本實驗室先前的研究發現在斑馬魚體內有兩型MRF4 (MRF4 a和MRF4 b)存在，兩者蛋白質序列相似度達97%，接著利用mrf4 a和mrf4 b特異性核酸探針進行原位雜交實驗偵測兩型mrf4表現位置發現mrf4 a表現位置在olfactory placode、三叉神經 (trigeminal nerve) 和RB神經元 ;mrf4 b表現位置在體節上。接著我利用核酸類似物胚胎抑制劑 (morpholino)去分別抑制兩型或同時抑制兩型mrf4，再利用各種標定物以了解兩型mrf4在斑馬魚體內分工情形。首先利用肌肉標定物F59、α-actin、tnnt1、tnnt3b，進行免疫螢光染色和原位雜交實驗，結果發現mrf4 a morphant慢肌纖維排列不受影響，α-actin表現正常，mrf4 b被抑制會造成慢肌纖維排列混亂，α-actin表現下降，tnnt1及tnnt3b則有表現下降及表現情形混亂。接著我使用神經標定物aat (anti-acetylated tubulin)、Znp1、α-bungarotoxin、Zn5和Zn12一級抗體對兩型mrf4 morphant進行染色，發現mrf4 a  morphant 上RB 神經元樹突生長數目變少且長度變短，二級運動神經發育受阻礙，而mrf4 b morphant中乙醯膽鹼接受體變少，一級運動神經生長受阻礙。综合以上結果我認為兩型mrf4功能有所不同，其中mrf4 a對於神經生長有較大相關性，而mrf4 b則和肌肉生長有較大相關性。

Our previous study shows that zebrafish has two MRF4 isoforms, MRF4 a and MRF4 b. The two MRF4 proteins exhibited a 97 % identity rate. To detect the spatiotemporal expression pattern of mrf4 a and mrf4 b at mRNA level, we used in situ hybridization. Our data showed mrf4 a expressed in trigeminal nerve, olfactory placode and RB (Rohon-beard) neuron. mrf4 b expressed in somite. To test the biological function of each mrf4, we first used anti-sense morpholino to knockdown mrf4 a and mrf4 b individually.Curved body were observed after knockdown. Then we used muscle markers F59, α-actin、tnnt1、tnnt3b，and neuron markers aat (anti-acetylated tubulin), Znp1, α-bungarotoxin, Zn5, Zn12 to detect morphological change of mrf4 morphant in skeletal muscle, slow muscle, fast muscle, motor neuron, innervations, acetylcholine receptor cluster and RB neuron. Our data shows mrf4 a knockdown led to RB neuron dendrite loss, motor neuron innervation, mrf4 b knockdown led to misalignment of slow muscle, α-actin, tnnt1, tnnt3b down reglation, secondary motorneuron defect. On the basis of our observations, we suggest that mrf4 a expression is involved in neuron development, mrf4 b expression is involved in  muscle development.

