Summary
Summary
One of the first crucial phases in the mammalian life is the early embryonic development,
where essential events and changes take place, necessitating highly regulated mechanisms and
genes. One of the first major events that take place after fertilization is the transition from
maternal to embryonic control, referred to as the maternal to zygotic transition. Both before
and after fertilization the embryonic development is controlled ..................
i
Acknowledgements
Acknowledgements
The experimental work for this master thesis was carried out in the laboratory of ...
I wish to thank ....
Furthermore, I would like to thank ....
ii
List of abbreviations
List of abbreviations
aa
amino acid
BLAST
Basic Local Alignment Search Tool
bp
base pair
ceZFR
C. elegans ZFR
.................................................................................
.................................................................................
ZFR-GFP
construct: fusion protein between ZFR and GFP
iii
List of abbreviations
iv
Table of contents
Table of contents (STARTs on an uneven page number p. 1)
Summary .................................................................................................................................... i
Acknowledgements ...................................................................................................................ii
List of abbreviations ................................................................................................................iii
Table of context......................................................................................................................... v
1
Introduction ....................................................................................................................... 1
1.1
Early development ....................................................................................................... 1
1.2
The maternal-to-zygotic transition............................................................................... 1
1.3
Zinc finger binding proteins ........................................................................................ 1
1.4
ZFR, a zinc finger RNA binding protein ..................................................................... 1
1.4.1
Knowledge about ZFR ......................................................................................... 1
1.4.2
The domains within ZFR ...................................................................................... 2
1.5
Conservation and orthologs functions ......................................................................... 2
1.5.1
Conservation from nematode to man.................................................................... 2
1.5.2
The human ZFR ortholog – interacts with a pre-mRNA splicing activator Error!
Bookmark not defined.
1.5.3
The ZFR ortholog in D. melanogaster – promotes productive splicing............... 2
1.5.4
Rodent ZFR – involved in nucleocytoplasmic shuttling ...................................... 2
1.5.5
ZFR ortholog in C. elegans – involved in axon guidance .................................... 2
1.6
MZT across species and worm as a model and the nervous system ............................ 2
1.6.1
Control of the earliest steps of development in different animal models ............. 2
1.6.2
The nematode C. elegans as a model organism and its nervous system .....Error!
Bookmark not defined.
2
Aim of study ....................................................................................................................... 3
3
Material and Methods ....................................................................................................... 5
3.1
Quantitative PCR ......................................................................................................... 5
3.1.1
Maintenance of N2 strain and harvesting of different worm stages ..................... 5
3.1.2
RNA isolation ....................................................................................................... 5
3.1.3
Reverse Transcription and Quantitative PCR ....................................................... 5
3.2
Localization study of ZFR C. elegans ......................................................................... 5
3.2.1
RNA isolation and RT-PCR ................................................................................. 5
v
Table of contents
3.2.2
Cloning of ZFR_C.elegans into pB.RN3P/eGFP vector ...................................... 5
3.2.3
mRNA preparation ............................................................................................... 5
3.2.4
Collection and Microinjection of embryos and analysis thereof .......................... 5
3.3
RNAi C. Elegans .......................................................................................................... 5
3.3.1
Cloning: generation of constructs for RNAi ........................................................ 5
3.3.2
RNAi feeding and screening ................................................................................ 6
3.4
In situ hybridization ..................................................................................................... 6
3.4.1
4
Generation of in situ hybridization probe and the experiment ............................. 6
Results ................................................................................................................................ 7
4.1
Expression profile of ZFR in C. elegans ..................................................................... 7
4.1.1
Test of primer pairs and their primer efficiency ................................................... 7
4.1.2
Zfr mRNA expression in the developing C. elegans ............................................ 7
4.2
Localization analysis of C. elegans ZFR in mouse embryos ....................................... 7
4.2.1
The C. elegans ZFR localizes to the nucleus and are associated with
chromosomes....................................................................................................................... 7
4.3
4.3.1
Generation of constructs ....................................................................................... 7
4.3.2
Knocking down ZFR by RNAi in wild type strain N2 ......................................... 7
4.3.3
Knocking down ZFR by RNAi in the hypersensitive strain VH715 .................... 7
4.3.4
Phenotype: Axon midline crossing ....................................................................... 7
4.3.5
Phenotype: Axon defasciculation ....................... Error! Bookmark not defined.
