The canonical Wnt pathway directly regulates NRSF/REST expression in chick spinal cord

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Abstract

Neural precursor cells actively proliferate in the ventricular zone to self-renew the stem cell population, and in parallel, maintain their undifferentiated state. This progenitor pool generates postmitotic cells that migrate to the mantle layer and differentiate into mature neurons. The growth of these stem cells is strictly controlled by the canonical Wnt signaling cascade, in part mediated by the direct regulation of Cyclin D1, a critical regulator of cell cycle progression. Here, we report that the canonical Wnt pathway directly controls the expression of NRSF/REST. The Wnt-activated β-catenin/TCF complex up-regulates this gene through a conserved element found in its exon 1a, a critical result obtained by a novel in ovo transcriptional assay. Hence, our data show that the canonical Wnt signaling cascade directly regulates the NRSF/REST and Cyclin D1 genes, thereby controlling the progenitor cells. In addition, we show that our in ovo transcription assay is a powerful way to analyze gene regulation in a natural in vivo context.

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Materials and methods

cDNA cloning and expression vectors. Full-length cDNAs of mouse Wnt1, β-catenin, and dominant-negative Lef1 were inserted into the pCAGGS expression vector [18]. Mouse full-length Wnt1 cDNA was amplified by RT-PCR based on the published sequence. Mouse β-catenin and Lef1 were gifts from Drs. H. Shibuya and S. Nakagawa, respectively. Chick Wnt1 cDNA was a gift from Dr. M. Wassef and was used for in situ hybridization. Chick Cyclin D1 cDNA was obtained by RT-PCR based on the published sequence.

Expression of the NRSF, Wnt1, and Cyclin D1 genes in developing chick spinal cord

In a search of putative factors that prevent neural differentiation of actively dividing progenitor cells in the chick spinal cord, we found that NRSF is expressed in the VZ (Fig. 1A), as reported in the mouse [12]. Expression of NRSF was not observed in the MZ, where mature neurons reside (Fig. 1A).

Previous studies have identified that dorsal–ventral growth of the spinal cord is controlled by a gradient of Wnt signaling emanating from the dorsal midline of the spinal cord. Among several Wnts,

The canonical Wnt pathway and its direct target, NRSF

Neural stem cells are located in the VZ and actively proliferate to renew the pool of progenitor cells. In parallel, the progenitor pool generates postmitotic cells that leave the VZ and migrate to the MZ to differentiate into neurons [1].

Several factors are known to regulate the growth of the spinal progenitor pool. Wnt1 and Wnt3a are the pivotal factors controlling this process [7]. These Wnt molecules transduce their signals through the canonical Wnt pathway, in which the β-catenin and the

Acknowledgements

We thank Drs. H. Shibuya, S. Nakagawa, M. Wassef, and S. Kitazawa for plasmids and probes. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Science, Sports and Culture of Japan (T.O.), and a Creative Basic Research grant from the Ministry of Education, Science, Sports and Culture of Japan (T.O.).

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