目錄

中文摘要----------------------------------------------------------------------------I
英文摘要---------------------------------------------------------------------------II
目    錄--------------------------------------------------------------------------III
圖表目錄--------------------------------------------------------------------------V
第一章 前言-----------------------------------------------------------------------1
第二章 材料與方法--------------------------------------------------------------7
    第一節 斑馬魚飼養及胚胎收集(Danio rerio)-------------------------8
    第二節 勝任細胞(Competent cell)製作及大腸桿菌之電轉型  
           (electroporation) --------------------------------------------------8
    第三節 小量質體萃取-----------------------------------------------------8
    第四節 胚胎固定及脫水--------------------------------------------------9
    第五節 morpholino knockdown實驗-----------------------------------9
    第六節 斑馬魚total RNA萃取-----------------------------------------10
    第七節 逆轉錄聚合酶鏈鎖反應---------------------------------------10
    第八節 Digoxigenin(DIG)標定核酸探針製作-----------------------10
    第九節 胚胎原位雜交---------------------------------------------------11
    第十節 免疫螢光染色---------------------------------------------------12
    第十一節 α-bungarotoxin (α-BTX)及Znp-1雙染色----------------12
    第十二節 冷凍切片------------------------------------------------------13
    第十三節 顯微照相系統------------------------------------------------13

第三章 結果---------------------------------------------------------------------14
    第一節 斑馬魚與其他脊椎動物MRF4ㄧ級結構分析及親緣關係
--------------------------------------------------------------------------------15
    第二節 以胚胎原位雜交(whole-mount in situ hybridization )偵測mrf4在斑馬魚體內生性表現時空分佈---------------------15
第三節 Mrf4 morpholino設計並以RT-PCR (reverse transcription
polymerase chain reaction) 確定mrf4 morpholino功能---------------------------------------------------------------------16
    第四節 各種Mrf4 morphant 於一般光源下生長型態-------------17
    第五節 不同濃度下各種mrf4 morpholino注射後對斑馬魚存活
           及畸形比例影響------------------------------------------------18
    第六節 mrf4表現下降對斑馬魚骨骼肌α-actin表現造成影響---19
    第七節 mrf4 morphant的tnnt 1表現情形----------------------------20
    第八節 mrf4 morphant的tnnt 3b表現情形---------------------------20
    第九節 mrf4 knockdown對慢肌纖維排列造成影響---------------22
    第十節 mrf4 morphant對RB(Rohon-Beard)神經元細胞樹突投射 
           (RB neuron peripheral process)造成影響-------------------22
    第十一節 mrf4 morphant一級運動神經與肌肉接點染色---------24
    第十二節 mrf4 morphant之二級運動神經染色---------------------26
第四章 討論---------------------------------------------------------------------27
    第一節 斑馬魚mrf4 alternative splicing假說------------------------28
    第二節 斑馬魚兩型mrf4時空分佈表現的不同---------------------28
    第三節 斑馬魚兩型mrf4表現與慢肌纖維生長發育的關係------29
    第四節 斑馬魚兩型mrf4在神經生長發育上造成不同影響------29

參考文獻--------------------------------------------------------------------------31
圖表--------------------------------------------------------------------------------37
附錄--------------------------------------------------------------------------------56
圖表目錄

Fig 1. 與其他脊椎動物MRF4胺基酸序列比對結果--------------------38
Fig 2. 斑馬魚與其他脊椎動物MRF4胺基酸序列演化樹--------------39
Fig 3. 斑馬魚mrf4表現時空上的分佈--------------------------------------40
Fig 4. 藉由反轉錄聚合酵素反應偵測斑馬魚mrf4剪接改變-----------43
Fig 5. 野生種與各種不同mrf4 morphant在可見光下觀察受精後36小時的胚胎型態-----------------------------------------------------------44
Fig 6. 野生種及各種mrf4 knockdown斑馬魚胚胎α-actin原位雜交染色--------------------------------------------------------------------------45
Fig 7. 野生種及各種mrf4 knockdown斑馬魚胚胎Tnnt 1原位雜交染色--------------------------------------------------------------------------46
Fig 8. 野生種及各種mrf4 knockdown斑馬魚胚胎Tnnt 3b原位雜交染色--------------------------------------------------------------------------47
Fig 9. 野生種及各種mrf4 knockdown胚胎F59免疫螢光染色，於斑馬魚胚胎受精後36小時慢肌纖維缺陷----------------------------48
Fig10. 各種mrf4 morphant aat成熟神經染色-----------------------------49
Fig 11. 各種mrf4 morphant Zn12 RB神經元染色------------------------ 51
Fig 12. 各種mrf4 morphant Znp 1及α-bungarotoxin雙染色，運動神經及運動神經肌肉接點分布情形------------------------------------52
Fig 13. 各種mrf4 morphant Zn 5 二級運動神經染色-------------------53
Table1. 各物種MRF4胺基酸序列相似度比較表-------------------------54
Table2. 注射不同濃度各種mrf4 morpholino後24hpf及36hpf斑馬魚存活率及畸形率統計 -----------------------------------------------55

1.	Couly, G.F., P.M. Coltey, and N.M. Le Douarin, The developmental fate of the cephalic mesoderm in quail-chick chimeras. Development, 1992. 114(1): p. 1-15.