4.3.6
Phenotype: cord commissures failed to reach target ............................................ 7
4.3.7
Other phenotypes .................................................................................................. 8
4.3.8
Verification of down-regulation of ZFR .............................................................. 8
4.4
5
RNAi studies in C. elegans .......................................................................................... 7
In situ hybridization ..................................................................................................... 8
Discussion ........................................................................................................................... 9
5.1
Part A studies: relative expression profile and localization of ceZfr ........................... 9
5.1.1
Expression profile of ZFR in C. elegans .............................................................. 9
5.1.2
Localization analysis of C. elegans ZFR in mouse embryos ............................... 9
5.2
Part B: Analysis of the function of ZFR in C. elegans ................................................ 9
5.2.1
Knock-out of Zfr by RNAi in C. elegans in wild type strain N2 ......................... 9
5.2.2
Knock-out of Zfr by RNAi in C. elegans in the hypersensitive strain VH715..... 9
vi
Table of contents
6
Conclusion ........................................................................................................................ 10
7
Future perspective ........................................................................................................... 11
8
References list .................................................................................................................. 12
9
Appendix .......................................................................................................................... 13
9.1
Appendix A – CLUSTALW alignement ................................................................... 13
9.2
Appendix B – Primer design ...................................................................................... 13
9.2.1
Quantitative PCR ................................................................................................ 13
9.2.2
RT-PCR .............................................................................................................. 13
9.2.3
In situ hybridization ............................................................................................ 13
9.3
Appendix C – vector maps......................................................................................... 14
9.3.1
Appendix C1: pTZ57R/T and pTZ57R_ZFR_Celegans_FL ............................. 14
9.3.2
Appendix C2: pB.RN3P/eGFP and pB.RN3P/eGFP-ZFR ................................. 14
9.3.3
Appendix C3: L4440 and RNAi constructs ........................................................ 14
9.4
Appendix D – quantitative PCR ................................................................................ 14
9.4.1
Appendix D1: Primer position overview ............................................................ 14
9.4.2
Appendix D2: Test data of primer pair ZFR1 – undiluted template .................. 14
9.4.3
Appendix D3: Test data of primer pair ZFR2 – undiluted template .................. 14
9.4.4
Appendix D4: Primer efficiency data – ZFR2 – all dilution data ...................... 14
9.4.5
Appendix D5: .................................................................................................... 14
9.4.6
Appendix D6: .................................................................................................... 14
9.4.7
Appendix D7: .................................................................................................... 14
9.4.8
Appendix D8: .................................................................................................... 14
9.4.9
Appendix D9: .................................................................................................... 14
9.4.10
Appendix D10: ................................................................................................... 14
9.4.11
Appendix D11: ................................................................................................... 14
9.4.12
Appendix D12: ................................................................................................... 14
9.4.13
Appendix D13: ................................................................................................... 14
9.4.14
Appendix D14: D ............................................................................................... 14
9.4.15
Appendix D15: ................................................................................................... 14
vii
Introduction
1 Introduction (STARTs on an uneven page number)
1.1 Early development
The beginning of a mammalian life start with the fertilization of an oocyte by a sperm, and
from this point forward the newly formed zygote will undergo enormous and dramatic
changes before it successfully becomes an embryo and subsequently develop into a new
individual. Some will say that this event is a miracle, which there are many reasons for.
Studies indicate that a total of 70% of all human conceptions are lost prior to live birth, where
the majority is lost before a pregnancy have been revealed [1]..............................
1.2 The maternal-to-zygotic transition
The time following fertilization and the first cleavage are crucial for further embryonic
development ........
