Repressor element-1 silencing transcription/neuron-restrictive silencer factor is required for neural sodium channel expression during development of Xenopus

Abstract

The ability of neurons to fire rapid action potential relies on the expression of voltage-gated sodium channels; the onset of the transcription of genes that encode these channels occurs during early neuronal development. The factors that direct and regulate the specific expression of ion channels are not well understood. Repressor element-1 silencing transcription/neuron-restrictive silencer factor (REST/NRSF) is a transcriptional regulator characterized as a repressor of the expression of NaV1.2, the gene encoding the voltage-gated sodium channel most abundantly expressed in the CNS, as well as of the expression of numerous other neuronal genes. In mammals, REST/NRSF is expressed mostly in non-neural cell types and immature neurons, and it is downregulated on neural maturation. To understand the mechanisms that govern sodium channel gene transcription and to explore the role of REST/NRSF in vivo, we inhibited REST/NRSF action in developing Xenopus laevis embryos by means of a dominant negative protein or antisense oligonucleotides. Contrary to what was expected, these maneuvers result in the decrease of the expression of the NaV1.2 gene, as well as of other neuronal genes in the primary spinal neurons and cranial ganglia, without overt perturbation of neurogenesis. These results, together with the demonstration of robust REST/NRSF expression in primary spinal neurons, suggest that REST/NRSF is required for the acquisition of the differentiated functional neuronal phenotype during early development. Furthermore, they suggest that REST/NRSF may be used to activate or repress transcription of neuronal genes in distinct cellular and developmental contexts.

Fig. 1.

NaV1.2 and REST/NRSF are expressed in neural tissues during development of X. laevis.a–d, ISH for xNaV1.2: expression is restricted to primary spinal neurons (sn), cranial ganglia primordia (cg), and olfactory placodes (op). In a section of a stage 18 embryo, xNaV 1.2 expression is observed in the lateral and ventral regions of the neural tube. a, Dorsal view, stage 18; b, lateral view, stage 18;c, anterior view, stage 24; d, central transversal section, stage 18; n, notochord;m, presomitic mesoderm; e,left, ISH showing diffuse expression of REST during neurulation (stage 18), including neural folds; right, a sense REST probe does not produce significant labeling in a stage 18 embryo; f, at stage 35, REST/NRSF expression is stronger in the anterior neural tissue; g, RT-PCR showing the coexpression of xNaV1.2 and REST/NRSF in dissected tissues at the stages annotated; np, neural plate; nt, neural tube; pm, presomitic mesoderm; s, somites. The constitutively expressed transcript EF1α is shown as a control.

Fig. 2.

DBD-xREST binds specifically to the RE-1 and inhibits xNaV1.2 expression. a, Electrophoretical mobility shift assay analysis of the complex formation by the use of protein extracts of noninjected embryos (lanes 1–2) and embryos injected with the DBD-xREST RNA (lanes 3–9). Two or 10 μg of total protein was used in each assay. DBD-xREST binds to RE-1 (lanes 3–6). The addition of antibody against the Myc epitope produces the super-retardation of the complex (lanes 7–9). The binding specificity of the complex was evaluated by the addition of 2- or 50-fold excess of cold probe (lanes 5–6, 8–9). The probe alone is shown inLane 10. Arrows indicate the complexes.b, Expression of xREST-DBD inhibits xNaV1.2 expression during neurulation. ISH for xNaV1.2; the injected side is marked by anasterisk. Notice a single line of xNaV1.2 labeling, in contrast with Figure 1a. c, Injection of a morpholino-modified antisense oligonucleotide targeting xREST/NRSF inhibits xNaV 1.2 expression at the time of neurulation.d, Detail of a noninjected embryo (top panel) and embryos injected with xREST-DBD RNA (middle panel) or the antisense oligonucleotide (bottom panel) in one of two cells.

Fig. 3.

xREST-DBD decreases the expression of other neuronal genes but does not perturb neural induction.a–c, ISH for N-tubulin; the injected side is marked by an asterisk; b, both lateral views;c, anterior view of the same embryo, where thearrowheads signal the label of the primary spinal neurons (sn) and cranial ganglia (cg).d, xREST-DBD represses SCG10 expression.Doublearrowheads signal the expected normal position of N-tubulin or SCG10 expression. The spotted labeling in the injected side (a) and thediffuse hue in c result from β-galactosidase activity staining. e–h, xREST-DBD does not affect neural induction and early differentiation. Expression patterns of Sox2 (e), Slug (f), Hox11L12 (g), and Pax-3 (h). e, h, Anterior views; f, g, dorsal views.