2.	Hacker, A. and S. Guthrie, A distinct developmental programme for the cranial paraxial mesoderm in the chick embryo. Development, 1998. 125(17): p. 3461-72.
3.	Hatta, K., et al., Specification of jaw muscle identity in zebrafish: correlation with engrailed-homeoprotein expression. Science, 1990. 250(4982): p. 802-5.
4.	Noden, D.M., The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am J Anat, 1983. 168(3): p. 257-76.
5.	Noden, D.M., et al., Differentiation of avian craniofacial muscles: I. Patterns of early regulatory gene expression and myosin heavy chain synthesis. Dev Dyn, 1999. 216(2): p. 96-112.
6.	Schilling, T.F. and C.B. Kimmel, Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo. Development, 1994. 120(3): p. 483-94.
7.	Tajbakhsh, S. and M. Buckingham, The birth of muscle progenitor cells in the mouse: spatiotemporal considerations. Curr Top Dev Biol, 2000. 48: p. 225-68.
8.	Rhodes, S.J. and S.F. Konieczny, Identification of MRF4: a new member of the muscle regulatory factor gene family. Genes Dev, 1989. 3(12B): p. 2050-61.
9.	Braun, T., et al., Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12. Embo J, 1990. 9(3): p. 821-31.
10.	Miner, J.H. and B. Wold, Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc Natl Acad Sci U S A, 1990. 87(3): p. 1089-93.
11.	Davis, R.L., H. Weintraub, and A.B. Lassar, Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell, 1987. 51(6): p. 987-1000.
12.	Wright, W.E., D.A. Sassoon, and V.K. Lin, Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell, 1989. 56(4): p. 607-17.
13.	Edmondson, D.G. and E.N. Olson, A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev, 1989. 3(5): p. 628-40.
14.	Braun, T., et al., A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. Embo J, 1989. 8(3): p. 701-9.
15.	Lu, J., et al., MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD. Proc Natl Acad Sci U S A, 1999. 96(2): p. 552-7.
16.	Murre, C., P.S. McCaw, and D. Baltimore, A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell, 1989. 56(5): p. 777-83.
17.	Blackwell, T.K. and H. Weintraub, Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science, 1990. 250(4984): p. 1104-10.
18.	Rudnicki, M.A., et al., Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell, 1992. 71(3): p. 383-90.
19.	Braun, T., et al., Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell, 1992. 71(3): p. 369-82.
20.	Rudnicki M, et al., MyoD or Myf-5 is required for the formation of skeletal muscle. Cell, 1993. 75: p. 1351-1359.
21.	Hasty, P., et al., Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature, 1993. 364(6437): p. 501-6.
22.	Nabeshima Y, et al., Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature, 1993. 364: p. 532-535.
23.	Braun, T. and H.H. Arnold, Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development. Embo J, 1995. 14(6): p. 1176-86.
24.	Patapoutian, A., et al., Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome. Development, 1995. 121(10): p. 3347-58.
25.	Zhang, W., R.R. Behringer, and E.N. Olson, Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes Dev, 1995. 9(11): p. 1388-99.
26.	Patapoutian, A., et al., Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice. Development, 1993. 118(1): p. 61-9.
27.	Saitoh, O., et al., Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene. Nucleic Acids Res, 1993. 21(10): p. 2503-9.
28.	Olson, E.N., et al., Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell, 1996. 85(1): p. 1-4.
29.	Yoon, J.K., et al., Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus. Dev Biol, 1997. 188(2): p. 349-62.
30.	Nasevicius, A. and S.C. Ekker, Effective targeted gene 'knockdown' in zebrafish. Nat Genet, 2000. 26(2): p. 216-20.
31.	Bober, E., et al., The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development. J Cell Biol, 1991. 113(6): p. 1255-65.
