢ ❣❡r♠ ❜❛♥❞ ❡①t❡♥s✐♦♥✱ t❤❡ ②♦❧❦ s❛❝ ✭❛ ♥✉tr✐❡♥t❢✉❧❧ ❜❛❣✮ ✐s ❛❧s♦ ❢♦r♠❡❞✱ ❛♥❞ ❝❡❧❧s ♣r❡❝✉rs♦rs t♦ t❤❡ ♥❡✉r❛❧ s②st❡♠ ✭♥❡✉r♦❜❧❛sts✮ st❛rt t♦ ♠✉❧t✐♣❧②✳ ❆t t❤✐s ♣♦✐♥t✱ t❤❡ ❡♠❜r②♦ ❤❛s ❛♣♣r♦①✐♠❛t❡❧② r❡❛❝❤❡❞ ✐ts ✜❢t❤ ❤♦✉r ♦❢ ❞❡✈❡❧♦♣♠❡♥t✳ ❉✉r✐♥❣ t❤❡ ❡❧❡✈❡♥t❤ st❛❣❡✱ t❤❡ ❡♠❜r②♦ ❡①♣❡r✐❡♥❝❡s ❛ ♠❛❥♦r ❣r♦✇t❤ ♣❤❛s❡✱ ✇❤✐❧❡ ♣❛r❛s❡❣♠❡♥t❛❧ ❣r♦♦✈❡s st❛rt t♦ ❢♦r♠✱ ❛♥❞ ♠❛♥❞✐❜✉❧❛✱ ❧❛❜✐✉♠ ❛♥❞ ♠❛①✲ ✐❧❧❛ ❜❡❣✐♥ t♦ ❛♣♣❡❛r✳ ❆t t❤✐s st❛❣❡✱ t❤❡ ●❡r♠ ❇❛♥❞ ❤❛s r❡❛❝❤❡❞ ✐ts ♠❛①✐♠❛❧ ❡①t❡♥s✐♦♥✳ ❆t st❛❣❡ t✇❡❧✈❡✱ t❤❡ ●❡r♠ ❇❛♥❞ st❛rts t♦ r❡tr❛❝t✱ ❞✉❡ t♦ ✐ts tr❛❝t✐♦♥ ❜② ❆♠♥✐♦s❡r♦s❛✱ ❛♥❞ ❜② ❛ ❧❡♥❣t❤❡♥✐♥❣ ♦❢ ❣❡r♠❜❛♥❞ ❝❡❧❧s ❛❧♦♥❣ t❤❡ ❉♦rs♦✲ ❱❡♥tr❛❧ ❛①✐s✱ ❛♥❞ ❛ s❤♦rt❡♥✐♥❣ ✐♥ t❤❡ ❆♥t❡r♦✲P♦st❡r✐♦r ❛①✐s✳ ❚❤❡ ❣❡r♠ ❜❛♥❞ ❤❡♥❝❡ ✇✐❞❡♥s ❉♦rs♦✲❱❡♥tr❛❧❧② ❞✉r✐♥❣ t❤❡ ♣r♦❝❡ss✱ ❛♥❞ t❤❡ ❆♠♥✐♦s❡r♦s❛ ❝❡❧❧s✱ ✇❤✐❝❤ ✇❡r❡ ❡❧♦♥❣❛t❡❞ ❛t ❡❛r❧② st❛❣❡ t✇❡❧✈❡✱ ✇✐❞❡♥ ❆♥t❡r♦✲P♦st❡r✐♦r❧②✳ ❚❤❡ ●❡r♠ ❇❛♥❞s ♠♦✈❡♠❡♥t ❝❛✉s❡s ♦♥❡ ♣❛rt ♦❢ t❤❡ ❣✉t ✭❤✐♥❞❣✉t✮ t♦ r❡❧♦❝❛t❡ ❛t t❤❡ ❞♦rs♦✲♣♦st❡r✐♦r ♣❛rt ♦❢ t❤❡ ❡♠❜r②♦✱ ✇❤✐❧❡ t❤❡ t✇♦ ♦t❤❡r ♣❛rts ✭❛♥t❡r✐♦r ❛♥❞ ♣♦st❡r✐♦r ♠✐❞❣✉t✮ ❢✉s❡✳ ❚❤❡ ❡♣✐❞❡r♠❛❧ s❡❣♠❡♥ts ❛❧s♦ ❢♦r♠ ❞✉r✐♥❣ t❤❡ ♣r♦❝❡ss✳ ❆t st❛❣❡ ✶✸✱ t❤❡ ●❡r♠ ❇❛♥❞ r❡tr❛❝t✐♦♥ ✐s ❝♦♠♣❧❡t❡✱ ❧❡❛✈✐♥❣ t❤❡ ❆♠✲ ♥✐♦s❡r♦s❛ t♦ ❝♦✈❡r t❤❡ ❞♦rs❛❧ ❤♦❧❡ ♣r❡✈✐♦✉s❧② ♦❝❝✉♣✐❡❞ ❜② t❤❡ ●❡r♠ ❇❛♥❞✳ ❚❤❡ ❍❡❛❞ ■♥✈♦❧✉t✐♦♥✱ ❛✐♠✐♥❣ ❛t ❝♦✈❡r✐♥❣ t❤❡ ❤❡❛❞ ✇✐t❤ ❡♣✐❞❡r♠✐s✱ ❛♥❞ ❉♦r✲ s❛❧ ❈❧♦s✉r❡✱ ❜♦t❤ st❛rt ❞✉r✐♥❣ st❛❣❡ t❤✐rt❡❡♥✳ ❉♦rs❛❧ ❈❧♦s✉r❡ ❝♦♥s✐sts ✐♥.

(24) 1.2. THE ACTORS OF DORSAL CLOSURE. 21. achieving the junction between the left and right sides of the epidermis, separated by the Amnioserosa at the time, on the dorsal midline of the embryo. During stage fourteen, these two event continue, while the anal plate moves ventrally from the posterior tip and the spiracles (external tracheal apertures) become evident. At