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- EXPERIMENTAL PROCEDURES
Zebrafish pou5f1, also known as pou2, encodes a POU-family transcription factor that is transiently expressed in the prospective midbrain and anterior hindbrain during gastrulation, governing brain development. In the present study, we found that the main regulatory elements reside in the proximal upstream DNA sequence from −2.2 to −0.12 kb (the −2.2/−0.1 region). The electrophoretic gel mobility shift assay (EMSA) revealed four functional octamer sequences that can associate with zebrafish Pou2/Pou5f1. The expression of mutated reporter constructs, as well as EMSA, suggested that these four octamer sequences operate in a cooperative manner to drive expression in the mid/hindbrain. We also identified a retinoic acid (RA) -responsive element in this proximal region, which was required to repress transcription in the posterior part of the embryo. These data provide a scheme wherein pou2/pou5f1 expression in zebrafish embryos is regulated by both an autoregulatory loop and repression by RA emanating from the posterior mesoderm. Developmental Dynamics 237:1373-1388, 2008. © 2008 Wiley-Liss, Inc.
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- EXPERIMENTAL PROCEDURES
The POU family of transcription factors is characterized by a structural motif called the POU domain, which is composed of the POU-specific domain and the POU homeodomain. Members of this family play important roles in many aspects of animal development, including brain formation and neurogenesis (Schonemann et al.,1998). Mouse Oct-3/4, also known as Pou5f1, belongs to class V of the POU family. It is expressed in pluripotent cells, such as inner cell mass (ICM) cells of the blastocyst, primordial germ cells (PGCs), and embryonic stem (ES) cells, and is thought to play pivotal roles in the maintenance of pluripotency (Ovitt and Schöler,1998; Pesce et al.,1998; Niwa,2007). Recently, Niwa and his colleagues showed in a series of experiments, in which Oct-3/4 expression was manipulated by a tetracycline-regulated transgene, that Oct-3/4 is a master regulator of pluripotency that controls lineage commitment (Niwa et al.,2000; Pesce and Schöler,2001).
Zebrafish pou2 was originally identified as a gene for a novel POU transcription factor with structural features of both class III and V factors (Takeda et al.,1994). Similar to Oct-3/4, the Pou2 protein associates with an octamer sequence (ATGCAAAT). Based on its structural characteristics and syntenic relationship, pou2 was recently suggested to be a homologue of Oct3/4/Pou5f1 (Belting et al.,2001; Burgess et al.,2002). Maternally derived mRNA transcripts for pou2 are present in unfertilized eggs, and maternal and/or zygotic expression is widely observed until gastrulation. By the end of epiboly, pou2 expression is restricted to the prospective midbrain and anterior hindbrain, and finally disappears from the brain during early somitogenesis (Takeda et al.,1994; Hauptmann and Gerster,1995). The overall temporal and spatial expression pattern of Oct-3/4 in mice is similar to that of pou2 in zebrafish. Oct-3/4 is also ubiquitously expressed in early embryos, both maternally and zygotically, after which it is restricted to ICM cells, then to epiblast cells, and finally to PGCs alone (Ovitt and Schöler,1998; Pesce et al.,1998).
Genetic studies showed that pou2 is disrupted in the spiel-ohne-grenzen (spg) mutant, which does not develop the midbrain–hindbrain boundary (MHB) /isthmus and cerebellum (Schier et al.,1996; Belting et al.,2001; Burgess et al.,2002). The MHB is a potent signaling center that organizes the development of the midbrain and cerebellum. Its positioning in the neural plate depends on the interaction of otx2 and gbx1/2, which are expressed in the future fore/midbrain and hindbrain, respectively (Wassef and Joyner,1997; Kikuta et al.,2003; Rhinn et al.,2003; Hidalgo-Sanchez et al.,2005). At the expression boundary of these two genes, Fgf8, Pax2, and Wnt1 are independently induced near the end of gastrulation; this event establishes the MHB and leads to the activation of downstream genes such as En and other MHB genes (Nakamura,2001; Rhinn and Brand,2001). This gene cascade, which is conserved in all vertebrates examined thus far, is thought to promote and maintain the formation of the MHB/isthmic region.
The expression of pou2 in the zebrafish embryonic brain overlaps that of pax2a around the MHB. Furthermore, pax2a expression is down-regulated in spg embryos, whereas pou2 expression is not affected in no isthmus embryos, which display a defect in pax2a. These results indicate that pou2 is required for pax2a activation in the prospective MHB (Belting et al.,2001; Burgess et al.,2002). Indeed, the MHB enhancer of mouse Pax2 is recognized by Oct-3/4 (Pfeffer et al.,2002). Meanwhile, spg mutant embryos show normal expression of otx2 and gbx1, suggesting that pou2 operates downstream of these genes and contributes to the establishment of the MHB by means of the direct regulation of pax2a.
In addition to defects in the MHB, spg mutant embryos show abnormal morphology and boundaries of rhombomeres in the hindbrain, indicating that spg/pou2 is also involved in hindbrain segmentation. Indeed, pou2 is transiently expressed in rhombomeres r2 and r4 near the end of epiboly (Hauptmann and Gerster,1995). In the prospective hindbrain, each rhombomere is specified by a combination of Hox genes (Hox code) and additional regulatory genes, such as krox20 and velentino (val)/mafB (Theil et al.,2002; Wiellette and Sive,2003). During the establishment of rhombomeres, pou2 may function upstream of krox20 and val, leading to the formation of a gene network that controls hindbrain segmentation (Hauptmann et al.,2002b).
In contrast, although a great deal is known regarding the role of Oct-3/4 in early mouse embryos and the maintenance of ES/EC cells, its role in brain formation remains unclear. In mouse embryos, Oct-3/4 is broadly expressed in the neural plate, and Oct-3/4 overexpression in zebrafish spg embryos restores MHB development (Schöler et al.,1990; Reim and Brand,2002). Interestingly, it was recently shown that class V genes of chick and Xenopus are expressed in the anterior brain similar to pou2 (Morrison and Brickman,2006; Lavial et al.,2007). These results raise a possibility that class-V POU genes of other vertebrates, including Oct-3/4, are also involved in brain development.
To fully understand a given regulatory network, we must elucidate the role of genes that occupy node positions. In the case of mouse Oct-3/4, several reports identified three regulatory regions (Ovitt and Schöler,1998; Niwa,2007): the proximal promoter, the proximal element (PE), and the distal element (DE). The proximal promoter mediates general activation by means of Sp1/Sp3 and repression by means of retinoic acid (RA). The PE drives Oct-3/4 expression in the epiblast and embryonal carcinoma (EC) cells, whereas the DE directs expression in the ICM, ES cells, and PGCs (Yeom et al.,1996; Niwa,2007). The DE also mediates regulation by the cooperative function of Oct-3/4 and Sox2, allowing for the formation of an autoregulatory loop (Okumura-Nakanishi et al.,2005), which is further suppressed by Cdx2 that is known to operate in the differentiation of the trophoectoderm (Niwa et al.,2005).
Despite the essential role played by pou2 in zebrafish brain formation, however, the regulatory mechanism governing its unique and dynamic expression pattern in the brain region is unknown. We used a combination of reporter analyses and electrophoretic mobility shift assays (EMSA) to identify flanking regulatory DNA sequences and determine their roles in pou2 regulation. Our results suggest that pou2 is regulated by means of a positive autoregulatory loop and RA-mediated repression.