Participación del Factor Silenciador Neuronal Restrictivo - Tesis ...

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ABSTRACT The RE-1 silencer of transcription/neural restrictive silencer factor (REST/NRSF) was initially identified as a repressor of neuronal genes containing a 23 base pair conserved motif sequence known as RE-1/NRSE. The expression pattern of the mRNA encoding REST/NRSF and an extensive molecular characterization of its function, led to the view of REST/NRSF as a factor that silences neuronal genes in non-neuronal cell types and immature neuronal precursors. Recently it has been shown that REST/NRSF plays distinct roles in the transit of embryonic stem cells to differentiated neurons, and has also been identified as a tumor suppressor gene, which is <strong>del</strong>eted in human neoplasias. Homozygous <strong>del</strong>etion of the REST/NRSF gene is lethal for mice around embryonic day 10. Examination of these embryos reveals derepression of some neuronal genes in non-neural tissues. Conversely, gain of function of REST/NRSF in developing chick spinal neurons results in repression of the expression of some neuronal genes and defects in axon navigation. In contrast, in Xenopus laevis embryos the interference with XREST/NRSF function from early stages of development does not lead to early lethality and results in repression of neuronal genes at late neurula stage and in the expansion of neural tissue. These results contrast with the body of knowledge that emerged from molecular studies and indicates that XREST/NRSF function is necessary for the expression of neuronal genes. Moreover, these data along with the observed in mouse embryos and in neural stem cells, suggest that REST/NRSF function could be required at early stages of neural development. In this thesis we studied the role of XREST/NRSF during Xenopus development, from the establishment of neuroectoderm to the neuronal differentiation. We find that impairment of XREST/NRSF function in Xenopus embryos from late blastula stage leads to the perturbation of neural tube, cranial ganglia and eye development. The origin of these defects is the abnormal patterning of the ectoderm during gastrulation and early neurulae. In Xenopus 3
laevis the ectoderm is patterned at gastrula stage by the gradient activity of BMP signaling. A high activity of BMP determines epidermis at ventral side, an intermediate neural crests and the lowest activity of BMP signaling at the dorsal side is necessary to determine neural plate. Interference of XREST/NRSF function during the late blastula stage leads to an expansion of the neural markers concomitant with lateral displacement and decrease of the expression of epidermal keratin and neural crest markers. Further, neurogenesis proceeds abnormally, with loss of the expression of proneural, neurogenic and neuronal genes. The interference of REST/NRSF mimics several features associated to a decreased BMP function in the ectoderm and counteracts some effects of Bmp- 4 misexpression. These results indicate that REST/NRSF function is required in vivo for the specification of ectodermic cell fates at least in part through the modulation of BMP signaling. Moreover, we showed that interference of XREST/NRSF function during differentiation of spinal neuronal is associated to an increase in the density of sodium currents; this observation is compatible with a role of this factor in the regulation of gene expression in post mitotic neurons. In conclusion in this thesis we have been able to show that XREST/NRSF participates in different stages of neural development in vivo. At early stages it is required to pattern the dorsal-ventral axis of the ectoderm and later, during neuronal differentiation, it modulates the acquisition of neuronal excitability probably by the regulation of neuronal voltage gated sodium channels. 4
Page 1 and 2: Universidad de Chile, Facultad de M
Page 3 and 4: Quisiera agradecer muy especialment
Page 5 and 6: Hipótesis……………………
Page 7 and 8: 6. Función de XCoREST en el la esp
Page 9 and 10: Índice de figuras Figura 1. Experi
Page 11 and 12: Lista de publicaciones - Olguín P,
Page 13: embriones de Xenopus desde la blás
Page 17 and 18: Posteriormente, en otros vertebrado
Page 19 and 20: 1.3 Inductores neurales y vías de
Page 21 and 22: formación de la placa neural y la
Page 23 and 24: Figura 3. Modelos de inducción neu
Page 25 and 26: destinos dependiendo de su localiza
Page 27 and 28: 16 Figura 4. Modelo de doble gradie
Page 29 and 30: 3.2 Neurogénesis primaria en Xenop
Page 31 and 32: adoptar el destino neural para fina
Page 33 and 34: neuronas ectópicas cuando es sobre
Page 35 and 36: Skarmeta et al., 1996) . Zic2 supri
Page 37 and 38: etrasan la diferenciación neuronal
Page 39 and 40: genes reporteros (ej. lacZ) que con
Page 41 and 42: manifiestan en la desacetilación d
Page 43 and 44: (Ballas et al., 2005) (Figura 11).