early stage fteen, Dorsal Closure and Head Involution are nished, and the guts have achieved a full fusion, and now enclose the yolk sac. During stage 16, the epidermis reaches the full segmentation that will be present in the larva. This segmented body plan is important: the segments formed during embryogenesis are closely related to those of the adult y, and each adult segment is specialized in forming particular structures (wings, legs...). At stage 17, the trachea are lled in with air, and the larva hatches. The. Drosophila. Embryonic Stages were presented here in a short and. simplied description of some of the processes required to form a healthy larva, i.e. to perform a proper development. Many events of a great importance weren't thoroughly introduced, the goal of this paragraph being to give an overview of. Drosophila 's embryogenesis.. In their globality, these. processes are of many types, and serve many purposes, from the acquisition. 1.2T heactorsofD orsalC losure. of cell and tissue identity to the shaping of the future larva. In the next section, we will present an event belonging to the latter type, event which is the subject of study of this thesis: Dorsal Closure.. As presented earlier, Dorsal Closure is an embryonic event occurring between stage 13 and 15, i.e. approximately between the tenth and the twelfth hour of development. It aims at fusing the lateral sides of the epidermis on the dorsal midline, in a process similar to wound healing. To reach a proper closure, and in order to achieve an accurate pairing of epidermal segments.

(25) ✷✷. ❋✐❣✉r❡ ✶✳✸✿. ❈❍❆P❚❊❘ ✶✳. ❚❛❜❧❡ ♦❢ s❡✈❡r❛❧ ❡♠❜r②♦♥✐❝ st❛❣❡s✳. ❡❧❡❝tr♦♥✐❝ ♠✐❝r♦s❝♦♣②✳ ❬✶✶❪. ■◆❚❘❖❉❯❈❚■❖◆. P✐❝t✉r❡s t❛❦❡♥ t❤❛♥❦s t♦.