32.	Sassoon, D., et al., Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature, 1989. 341(6240): p. 303-7.
33.	Hinterberger, T.J., et al., Expression of the muscle regulatory factor MRF4 during somite and skeletal myofiber development. Dev Biol, 1991. 147(1): p. 144-56.
34.	Summerbell, D., C. Halai, and P.W. Rigby, Expression of the myogenic regulatory factor Mrf4 precedes or is contemporaneous with that of Myf5 in the somitic bud. Mech Dev, 2002. 117(1-2): p. 331-5.
35.	Ott, M.O., et al., Early expression of the myogenic regulatory gene, myf-5, in precursor cells of skeletal muscle in the mouse embryo. Development, 1991. 111(4): p. 1097-107.
36.	Kassar-Duchossoy, L., et al., Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature, 2004. 431(7007): p. 466-71.
37.	Della Gaspera, B., et al., Spatio-temporal expression of MRF4 transcripts and protein during Xenopus laevis embryogenesis. Dev Dyn, 2006. 235(2): p. 524-9.
38.	Gundersen, K., Determination of muscle contractile properties: the importance of the nerve. Acta Physiol Scand, 1998. 162(3): p. 333-41.
39.	DiMario, J.X. and F.E. Stockdale, Both myoblast lineage and innervation determine fiber type and are required for expression of the slow myosin heavy chain 2 gene. Dev Biol, 1997. 188(1): p. 167-80.
40.	Washabaugh, C.H., et al., Role of the nerve in determining fetal skeletal muscle phenotype. Dev Dyn, 1998. 211(2): p. 177-90.
41.	Leberer, E., U. Seedorf, and D. Pette, Neural control of gene expression in skeletal muscle. Calcium-sequestering proteins in developing and chronically stimulated rabbit skeletal muscles. Biochem J, 1986. 239(2): p. 295-300.
42.	Misgeld, T., et al., Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron, 2002. 36(4): p. 635-48.
43.	Yang, X., et al., Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation. Neuron, 2001. 30(2): p. 399-410.
44.	Lin, W., et al., Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature, 2001. 410(6832): p. 1057-64.
45.	Catterall, W.A., From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron, 2000. 26(1): p. 13-25.
46.	Kallen, R.G., S.A. Cohen, and R.L. Barchi, Structure, function and expression of voltage-dependent sodium channels. Mol Neurobiol, 1993. 7(3-4): p. 383-428.
47.	Caldwell, J.H., Clustering of sodium channels at the neuromuscular junction. Microsc Res Tech, 2000. 49(1): p. 84-9.
48.	Awad, S.S., et al., Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effects of muscle activity. J Neurosci, 2001. 21(21): p. 8456-63.
49.	Thompson, A.L., et al., A selective role for MRF4 in innervated adult skeletal muscle: Na(V) 1.4 Na+ channel expression is reduced in MRF4-null mice. Gene Expr, 2005. 12(4-6): p. 289-303.
50.	Weis, J., et al., Denervation induces a rapid nuclear accumulation of MRF4 in mature myofibers. Dev Dyn, 2000. 218(3): p. 438-51.
51.	Zhou, Z. and A. Bornemann, MRF4 protein expression in regenerating rat muscle. J Muscle Res Cell Motil, 2001. 22(4): p. 311-6.
52.	Plaghki, L., Regeneration and myogenesis of striated muscle. J Physiol (Paris), 1985. 80(2): p. 51-110.
53.	Fuchtbauer, E.M. and H. Westphal, MyoD and myogenin are coexpressed in regenerating skeletal muscle of the mouse. Dev Dyn, 1992. 193(1): p. 34-9.