1.3 Zinc finger binding proteins
One of the most abundant protein families in the human genome are zinc finger proteins (Zfp)
[27-29], .........
1.4 ZFR, a zinc finger RNA binding protein
1.4.1 Knowledge about ZFR
As mentioned above, zinc finger proteins are one of the most abundant classes of proteins in
the mammalian genome and a major class of growth and development regulatory protein [27,
28, 45] .......
...... carried out in the XXX laboratory, see figure 3A, (Doğanlı*, Kjærgaard*, Olsen, Oxvig,
Füchtbauer, and Lykke-Hartmann. ”Developmental Expression and Subcellular Localization of Mouse ZFR
compared to C. elegans and zebrafish ZFR orthologs in Early Development”. Submitted to the
International Journal of Developmental Biology).
1
Introduction
1.4.2 The domains within ZFR
Little is known about the function of ..................
1.5 Conservation and orthologs functions
The knowledge about the function of ZFR ......
1.5.1 Conservation from nematode to man
ZFR is reported to be a highly conserved protein ........
1.5.2 The ZFR ortholog in D. melanogaster – promotes productive splicing
Analogous to the hZFR, D. melanogaster ZFR (Zn72D) was also shown to be .......
1.5.3 Rodent ZFR – involved in nucleocytoplasmic shuttling
In rat hippocampus neurons ZFR seems to have a different role, where .....
1.5.4 ZFR ortholog in C. elegans – involved in axon guidance
In nervous system development, the navigation of axons ....
1.6 MZT across species and worm as a model and the nervous system
As described above we have only seen the tip of the iceberg regarding ............
1.6.1 Control of the earliest steps of development in different animal models
At first, the early embryonic development in commonly used animal models ........
Figure 1: The life cycle of C. elegans. From fertilized egg to adult
hermaphrodite on about 60 hours, through the larva stages L1-L4 (figure
adapted from Altun et al. 2009 [114]).
2
Aim of study
2 Aim of study (STARTs on an uneven page number)
The aim of this thesis is to compare ........................
3
Aim of study
4
Material and Methods
3 Material and Methods (STARTs on an uneven page number)
3.1 Quantitative PCR
3.1.1 Maintenance of N2 strain and harvesting of different worm stages
The Bristol strain N2 was used as a standard wild type strain ...............
3.1.2 RNA isolation
RNA was isolated from worms by using .............
3.1.3 Reverse Transcription and Quantitative PCR
SuperScriptTM III Reverse Transcriptase ...................
3.2 Localization study of ZFR C. elegans
3.2.1 RNA isolation and RT-PCR
RNA total was isolated for a sample containing worms .....
3.2.2 Cloning of ZFR_C.elegans into pB.RN3P/eGFP vector
Preparation of pB.RN3P/eGFP vector [126], see appendix C2......
3.2.3 mRNA preparation
The ZFR-GFP template, to generate in vitro transcribed Mrna .......
3.2.4 Collection and Microinjection of embryos and analysis thereof
F1 (C57BL/6CBA) female were injected with ......
3.3 RNAi C. Elegans
3.3.1 Cloning: generation of constructs for RNAi
Three constructs were generated for specific RNA-meditated interference .....
5
Material and Methods
3.3.2 RNAi feeding and screening
The strains used for the RNAi experiments were N2 .....
3.4 In situ hybridization
3.4.1 Generation of in situ hybridization probe and the experiment
Primers were designed to amplify a 300 bp long fragment .....
.
6
Results
4 Results (STARTs on an uneven page number)
4.1 Expression profile of ZFR in C. elegans
4.1.1 Test of primer pairs and their primer efficiency
Several studies have shown that ZFR is expressed .....
4.1.2 Zfr mRNA expression in the developing C. elegans
Due to the very little is ......
4.2 Localization analysis of C. elegans ZFR in mouse embryos
4.2.1 The C. elegans ZFR localizes to the nucleus and are associated with chromosomes
4.3 RNAi studies in C. elegans
4.3.1 Generation of constructs
Several C. elegans RNAi libraries exist .................... .
