Hox genes and the regulation of programmed cell death in the embryonic central nervous system of Drosophila melanogaster [Elektronische Ressource] / Ana Rogulja-Ortmann

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

Hox genes and the regulation of programmed
cell death in the embryonic central nervous
system of Drosophila melanogaster




Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften


Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz





Ana Rogulja-Ortmann
Mainz, März 2009
























Dekan:

1. Berichterstatter:
2. Berichterstatter:

Datum der mündlichen Prüfung: 30.04.2009
_ Chapter index

Chapter index

1. Introduction...................................................................................... 1
1.1. Development of the Drosophila central nervous system............................ 1
1.2. Hox genes in CNS development................................................................ 5
1.2.1. Early function of Hox genes 6
1.2.2. Late functions of Hox genes............................... 8
1.3. Programmed cell death.............................................................................. 8
1.4. Aims ......................................... 11

2. Materials and methods.................................................................. 12
2.1. Fly food media ......................................................................................... 12
2.1.1. Fly stock maintenance...................................... 12
2.1.2. Apple juice agar................ 12
2.2. Fly stocks ................................................................. 12
2.3. Genetic crosses....................... 14
2.4. Ectopic gene expression.......................................... 14
2.4.1. The Gal4/UAS system ...................................... 14
2.4.2. The heat shock system..... 15
2.5. Embryo collection..................................................................................... 15
2.6. Heat-shock procedure.............. 16
2.7. Immunohistochemistry............. 16
2.7.1. Antibodies......................................................................................... 16
2.7.2. Fixation of embryos for antibody staining and in situ RNA
hybridization..................... 18
2.7.3. Preparation and fixation of L1 larval CNS......................................... 19
2.7.4. Incubation in antibody solutions........................................................ 19
2.7.4.1. Fluorescent staining.. 19
2.7.4.2. Color staining............................................................................. 20
2.8. RNA in situ hybridization.......... 20
2.8.1. Generation of a reaper riboprobe..................... 21
2.8.2. Hybridization................................................................ 21
2.8.3. Signal detection................ 22
I _ Chapter index

2.9. Image detection and documentation........................................................ 23
2.10. Chemicals and solutions.......................................... 23
2.11. Equipment and software.......................................... 25

3. Results .......................................................................................... 27
A. Programmed cell death in the developing embryonic central
nervous system………………….………….…….……..……………27
3.1. CNS morphology of apoptosis-deficient embryos.................................... 28
3.2. Identification of apoptotic cells in the CNS of wild type embryos............. 31
3.2.1. Markers with broad expression domains.......... 33
3.2.2. Markers expressed in small groups of cells...................................... 35
B. Regulation of apoptosis in identified dying neurons in the
embryonic CNS……………………………………….………………42
3.3. Segment-specific apoptosis of U motoneurons ....................................... 42
3.3.1. Expression of Antennapedia in U motoneurons ............................... 42
3.3.2. Expression of the bithorax complex genes in U motoneurons.......... 43
3.4. Segment-specific apoptosis of the GW and the NB2-4t anterior
motoneuron.............................................................................................. 45
3.4.1. The NB7-3 lineage............................................ 45
3.4.2. The NB2-4t lineage........................................... 50
3.5. Expression pattern of Ultrabithorax in the NB7-3 and NB2-4t lineages... 53
3.5.1. Ubx expression in NB7-3.................................. 53
3.5.2. Ubx and abdA determine segment-specific NB7-3 identity .............. 55
3.5.3. Ubx expression in NB2-4.................................. 58
3.5.4. How is the differential regulation of Ubx in the NB7-3 lineage of
segment T2 achieved?..................................... 58
3.6. Ubx is necessary and sufficient to induce apoptosis in the GW and MNa
motoneurons............................................................................................ 61
3.6.1. Exploring the cell context-specific effect of Ubx ............................... 66
3.7. Initiation of apoptosis is a late function of Ubx......................................... 70
3.8. Antp is necessary and sufficient for survival of the GW motoneuron....... 73
3.9. Ubx prevents Antp from promoting survival of the GW motoneuron........ 77

II _ Chapter index

4. Discussion..................................................................................... 86
4.1. The CNS of apoptosis-deficient embryos does not appear grossly
perturbed ................................................................................................. 87
4.2. Specification of supernumerary neural cells in apoptosis-deficient
embryos... 87
4.2.1. Supernumerary cells cannot be specified as glia ............................. 87
4.2.2. Some supernumerary cells can differentiate into neurons................ 88
4.2.3. On the origins of supernumerary cells in apoptosis-deficient
embryos…………………………………………………………….……..89
4.3. Involvement of Hox genes in developmental apoptosis in the CNS ........ 90
4.3.1. Abdominal-B may be involved in apoptosis of U motoneurons.......... 91
4.4. A dual requirement for Ubx in the development of the NB7-3 and NB2-4
lineages ................................................................................................... 92
4.5. The ability of Ubx to induce apoptosis is context dependent................... 93
4.6. Ubx counteracts Antp to induce programmed cell death ......................... 96
4.7. Hox gene dependent apoptosis as a mechanism for CNS patterning ..... 98

5. Summary..................................................................................... 100

6. References.................................................................................. 102

7. Appendix ..................................................................................... 112
Abbreviation index…………………………………………………...………112
Declaration……………………………………………………...…………….114

III _ Introduction
1. Introduction


The body plan of many animals is composed of groups of homologous
structures (e.g. somites in vertebrates or body segments in the fruitfly Drosophila
melanogaster) that, over the course of development, achieve amazing
morphological and functional diversity along the body axis. The organ with the
highest degree of complexity and cellular diversity is the central nervous system,
as here diverse regional requirements for control of locomotion, respiration,
reproduction etc. need to be fulfilled. Understanding how this morphological and
functional pattern diversity arises during development is one of the fundamental
challenges in biology. We know today that it requires a precisely controlled balance
between cell proliferation, differentiation and death. However, the regulatory
mechanisms controlling these processes and how they are integrated to allow
regional specification are not well understood.
Drosophila melanogaster represents a widely used model organism for
investigations into patterning mechanisms. During my thesis work, I have
attempted to add a small piece to the puzzle of central nervous system patterning
by examining what role programmed cell death, or apoptosis, plays in the
generation of segmental diversity in the Drosophila embryonic central nervous
system, and by investigating which developmental regulators are involved in
controlling it.

1.1. Development of the Drosophila central nervous
system

The nervous system of Drosophila is composed of the central nervous system
(CNS), comprising the ventral nerve cord (VNC)