(26) ✶✳✷✳. ❚❍❊ ❆❈❚❖❘❙ ❖❋ ❉❖❘❙❆▲ ❈▲❖❙❯❘❊. ✷✸. ✭❡❛❝❤ s❡❣♠❡♥t ❤❛s t♦ ❢✉s❡ ✇✐t❤ t❤❡ ❝♦rr❡s♣♦♥❞✐♥❣ ♦♥❡ ♦♥ t❤❡ ♦t❤❡r s✐❞❡ ♦❢ t❤❡ ❡♣✐❞❡r♠✐s✮✱ s❡✈❡r❛❧ ♠♦t♦rs ♦❢ ❝❧♦s✉r❡ ❛r❡ r❡q✉✐r❡❞✳ ❚❤❡r❡ ❛r❡ t❤r❡❡ ♦❢ t❤❡♠✿ • ❚❤❡ ❩✐♣♣✐♥❣✿ ❛t t❤❡ ❈❛♥t❤✐ ✭✐✳❡✳ t❤❡ ❛♥t❡r♦✲♣♦st❡r✐♦r ❡①tr❡♠✐t✐❡s ♦❢. t❤❡ ❡②❡✲s❤❛♣❡❞ ❉♦rs❛❧ ❍♦❧❡✮✱ ♣r♦tr✉s✐♦♥s ✭✜❧♦♣♦❞✐❛✮ s❡♥t ❜② t❤❡ ❞♦rs❛❧✲ ♠♦st ❡♣✐❞❡r♠❛❧ ❝❡❧❧s ✭▲❡❛❞✐♥❣ ❊❞❣❡ ❝❡❧❧s✮ ♦❢ ❜♦t❤ s✐❞❡s ♦❢ t❤❡ ❡♣✐❞❡r♠✐s ❢✉s❡ ✇✐t❤ t❤❡ ♦♣♣♦s✐t❡ ▲❡❛❞✐♥❣ ❊❞❣❡✱ ❛♥❞ ♣✉❧❧ ❜♦t❤ s✐❞❡s ♦❢ t❤❡ ❡♣✐✲ ❞❡r♠✐s t♦✇❛r❞ ❡❛❝❤ ♦t❤❡r✳ ❋✐❧♦♣♦❞✐❛ ❛❧s♦ ❛❧❧♦✇ s❡❣♠❡♥t r❡❝♦❣♥✐t✐♦♥✳ ❚❤❡② ♦♥❧② ❢✉s❡ ✇✐t❤ ❝❡❧❧s ♦❢ t❤❡ s❛♠❡ s❡❣♠❡♥t t❤❛♥ t❤❡ ❝❡❧❧ ❢r♦♠ ✇❤✐❝❤ t❤❡② ♦r✐❣✐♥❛t❡✱ ❛❧❧♦✇✐♥❣ t♦ ❛❝❤✐❡✈❡ ❛ ♣r♦♣❡r s❡❣♠❡♥t ♠❛t❝❤✐♥❣ ❬✶✷❪✳. • ❚❤❡ ❆❝t✐♥ ❈❛❜❧❡✿ ❛ tr❛♥s❝❡❧❧✉❧❛r str✉❝t✉r❡ ♣r♦❞✉❝❡❞ ❜② t❤❡ ▲❡❛❞✐♥❣. ❊❞❣❡ ❝❡❧❧s✱ ♠❛❞❡ ♦❢ ❛❝t✐♥ ✭❛ ✜❧❛♠❡♥t♦✉s ♣r♦t❡✐♥✮ ❛♥❞ ♥♦♥✲♠✉s❝✉❧❛r ▼②♦s✐♥ ■■ ✭❛ ❝♦♥tr❛❝t✐❧❡ ♣r♦t❡✐♥✮✳ ❚❤❡ ❆❝t✐♥ ❈❛❜❧❡ s✉rr♦✉♥❞s t❤❡ ✇❤♦❧❡ ❞♦rs❛❧ ❤♦❧❡✱ ❛♥❞✱ ❝♦♥tr❛❝t✐♥❣✱ t❡♥❞s t♦ r❡❞✉❝❡ t❤❡ ❤♦❧❡✬s s✉r❢❛❝❡✳. • ❚❤❡ ❆♠♥✐♦s❡r♦s❛✿ ❆♠♥✐♦s❡r♦s❛ ✐s ❛♥ ❡①tr❛✲❡♠❜r②♦♥✐❝ t✐ss✉❡ ✭✐✳❡✳ ❛. t✐ss✉❡ t❤❛t ✇✐❧❧ ♥♦t ❢♦r♠ ❛♥② t✐ss✉❡ ♥♦r ♦r❣❛♥ ✐♥ t❤❡ ❧❛r✈❛✮ t❤❛t ❝♦✈❡rs t❤❡ ❉♦rs❛❧ ❍♦❧❡✳ ❋♦r♠✐♥❣ ❛❝t♦✲♠②♦s✐♥ ❝♦♠♣❧❡①❡s✱ ❆♠♥✐♦s❡r♦s❛ ❝❡❧❧s ♣✉❧❧ ❜♦t❤ s✐❞❡s ♦❢ t❤❡ ❡♣✐❞❡r♠✐s t♦✇❛r❞ t❤❡ ❞♦rs❛❧ ♠✐❞❧✐♥❡✳ ❚❤❡ t✐ss✉❡ ✐s ❡❧✐♠✐♥❛t❡❞ ❞✉r✐♥❣ t❤❡ ♣r♦❝❡ss t❤r♦✉❣❤ ❛r❡❛ r❡❞✉❝t✐♦♥ ❛♥❞ ❝❡❧❧ ❞❡❛t❤✳. ❚❤♦s❡ str✉❝t✉r❡s ❛♥❞ t✐ss✉❡s ❛r❡ ♦❢ ❣r❡❛t ✐♠♣♦rt❛♥❝❡ ❢♦r t❤❡ ♣r♦♣❡r ❛❝❤✐❡✈❡♠❡♥t ♦❢ ❉♦rs❛❧ ❈❧♦s✉r❡✳ ■♥ t❤❡ ❢♦❧❧♦✇✐♥❣ s❡❝t✐♦♥s✱ ✇❡ ✇✐❧❧ ❞❡t❛✐❧ t❤❡✐r r♦❧❡s ❛♥❞ ❦♥♦✇♥ ❜❡❤❛✈✐♦rs✳ ✶✳✷✳✶. ❚❤❡ ❩✐♣♣✐♥❣. ❚❤❡ ③✐♣♣✐♥❣ ✐s ♠♦st ✐♠♣♦rt❛♥t ✐♥ t❤❡ ❧❛st st❡♣s ♦❢ ❉♦rs❛❧ ❈❧♦s✉r❡✱ ✇❤❡♥ ❡♣✐❞❡r♠❛❧ ❝❡❧❧s ❛r❡ ❝❧♦s❡ ❡♥♦✉❣❤ t♦ ❡❛❝❤ ♦t❤❡r✳ ❊✈❡r② ❝❡❧❧ ♦❢ t❤❡ ▲❡❛❞✐♥❣ ❊❞❣❡ s❡♥❞s ♣r♦tr✉s✐♦♥s✱ ❝❛❧❧❡❞ ✜❧♦♣♦❞✐❛✱ t♦✇❛r❞ t❤❡ ❝❡♥t❡r ♦❢ t❤❡ ❞♦rs❛❧ ❤♦❧❡✳ ❋✐❧♦♣♦❞✐❛ ❤❛✈❡ ❛ ❧✐♠✐t❡❞ r❛♥❣❡✱ ❛♥❞ ❤❡♥❝❡ ♠❛♥❛❣❡ t♦ ❝♦♥♥❡❝t ✇✐t❤ t❤❡ ♦♣♣♦s✐t❡ ▲❡❛❞✐♥❣ ❊❞❣❡ ♦♥❧② ✇❤❡♥ ✐t ✐s ❝❧♦s❡ ❡♥♦✉❣❤✱ ✐✳❡✳ ❛t t❤❡ ❝❛♥t❤✐✱ ♦r.

