The Experimental Work for This Master Thesis Was Carried out in the Laboratory Of

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 ......

vii

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 ....

vii

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

vii

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 2

1.3 Zinc finger binding proteins 3

1.4 ZFR, a zinc finger RNA binding protein 4

1.4.1 Knowledge about ZFR 4

1.4.2 The domains within ZFR 6

1.5 Conservation and orthologs functions 8

1.5.1 Conservation from nematode to man 9

1.5.2 The human ZFR ortholog – interacts with a pre-mRNA splicing activator 10

1.5.3 The ZFR ortholog in D. melanogaster – promotes productive splicing 11

1.5.4 Rodent ZFR – involved in nucleocytoplasmic shuttling 11

1.5.5 ZFR ortholog in C. elegans – involved in axon guidance 14

1.6 MZT across species and worm as a model and the nervous system 16

1.6.1 Control of the earliest steps of development in different animal models 17

1.6.2 The nematode C. elegans as a model organism and its nervous system 18

2 Aim of study 21

3 Material and Methods 22

3.1 Quantitative PCR 22

3.1.1 Maintenance of N2 strain and harvesting of different worm stages 22

3.1.2 RNA isolation 22

3.1.3 Reverse Transcription and Quantitative PCR 23

3.2 Localization study of ZFR C. elegans 24

3.2.1 RNA isolation and RT-PCR 24

3.2.2 Cloning of ZFR_C.elegans into pB.RN3P/eGFP vector 24

3.2.3 mRNA preparation 25

3.2.4 Collection and Microinjection of embryos and analysis thereof 26

3.3 RNAi C. Elegans 27

3.3.1 Cloning: generation of constructs for RNAi 27

3.3.2 RNAi feeding and screening 28

3.4 In situ hybridization 29

3.4.1 Generation of in situ hybridization probe and the experiment 29

4 Results 30

4.1 Expression profile of ZFR in C. elegans 30

4.1.1 Test of primer pairs and their primer efficiency 30

4.1.2 Zfr mRNA expression in the developing C. elegans 33

4.2 Localization analysis of C. elegans ZFR in mouse embryos 34

4.2.1 The C. elegans ZFR localizes to the nucleus and are associated with chromosomes 34

4.3 RNAi studies in C. elegans 36

4.3.1 Generation of constructs 36

4.3.2 Knocking down ZFR by RNAi in wild type strain N2 37

4.3.3 Knocking down ZFR by RNAi in the hypersensitive strain VH715 37

4.3.4 Phenotype: Axon midline crossing 38

4.3.5 Phenotype: Axon defasciculation 40

4.3.6 Phenotype: cord commissures failed to reach target 41

4.3.7 Other phenotypes 42

4.3.8 Verification of down-regulation of ZFR 42

4.4 In situ hybridization 42

5 Discussion 43

5.1 Part A studies: relative expression profile and localization of ceZfr 43

5.1.1 Expression profile of ZFR in C. elegans 43

5.1.2 Localization analysis of C. elegans ZFR in mouse embryos 44

5.2 Part B: Analysis of the function of ZFR in C. elegans 45

5.2.1 Knock-out of Zfr by RNAi in C. elegans in wild type strain N2 45

5.2.2 Knock-out of Zfr by RNAi in C. elegans in the hypersensitive strain VH715 45

6 Conclusion 47

7 Future perspective 48

8 References list 50

9 Appendix 58

9.1 Appendix A – CLUSTALW alignement 58

9.2 Appendix B – Primer design 61

9.2.1 Quantitative PCR 61

9.2.2 RT-PCR 62

9.2.3 In situ hybridization 62

9.3 Appendix C – vector maps 63

9.3.1 Appendix C1: pTZ57R/T and pTZ57R_ZFR_Celegans_FL 63

9.3.2 Appendix C2: pB.RN3P/eGFP and pB.RN3P/eGFP-ZFR 64

9.3.3 Appendix C3: L4440 and RNAi constructs 65

9.4 Appendix D – quantitative PCR 66

9.4.1 Appendix D1: Primer position overview 66

9.4.2 Appendix D2: Test data of primer pair ZFR1 – undiluted template 67

9.4.3 Appendix D3: Test data of primer pair ZFR2 – undiluted template 68

9.4.4 Appendix D4: Primer efficiency data – ZFR2 – all dilution data 69

9.4.5 Appendix D5: 70

9.4.6 Appendix D6: 71

9.4.7 Appendix D7: 72

9.4.8 Appendix D8: 73

9.4.9 Appendix D9: 74

9.4.10 Appendix D10: 75

9.4.11 Appendix D11: 76

9.4.12 Appendix D12: 77

9.4.13 Appendix D13: 78

9.4.14 Appendix D14: D 79

9.4.15 Appendix D15: 80

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 InternationalJournal of Developmental Biology).

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 9: 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]).

Aim of study

2 Aim of study (STARTs on an uneven page number)

The aim of this thesis is to compare ......

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 .....

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 .....

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 ......

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 ....

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 ...

Conclusion

6 Conclusion (STARTs on an uneven page number)

In conclusion, this study we have shown that ....

Future perspective

7 Future perspective (STARTs on an uneven page number)

As mentioned, we only have limited knowledge of the function of ZFR ....

References list

8 References list (STARTs on an uneven page number)

1. 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.

2. Wang H, Dey SK: Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 2006, 7(3):185-199.

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’-.....

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: ......