A STUDY OF THE RECOMBINATION ACTIVATING GENE 1
IN THE ZEBRAFISH NERVOUS SYSTEM
FENG BO
A THESIS SUBMITTED FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
TEMASEK LIFE SCIENCES LABORATORY
NATIONAL UNIVERSITY OF SINGAPORE
2006
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ACKNOWLEDGMENTS
I would like to thank my supervisor, Dr Suresh Jesuthasan. Without his constant support
and guidance over these years, this dissertation would not have been possible. His
patience and encouragement carried me on through difficult times, his insights and
suggestions helped to shape my research skills, and his valuable feedback contributed
greatly to this dissertation.
I thank my thesis committee members: Dr. Vladimir Korzh, Dr. Patrick Tan and Dr. Wen
Zilong. Their valuable feedback helped me to improve this study in many ways.
I am grateful to Dr. Ding Shouwei, Dr. Liu Dingxiang for their guidance during the
rotation period in my first year in IMA. In their labs I touched and learnt a lot of
molecular techniques and knowledge that are very helpful to the work described in my
thesis.
Many of my thanks also go to my friends who have given me various help during my
graduate career. They are Mahendra Wagle, Cristiana Barzaghi, Caroline Kibat, Sylvie Le
Guyader, Jasmine D'souza, Micheal Hendricks and Sarada Bulchand. I enjoyed all the
vivid discussions we had on various topics and had lots of fun being a member of this
fantastic group.
Last but not least, I thank my family for their understanding and supporting through all
these years.
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TABLE OF CONTENTS
Title page

2.6.2 Immunofluorescence on cryo-sectioned tissue 27
2.6.3 Immunofluorescence on neurons from retina 28
2.6.4 Immunofluorescence on olfactory neurons 28
2.6.5 Immunofluorescent labeling of glomeruli 29
2.7 in situ hybridization 29
2.7.1 Probe synthesis 29
2.7.2 Whole-mount in situ hybridization 29
2.7.3 TSA modification 31
2.8 Microinjection 31
2.9 PCR 32
2.10 Electrophoresis 32
2.11 Electroporation 33
2.12 Storage of glyceral stock 34
2.13 Genotyping 34
2.13.1 Genotyping of the Rag1 mutant zebrafish 34
2.13.2 DNA isolation from individual embryos 34
2.13.3 DNA isolation from clipped caudal fins 35
2.13.4 Allele-specific PCR 35
2.13.5 Direct sequencing from PCR products 36
2.14 RNA isolation: 36
2.15 RT-PCR 37
2.15.1 DNase I treatment 37
2.15.2 First strand cDNA synthesis 38
2.15.3 Semi-quantitative RT-PCR 38
2.15.4 5’ RACE for 12158 38
2.15.5 Real-time RT-PCR 39
2.15.6 RT-PCR with DEG kit 39
2.16 Microarray 40
2.16.1 Construction and hybridization of the zebrafish microarray 40
2.16.2 Microarray data analysis 41

3.6.1 Depletion of RAG1 doesn’t affect the axon targeting of the
GFP positive OSNs 85
3.6.1.1 Effect of knocking-down RAG1 by morpholinos 85
3.6.1.2 Analysis of zebrafish Rag1 mutant 89
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3.6.2 No other neuronal defect was detected in Rag1 mutant fish 92
3.7 Conclusions 94
CHAPTER 4 RESULTS_PART 2 Searching for Rag1
Downstream Genes in the Nervous System by Microarray
4.1 Two sets of microarray experiments were done to search for
Rag1-downstream genes in the nervous system 96
4.2 Data normalization and statistical analysis 97
4.2.1 Data preprocess and normalization 97
4.2.2 Statistical significance analysis 104
4.3 Interpretation of the adult OE microarray result 109
4.3.1 Expression alteration in the Rag1 mutant fish was detected
at different regulation levels. 111
4.3.2 Innate immunity was largely up-regulated in the Rag1 mutant fish 113
4.3.3 Expression of a large group of neuronal genes decreased
in the Rag1 mutants 118
4.3.4 Other alterations in the Rag1 mutant fish 120
4.3.5 Summary 123
4.4 Characterization of 12158, a candidate downstream gene of Rag1 125
4.4.1 Two versions of 12158 were cloned 125
4.4.2 12158B might be evolved from transposition of a LINE element
in the 12158A allele 128
4.4.3 The two versions of 12158 are two alleles in the same locus 133
4.4.4 12158 transcript is down-regulated in Rag1 mutant fish 136
4.5 Summary 136
CHAPTER 5 DISCUSSION

