臺灣博碩士論文加值系統

斑馬魚為一種脊椎模式動物，常被用來研究疾病發育，但是目前對斑馬魚的性別決定以及性腺分化的調控機制仍不清楚。最近有一個轉錄因子Foxl3，被認為會促進稻田魚的卵子形成和抑制生殖細胞分化成為精子。因此想探討Foxl3對斑馬魚的功能與表現。本研究先透過原位雜交法來觀察斑馬魚性腺上foxl3 mRNA的表現，斑馬魚在出生後第八天性腺會開始表現出foxl3，並且持續表現在有經歷過增生的生殖群細胞，然而雄性在出生一個月後性腺的foxl3表現量會大幅下降並且消失，但在雌性卵巢裡的生殖細胞仍保有foxl3訊號，但是當這群細胞進入到減數分裂zygotene I時期之後的過程中foxl3會消失。另外，為了探討foxl3基因的功能，我透過CRISPR-Cas9系統建立出foxl3的突變魚，並且得到四種突變基因型的F1突變魚，初步看到破壞foxl3的F0突變魚和F0突變魚互相交配的F1子代會擁有比較高比例的雄性。還有為了觀察foxl3+生殖細胞的細胞分裂情形，利用Tol2系統來建立出在foxl3+細胞帶有表現EGFP的轉基因魚，初步看到在F0魚的性腺上有綠色螢光的表現。這些結果顯示出斑馬魚的foxl3會參與在生殖細胞分化成卵子的過程。

Zebrafish (Danio rerio) is a vertebrate model animal. It has been widely used studies in development and disease. However, the mechanisms of sex determination and gonadal differentiation in zebrafish still remain unclear. Forkhead box L3 (Foxl3) is a transcription factor that suppresses germ cell from entering spermatogenesis in medaka. Here, I examined the mRNA expression of foxl3 in gonad of zebrafish by in situ hybridization. foxl3 transcripts can be detected in the gonad starting from 8 days post fertilization and continued to be expressed in some proliferating germ cell that divide synchronously forming a cyst in bi-potential gonad. At one month post fertilization, foxl3 signal was declined in testis, but the signal remained in cystic germ cells of ovary. After cystic germ cells entered meiosis, foxl3 signal disappeared after the zygotene stage of prophase meiosis I during oogenesis. To investigate foxl3 function, I generated foxl3 mutant by CRISPR-Cas9 system, and obtained four types of F1 mutants. My initial results indicated that disruption of foxl3 may lead to more male fishes in F0 mutant and F0-incross offspring. To observe the division of foxl3+ germ cells during gonad development, I also generated transgenic fish via Tol2 system which enables EGFP expression under the control of foxl3 promoter. F0 fish appeared to exhibit green fluorescence in the gonad. In summary, these results show that foxl3 of zebrafish may be involved in the differentiation of germ cells into oocytes.

摘要 I
Abstract II
Table of Contents III
List of Tables V
List of Figures VI
Chapter 1 Introduction 1
1.1 Sex determination 1
1.2 Gonadal differentiation by antagonistic signals 2
1.3 Gonadal differentiation involve in gametogenesis 4
Mitosis in oocyte and spermatocyte development 4
Meiosis in oocyte and spermatocyte development 5
The mechanism of meiosis 6
1.4 Stages of oocyte in zebrafish during oogenesis 7
Oogonia (diameter=7-20 μm) 7
Primary growth stage (stage I diameter=7-140 μm) 7
Cortical alveolus stage (stage II diameter=140-340 μm) 7
Vitellogenesis stage (stage III diameter=340-690 μm) 8
Maturational stage (stage IV diameter=690-730 μm) 8
Complete egg (stage V diameter=730-750 μm) 8
1.5 Forkhead box (Fox) family 8
Forkhead box L2 (FOXL2) 9
Forkhead box L3 (Foxl3) 10
1.6 Motivation 11
Chapter2 Materials and methods 12
2.1 Zebrafish and husbandry 12
2.2 Measuring of the body length of zebrafish 12
2.3 cDNA synthesis 12
2.4 Reverse-transcriptional PCR (RT-PCR) 13
2.5 Plasmid cloning 13
2.6 RNA probe synthesis 14
2.7 Whole-mount in situ hybridization 15
2.8 EdU treatment 16
2.9 Generation of knockout fish by CRISPR-Cas9 16
2.10 Capillary Electrophoresis 17
2.11 Generation of Tg(foxl3:EGFP) transgenic fish 17
2.12 Genotyping 17
2.13 Photography 18
2.14 Statistical analysis 18
Chapter 3 Results 19
3.1 foxl2a expresses in gonad during gonadal development 19
3.2 foxl3 expresses in gonad during gonadal development 19
3.3 Sexual dimorphic expression of foxl3 between adult gonads 20
3.4 foxl3 is expressed in proliferating progenitor germ cell 21
3.5 foxl3 is disappeared after germ cell entry into meiosis 22
3.6 Generation of foxl3 knockout zebrafish 23
Evaluation and screening of gRNA in zebrafish 23
Analysis of F1 heterozygous knockout fish 24
3.7 Establishment of Tg(foxl3:EGFP) transgenic zebrafish 25
Proximal promoter region of foxl3 25
Chapter 4 Discussions 26
4.1 Expression of foxl2 and foxl3 in gonads 26
4.2 The expression of foxl3 during gonadal differentiation 27
4.3 The phenotype of Foxl3 knockout fish 28
Future work 30
References 31
Tables 38
Figure legends 43
Figures 48

List of Tables
Table 1. The standard body lengths in zebrafish at different days post fertilization 38
Table 2. The oligonucleotide sequences used in this study 39
Table3. The list of plasmids used in this study 40
Table 4. The duration of proteinase K treatment at different stage zebrafish for in situ hybridization 41
Table 5. The batch of foxl3 knockout fish. 42

List of Figures
Introductory Fig. I1. Oocyte development in vertebrates. 48
Introductory Fig. I2. Phase of meiosis and gametogenesis 49
Introductory Fig. I3. Gametogenesis in mammals between male and female. 50
Introductory Fig. I4. Stages of early oocytes in zebrafish. 51
Introductory Fig. I5. Stages of oocyte development. 52
Introductory Fig. I6. Domain organization of Foxl1, Foxl2 and Foxl3 in zebrafish. 53
Introductory Fig. I7. Phylogenetic tree of Foxl family in different species. 54

Method Fig. M1. Generation of GFP+: gSAlzGFFM789A transgenic fish by gene trap 55
Method Fig. M2. Maps of foxl2, foxl3, nanos2 and egfp plasmids. 56
Method Fig. M3. pT7-gRNA construct. 57
Method Fig. M4. foxl3 transgenic construct. 58

Fig. 1. The expression of foxl2 appears in gonad of TL strain from 8dpf. 59
Fig. 2. foxl3 expression pattern in TL and Nadia strain. 60
Fig. 3. foxl3 consistently expresses in ovary instead of testis. 61
Fig. 4. foxl3 is expressed in a subset of pre-meiotic germ cell. 62
Fig. 5. foxl3+ germ cells are in proliferating cyst. 63
Fig. 6. foxl3 disappears after the leptotene stage of meiosis I prophase. 64
Fig. 7. Generation of foxl3 knockout fish. 65
Fig. 8. Detection of green fluorescence in foxl3:EGFP transgenic fish. 66
Fig. 9. Expression of foxl3 in zebrafish during germ cell development. 67

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