54.	Grounds, M.D., et al., Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes. Cell Tissue Res, 1992. 267(1): p. 99-104.
55.	Kami, K., K. Noguchi, and E. Senba, Localization of myogenin, c-fos, c-jun, and muscle-specific gene mRNAs in regenerating rat skeletal muscle. Cell Tissue Res, 1995. 280(1): p. 11-9.
56.	Marsh, D.R., et al., Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J Appl Physiol, 1997. 83(4): p. 1270-5.
57.	Mendler, L., et al., mRNA levels of myogenic regulatory factors in rat slow and fast muscles regenerating from notexin-induced necrosis. Neuromuscul Disord, 1998. 8(8): p. 533-41.
58.	Rantanen, J., et al., Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: evidence for two different populations of satellite cells. Lab Invest, 1995. 72(3): p. 341-7.
59.	Sakuma, K., et al., The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles. Biochim Biophys Acta, 1999. 1428(2-3): p. 284-92.
60.	Launay, T., et al., Expression and neural control of myogenic regulatory factor genes during regeneration of mouse soleus. J Histochem Cytochem, 2001. 49(7): p. 887-99.
61.	Nicolas, N., et al., Localization of Myf-5, MRF4 and alpha cardiac actin mRNAs in regenerating Xenopus skeletal muscle. C R Acad Sci III, 1998. 321(5): p. 355-64.
62.	Christel, B., et al., Expression of MRF4 protein in adult and in regenerating muscle in xenopus. Dev Dyn, 2003(227): p. 445-449.
63.	Sunyer, T. and J.P. Merlie, Cell type- and differentiation-dependent expression from the mouse acetylcholine receptor epsilon-subunit promoter. J Neurosci Res, 1993. 36(2): p. 224-34.
64.	Jennings, C.G., Expression of the myogenic gene MRF4 during Xenopus development. Dev Biol, 1992. 151(1): p. 319-32.
65.	Nicolas, N., et al., Long-term denervation modulates differentially the accumulation of myogenin and MRF4 mRNA in adult Xenopus muscle. Neurosci Lett, 1999. 277(2): p. 107-10.
66.	Nicolas, N., et al., Neural and hormonal control of expression of myogenic regulatory factor genes during regeneration of Xenopus fast muscles: myogenin and MRF4 mRNA accumulation are neurally regulated oppositely. Dev Dyn, 2000. 218(1): p. 112-22.
67.	Devoto, S.H., et al., Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development, 1996. 122(11): p. 3371-80.
68.	Downes, G.B. and M. Granato, Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability. Dev Biol, 2004. 270(1): p. 232-45.
69.	Svoboda, K.R., A.E. Linares, and A.B. Ribera, Activity regulates programmed cell death of zebrafish Rohon-Beard neurons. Development, 2001. 128(18): p. 3511-20.
70.	Williams, J.A., et al., Programmed cell death in zebrafish rohon beard neurons is influenced by TrkC1/NT-3 signaling. Dev Biol, 2000. 226(2): p. 220-30.
71.	Delalande, J.M. and P.Y. Rescan, Differential expression of two nonallelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss). Dev Genes Evol, 1999. 209(7): p. 432-7.
72.	Tajbakhsh, S., D. Rocancourt, and M. Buckingham, Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature, 1996. 384(6606): p. 266-70.
73.	Venuti, J.M., et al., Myogenin is required for late but not early aspects of myogenesis during mouse development. J Cell Biol, 1995. 128(4): p. 563-76.
74.	Voytik, S.L., et al., Differential expression of muscle regulatory factor genes in normal and denervated adult rat hindlimb muscles. Dev Dyn, 1993. 198(3): p. 214-24.
75.	Adams, L., et al., Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF4 gene expression to long-term muscle denervation. J Cell Biol, 1995. 131(5): p. 1341-9.
76.	Beattie, C.E., Control of motor axon guidance in the zebrafish embryo. Brain Res Bull, 2000. 53(5): p. 489-500.