neuronal genes 157
5.4.1.2 Gene expression beyond the tissue restriction 159
5.4.2 Microarray with zebrafish 159
5.5 Abundant polymorphism in zebrafish genome 160
5.5.1 Abundant nucleotide sequence polymorphism revealed by
GeneFishing technology 160
5.5.2 A repetitive element generated polymorphism was found in
12158 locus 161
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5.6 Overall conclusion 161
REFERENCES 164
APPENDIXES
1. Solutions 186
2. Primers for Rag1 and Rag2 genes 188
3. Primers for general use 189
4. Primers for 12158 and MHC Class I genes 190
5. The 341 significants in adult OE microarray 191
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SUMMARY
Rag1 (recombination activating gene 1) plays a key role in V(D)J recombination and
vertebrate adaptive immunity. Besides immune organs, Rag1 transcripts have also been
detected in the nervous system of vertebrates, where its function is not known. To
investigate whether Rag1 is functional and what role it could play in the nervous system,
we initiated a study with zebrafish.
Firstly, we examined fluorescent transgenic zebrafish with laser scanning confocal
microscopy, to document the expression of Rag1 at single cell resolution.
Using a Rag1:GFP line, we found that Rag1 was selectively expressed in many parts of
the nervous system. The strongest expression appeared in the olfactory system, where
Rag1-driven GFP was restricted only to a subset of microvillous OSNs (olfactory sensory
neurons), which projected their axons to the lateral olfactory bulb. Experiments on RAG1

Table 1. Microarray experiments design. 100
Table 2. Summary of microarray data analysis. 101
Table 3. Expression changes of genes involved in different level of
regulations. 112
Table 4. Expression alteration of immunity-relevant genes in the
Rag1 mutants. 116
Table 5. Expression alteration of neuronal genes revealed in the adult OE
microarray. 121
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LIST OF FIGURES
Figure 1-1. Immunoglobulin (Ig) and T cell receptor (TCR). 2
Figure 1-2. An example of V(D)J recombination: the V-J joining process involved
in making a ț light chain of immunoglobulin in mouse. 3
Figure 1-3. DNA cleavage by RAG proteins. 6
Figure 1-4. NHEJ proteins repair and join RAG-liberated coding and signal ends. 7
Figure 1-5. Schematic representation of murine Rag locus and full-length RAG
proteins. 9
Figure 3-1. Rag transcripts were detected in the zebrafish early embryo. 44
Figure 3-2. Expression of Rag-driven reporters in the zebrafish early embryo. 46
Figure 3-3. Rag genes were detected in the nervous system of zebrafish larvae
by RT-PCR and in situ hybridization. 48
Figure 3-4. The organization of zebrafish olfactory system. 52
Figure 3-5. Expression of Rag1-driven GFP in the embryonic zebrafish olfactory
system. 54
Figure 3-6. Expression of Rag1-driven GFP in the adult zebrafish olfactory
system. 56
Figure 3-7. The DiI/Di8-labeled olfactory system of Rag1:GFP fish. 57
Figure 3-8. Rag1:GFP-positive OSNs do not express OMP. 59
Figure 3-9. Expression of G alpha subunits in larval olfactory neurons. 60
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Figure 4-5. The expression changes of the 341 significants in wt and
Rag1 mutants. 107
Figure 4-6. SAM analysis result for the adult OE microarray. 108
Figure 4-7. A summary of the 341 significants produced in ANOVA analysis
from the adult OE microarray. 110
Figure 4-8. The distribution of immune genes and neuronal genes in the
341-gene tree. 124
Figure 4-9. 5’ RACE of clone 12158. 126
Figure 4-10. Clone 12158B matches to BAC clone CH211-206E6. 129
Figure 4-11. The 5’ part and 3’ part of 12158B are unequally transcribed. 131
Figure 4-12. 12158A matches to the flanking regions of the CR1-1 element in
BAC CH211-206E6. 132
Figure 4-13. Blast result of the CR1-1 and flanking regions. 134
Figure 4-14. 12158A and 12158B are single-copy alleles and locate in the
same locus of zebrafish genome. 135
Figure 5-1. Over expression of Rag1 in early zebrafish embryo. 154
Figure 5-2. Polymorphism revealed by the GeneFishing DEG kit. 162
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LIST OF ABBREVIATIONS
A/P Anterior/posterior
abcb3 ATP-binding cassette, subfamily B member 3
ACP Annealing Control Primer
ANOVA Analysis of Variance
AOB Accessory Olfactory Bulb
AP Alkline Phosphatase
ATM Ataxia Telangiectasia Mutated Protein
BAC Bacterial Artificial Chromosome
BBB Brain blood barrier
Bcl B-cell leukemia/lymphoma 1
BSA Bovine Serum Albumin