(27) ✷✹. ❈❍❆P❚❊❘ ✶✳. ■◆❚❘❖❉❯❈❚■❖◆. t♦✇❛r❞ t❤❡ ❡♥❞ ♦❢ ❞♦rs❛❧ ❝❧♦s✉r❡✳ ❚❤❡r❡❢♦r❡✱ ③✐♣♣✐♥❣ ✐s ♠❛✐♥❧② r❡s♣♦♥s✐❜❧❡ ♦❢ t❤❡ ❡②❡✲s❤❛♣❡❞ ❢♦r♠ ♦❢ t❤❡ ❞♦rs❛❧ ❤♦❧❡✿ t❤❡ ❞♦rs❛❧ ❡❞❣❡ ♦❢ t❤❡ ❡♣✐❞❡r♠✐s ✐s ❝♦♥✈❡① ❛t t❤❡ ❡①tr❡♠✐t✐❡s✱ ❛♥❞ ❝♦♥❝❛✈❡ ✐♥ ✐ts ❝❡♥t❡r✳ ❚❤❡ ③✐♣♣✐♥❣ ❛❧s♦ s❡r✈❡ ❛ s❡❝♦♥❞ ❢✉♥❝t✐♦♥✿ s❡❣♠❡♥t r❡❝♦❣♥✐t✐♦♥✳ ■♥❞❡❡❞✱ ❡♣✐❞❡r♠❛❧ ❝❡❧❧s ❤❛✈❡ ✈❛r✐♦✉s ✐❞❡♥t✐t✐❡s✱ ❛♥❞ t❤❡ ❧♦❝❛t✐♦♥ ♦❢ t❤❡s❡ ✐❞❡♥t✐t✐❡s ❢♦❧❧♦✇s ❛ s♣❡❝✐✜❝ s♣❛t✐❛❧ ♣❛tt❡r♥✳ ■t ✐s ✈✐s✐❜❧❡ ♦♥ t❤❡ ❧❛st t❤r❡❡ ♣✐❝t✉r❡s ♦❢ ❋✐❣✉r❡ ✶✳✸ t❤❛t t❤❡ ❡♣✐❞❡r♠✐s ♦❢ t❤❡ ❡♠❜r②♦ ❤❛s ❛r❡❛s s❡♣❛r❛t❡❞ ❜② ❣r♦♦✈❡s✿ t❤❡② ❛r❡ t❤❡ s❡❣♠❡♥ts✳ ❚❤❡s❡ s❡❣♠❡♥ts✬ ❛♣♣❡❛r❛♥❝❡ ✐s ❞✉❡ t♦ ❝♦♠♣❧❡① ❣❡♥❡ ✐♥t❡r❛❝t✐♦♥s ❞✉r✐♥❣ ❡♠❜r②♦❣❡♥❡s✐s✳ ❙❡❣♠❡♥t ❢♦r♠❛t✐♦♥ ❜❡❣✐♥s ✇✐t❤ t❤❡ ❞✐✛✉s✐♦♥ ♦❢ ♠❛t❡r♥❛❧ ❢❛❝t♦rs ❧✐❦❡ ❜✐✲ ❝♦✐❞ ♦r ❝❛✉❞❛❧ ❢r♦♠ t❤❡ ❛♥t❡r✐♦r ❛♥❞ ♣♦st❡r✐♦r ❡①tr❡♠✐t✐❡s ♦❢ t❤❡ ❡♠❜r②♦ ❛♥❞ ✐♥✇❛r❞✳ ❚❤❡s❡ ❢❛❝t♦rs ❛❝t✐✈❛t❡ t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♦❢ ❛ ❣r♦✉♣ ♦❢ ❣❡♥❡s ❝❛❧❧❡❞ ✧●❛♣ ❣❡♥❡s✧ ✭❤✉♥❝❤❜❛❝❦✱ ❦♥✐r♣s ❢♦r ✐♥st❛♥❝❡✮ ❝♦❞✐♥❣ tr❛♥s❝r✐♣t✐♦♥ ❢❛❝t♦rs✳ ❊❛❝❤ ♦❢ t❤❡s❡ ❣❛♣ ❣❡♥❡s ❡♥❝♦♠♣❛ss ✇✐❞❡ ③♦♥❡s ♦❢ t❤❡ ❡♣✐❞❡r♠✐s✱ ❝♦✈❡r✐♥❣ ❛r❡❛s t❤❛t ✇✐❧❧ ❣✐✈❡ r✐s❡ t♦ s❡✈❡r❛❧ s❡❣♠❡♥ts✳ ❚❤❡s❡ ❣❡♥❡s ❛❝t ❛s r❡❣✉❧❛t♦rs ❢♦r ❡❛❝❤ ♦t❤❡r✱ ❛s ✇❡❧❧ ❛s ❢♦r ❛♥♦t❤❡r s❡t ♦❢ ❣❡♥❡s✿ t❤❡ ♣❛✐r✲r✉❧❡ ❣❡♥❡s✳ ❚❤❡ ♣❛✐r✲r✉❧❡ ❣❡♥❡s ✭s✉❝❤ ❛s ❡✈❡♥✲s❦✐♣♣❡❞ ♦r ❤❛✐r②✮ ❛r❡ ♣r❡s❡♥t ✐♥ s❡✈❡♥ str✐♣❡s ♦♥ t❤❡ ❡♠❜r②♦✱ ❡❛❝❤ str✐♣❡ ❝♦✈❡r✐♥❣ ❡✈❡r② ♦t❤❡r s❡❣♠❡♥t✳ ❚❤❡② ❛r❡ t♦ s❡t t❤❡ tr❛♥s❝r✐♣t✐♦♥ ♦❢ ❛♥♦t❤❡r ❝❧❛ss ♦❢ ❣❡♥❡s✿ t❤❡ s❡❣♠❡♥t ♣♦❧❛r✐t② ❣❡♥❡s ✭✇✐♥❣❧❡ss✱ ❤❡❞❣❡❤♦❣✱ ❡♥❣r❛✐❧❡❞✳✳✳✮✳ ▼♦st ♦❢ t❤❡s❡ ❣❡♥❡s ❛r❡ ❡①♣r❡ss❡❞ ✐♥ t❤❡ ❢♦✉rt❡❡♥ ♣r❡✲s❡❣♠❡♥t❛r② str✐♣❡s✳ ❆s t❤❡✐r ♥❛♠❡s s✉❣❣❡sts✱ t❤❡② ❣✐✈❡ ❛ ♣♦❧❛r✐t② t♦ t❤❡ s❡❣♠❡♥ts✱ ✐✳❡✳✱ ❜❡t✇❡❡♥ t❤❡ ❣r♦♦✈❡s s❡♣❛r❛t✐♥❣ ❡❛❝❤ s❡❣♠❡♥t✱ ❞✐✛❡r❡♥t ❝❡❧❧ t②♣❡s ❛r❡ ❢♦✉♥❞ ✐♥ t❤❡ ❛♥t❡r✐♦r ❛♥❞ ♣♦st❡r✐♦r ❤❛❧✈❡s✳ ❚❤❡ r❡s✉❧t ♦❢ t❤✐s ❝❛s❝❛❞❡ ✐s ❛♥ ❡♠❜r②♦ ✇✐t❤ s♣❛t✐❛❧❧② r❡♣❡❛t❡❞ ❡①♣r❡ss✐♦♥ ♣❛tt❡r♥s✱ ❛♥❞ ❝❡❧❧ ✐❞❡♥t✐t✐❡s ❬✶✸❪✳ ❚❤❡r❡❢♦r❡✱ ❞✐✛❡r❡♥t ❝❡❧❧✉❧❛r ✐❞❡♥t✐t✐❡s ❛r❡ ♣r❡s❡♥t ✐♥ t❤❡ ❡♣✐❞❡r♠✐s✱ ✇✐t❤ ❛ s♣❛t✐❛❧❧② ♣❡r✐♦❞✐❝ ♣❛tt❡r♥✳ ■♥ ❛❞❞✐t✐♦♥✱ ❡❛❝❤ s❡❣♠❡♥t ✇✐❧❧ ❤❛✈❡ ❛ ♣❛rt✐❝✉❧❛r ♣✉r♣♦s❡ t♦ ❢♦r♠ t❤❡ ❛❞✉❧t ✢②✳ ❖♥ ♦♥❡ ✇✐❧❧ ❜❡ ❢♦r♠❡❞ t❤❡ ✇✐♥❣s✱ ♦♥ ❛♥♦t❤❡r ♦♥❡ ♣❛✐r ♦❢ ❧❡❣s✳✳✳ ❍❡♥❝❡✱ ✐t ✐s ✐♠♣♦rt❛♥t t❤❛t✱ ✇❤❡♥ ❥♦✐♥✐♥❣ ❜♦t❤ s✐❞❡s ♦❢ t❤❡.