Page 45 and 46: corticales aislados y tratados con
Page 47 and 48: 4.2.4 Función de REST/NRSF en neur
Page 49 and 50: 1/NRSE mutado se expresa en gran pa
Page 51 and 52: silvestres. Sin embargo, en estadio
Page 53 and 54: tejido neural, como también en la
Page 55 and 56: participar en la neurogénesis a tr
Page 57 and 58: -Pinzas nº 5 (A. DUMONT y FILS, Su
Page 59 and 60: -MEMPFA (MOPS 0,1 M pH 7,0 EGTA 2 m
Page 61 and 62: -Bromuro de Etidio (GIBCO BRL, Cali
Page 63 and 64: concentración de mRNA a inyectar s
Page 65 and 66:
Recolección de embriones. Para el
Page 67 and 68:
Técnicas de Biología Molecular. A
Page 69 and 70:
histona H3 fosforilada (Upstate) en
Page 71 and 72:
Síntesis de ribosondas para hibrid
Page 73 and 74:
Hibridación in situ (ISH). Los emb
Page 75 and 76:
RESULTADOS 1. Clonamiento del cDNA
Page 77 and 78:
Figura 14. Alineamiento de la secue
Page 79 and 80:
3. Participación de XREST/NRSF en
Page 81 and 82:
Figura 16. La interferencia de la f
Page 83 and 84:
3.2 La interferencia de la función
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74 Figura 17. La interferencia de l
Page 87 and 88:
4. Participación de XREST/NRSF en
Page 89 and 90:
Figura 19. La interferencia de la f
Page 91 and 92:
4.2 La sobreexpresión de XREST/NRS
Page 93 and 94:
4.3 Los fenotipos ectodérmicos aso
Page 95 and 96:
4.4 La sobreexpresión de XREST1 no
Page 97 and 98:
4.5 La interferencia de la función
Page 99 and 100:
4.6 XREST/NRSF participa en la form
Page 101 and 102:
Figura 24. Los fenotipos asociados
Page 103 and 104:
Para poner a prueba esta idea se co
Page 105 and 106:
6. XCoREST participa en el establec
Page 107 and 108:
Figura 26. XCoREST participa en el
Page 109 and 110:
1. XREST/NRSF regula la expresión
Page 111 and 112:
100 2. La interferencia de la funci
Page 113 and 114:
102 actividad de Wnt y FGF inducen
Page 115 and 116:
104 letalidad, probablemente porque
Page 117 and 118:
106 XREST/NRSF neuraliza los explan
Page 119 and 120:
108 mutada en los dominios de fosfo
Page 121 and 122:
110 5. XREST/NRSF modula la adquisi
Page 123 and 124:
112 cuenta de un potencial de acci
Page 125 and 126:
114 al neuroectodermo. La inhibici
Page 127 and 128:
116 embargo, estos resultados deben
Page 129 and 130:
118 Ballas N, Battaglioli E, Atouf
Page 131 and 132:
120 Chitnis AB. (1999). Control of
Page 133 and 134:
122 Godsave SF and Slack JM. (1989)
Page 135 and 136:
124 Kallunki P, Edelman GM & Jones
Page 137 and 138:
126 Luther WH. (1935). Entwicklungs
Page 139 and 140:
128 Oppenheimer JM. (1936b). Transp
Page 141 and 142:
130 Spemann H & Mangold H. (1924).
Page 143 and 144:
132 Wilson PA, Hemmati-Brivanlou A.
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Participación del Factor Silenciador Neuronal Restrictivo - Tesis ...

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