4.3.2
Knocking down ZFR by RNAi in wild type strain N2
A handful of phenotypes are reported concerning development ......
4.3.3 Knocking down ZFR by RNAi in the hypersensitive strain VH715
Some studies indicate that there is a decreased ........
4.3.4 Phenotype: Axon midline crossing
The midline crossing defect in the ventral nerve cord phenotype .....
4.3.5 Phenotype: Axon defasciculation
The axon defasciculation in the dorsal nerve cord, is a phenotype ....
4.3.6 Phenotype: cord commissures failed to reach target
The cord commissures failing to reach ......
7
Results
4.3.7 Other phenotypes
As mentioned earlier ventral cord patterning variation ........
4.3.8 Verification of down-regulation of ZFR
Adult hermaphrodites from the different generation ....
4.4 In situ hybridization
A preliminary and during the final part of this thesis, an attempt was ....
8
Discussion
5 Discussion (STARTs on an uneven page number)
In this master thesis the expression profile of .........
5.1 Part A studies: relative expression profile and localization of ceZfr
5.1.1 Expression profile of ZFR in C. elegans
The expression profile of ceZfr mRNA ....
5.1.2 Localization analysis of C. elegans ZFR in mouse embryos
The intracellular distribution of the C. elegans ZFR was analyzed by .....
5.2 Part B: Analysis of the function of ZFR in C. elegans
5.2.1 Knock-out of Zfr by RNAi in C. elegans in wild type strain N2
To gain more knowledge about the function of ZFR in ....
5.2.2 Knock-out of Zfr by RNAi in C. elegans in the hypersensitive strain VH715
Several studies have shown that RNAi ...
9
Conclusion
6 Conclusion (STARTs on an uneven page number)
In conclusion, this study we have shown that ....
10
Future perspective
7 Future perspective (STARTs on an uneven page number)
As mentioned, we only have limited knowledge of the function of ZFR ....
11
References list
8 References list (STARTs on an uneven page number)
1.
2.
Macklon NS, Geraedts JP, Fauser BC: Conception to ongoing pregnancy: the 'black
box' of early pregnancy loss. Hum Reprod Update 2002, 8(4):333-343.
Wang H, Dey SK: Roadmap to embryo implantation: clues from mouse models.
Nat Rev Genet 2006, 7(3):185-199.
12
Appendix
9 Appendix (STARTs on an uneven page number)
9.1 Appendix A – CLUSTALW alignement
9.2 Appendix B – Primer design
9.2.1 Quantitative PCR
Primer pair ZFR1...
9.2.2 RT-PCR
ZFR_BglII_F_Celeg...
9.2.3 In situ hybridization
ZFRpC_NcoI1336_F:
5’-.....
13
Appendix
9.3 Appendix C – vector maps
9.3.1
Appendix C1: pTZ57R/T and pTZ57R_ZFR_Celegans_FL
9.3.2 Appendix C2: pB.RN3P/eGFP and pB.RN3P/eGFP-ZFR
9.3.3 Appendix C3: L4440 and RNAi constructs
9.4 Appendix D – quantitative PCR
9.4.1 Appendix D1: Primer position overview
9.4.2 Appendix D2: Test data of primer pair ZFR1 – undiluted template
9.4.3 Appendix D3: Test data of primer pair ZFR2 – undiluted template
9.4.4 Appendix D4: Primer efficiency data – ZFR2 – all dilution data
9.4.5 Appendix D5: Test data of primer pair ZFR3 – undiluted template
9.4.6 Appendix D6: ................................
9.4.7 Appendix D7: ................................
9.4.8 Appendix D8: ................................
9.4.9 Appendix D9: ................................
9.4.10 Appendix D10: ................................
9.4.11 Appendix D11: ................................
9.4.12 Appendix D12: ................................
9.4.13 Appendix D13: ................................
9.4.14 Appendix D14: ................................
9.4.15 Appendix D15: ................................
14