GFP Green Fluorescent Protein
HMG1/2 High-Mobility-Group Protein 1/2
hpf Hours post fertilization
HRP Horseradish peroxidase
HuC Hu protein C
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Ig Immunoglobulin
IgH Immunoglobulin heavy chain
IgL Immunoglobulin light chain
INL Inner Nuclear Layer
IPTG Isopropyl-1-thio-ȕ-D-galactoside
Ku70/80 70 and 80 kD subunits of Ku antigen
LacZ Bacterial ȕ-galactosidase
LCR Locus Control Region
LG Linkage Group
log Logarithm
LOWESS Locally weighted scatterplot smoothing normalization
MHC Major Histocompatibility Complex
MOE Main Olfactory Epithelium
mRNA Messenger RNA
NCBI National Center for Biotechnology Information
NHEJ Non-Homologous DNA End Joining
NWC "very interesting" in Polish
OB Olfactory Bulb
°C Degree Celsius
OCAM Olfactory cell adhesion molecule
OE Olfactory Epithelium
OMP Olfactory Marker Protein
ONL Outer Nuclear Layer
OR Odorant Receptor

SSC Sodium chloride-sodium citrate buffer
SV2 Synaptic vesicle-2 protein
TAP2B Transporter associated with antigen processing 2 b
TCR T-cell receptor
TCRĮ T-cell receptor Į chain
TCRȕ T-cell receptor ȕ chain
TdT Terminal deoxynuleotidyl transferase
TIGR The Institute for Genomic Research
Tris Tris (hydroxymethyl) aminomethane
Tris-HCl Tris hydrochloride
TRPC2 Transient receptor potential channel C2
TSA Tyramide signal amplification
TUNEL Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick
End Labeling
UAS Upstream Activating Sequence
UTR Untranslated region
V2R Vasopressin type 2 receptor
VNO Vomeronasal Organ
wt Wild type
X-gal 5-bromo-4-chloro-3-indolyl-ȕ-D-galactoside
XRCC4 X-ray repair complementing defective repair in Chinese
hamster cells 4
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PUBLICATIONS
Feng, B., Bulchand, S., Yaksi, E., Friedrich, R. W. and Jesuthasan, S. (2005). The
recombination activation gene 1 (Rag1) is expressed in a subset of zebrafish
olfactory neurons but is not essential for axon targeting or amino acid
detection. BMC Neurosci 6, 46.
Feng, B., Schwarz, H. and Jesuthasan, S. (2002). Furrow-specific endocytosis during
cytokinesis of zebrafish blastomeres. Exp Cell Res 279, 14-20.

cells and T cells rearrange specifically the immunoglobulin and T cell receptor genes
respectively. The assembly of TCRȕ genes happens earlier than TCRĮ genes during T
cell development; IgH genes are assembled before IgL genes in developing B cells
(Bassing et al., 2002). And, all of the rearrangements occur in the context of allelic
exclusion. For example, a mature B cell expresses only one of its two IgH and one of its
multiple IgL alleles (Gorman and Alt, 1998). This ensures that any mature T cell or B cell
expresses only one type of antigen receptor.
V(D)J recombination is targeted by specific recombination signal sequences (RSSs) that
lie adjacent to each gene segment. These RSSs consist of conserved heptamer (consensus
5’-CACAGTG) and nonamer elements (consensus 5’-ACAAAAACC) separated by a
poorly conserved 12 or 23 nucleotides spacer. According to the length of its non-
conserved spacer, an RSS is referred as 12-RSS or 23-RSS. Efficient V(D)J
recombination take place only between a 12-RSS and a 23-RSS, a phenomenon known as
the 12/23 rule (Fig. 1-3) (Fugmann et al., 2000; Gellert, 2002).
The process of V(D)J recombination can be considered as two phases, cleavage and
joining. In the first phase, the two RSSs are recognized by the recombination machinery
and form the synaptic complex, where DNA is cleaved precisely between the RSSs and
their flanking coding element. In this process, the recognition of two RSSs and the
cleavage of double strands DNA are mainly processed by RAG1 and RAG2 proteins;
high-mobility-group protein 1 and 2 (HMG1/2) enhance the formation of synapsis and
DNA cleavage. To cleave the DNA, RAG proteins bind to both RSSs and introduce a
nick precisely at the 5’ border of the heptamer of each RSS. This leads to the exposure of
5
a 3’-hydroxyl group on the coding flank, which subsequently attacks a phosphodiester
bond on the other DNA strand and produces a covalently sealed hairpin coding end. The
other side of the break remains as 5’ phosphorylated blunt end, which terminates in the
heptamer of the RSS and is referred as the signal end (Fig. 1-3) (Gellert, 2002). The
second phase is a joining phase. Initially the four RAG-liberated DNA ends remain
associated with RAG in a stable post-cleavage synaptic complex (PSC), which is
important for coupling the cleavage and joining stages of V(D)J recombination (Ramsden