Cloning and analysing of 5' flanking region of Xenopus organizer gene noggin

    Xenopus organizer specific gene Noggin possesses nearly all the characterestic properties of the action of organizer to specify the embryonic body axis. To analyze how the maternal inherited factors control its expression pattern, we cloned the 5' regulatory region of noggin gene. The 1.5 kb upstream sequense could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragmet is essential for activating noggin expression. We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components.

Key words: Xenopus, organizer specific gene, noggin, regulatory region

    The dorsal blastopore lip of Xenopus early gastrulae plays a key role in specifying the body axis. Transplanting a small piece of dorsal lip into the ventral side of another embryo causes the formation of a secondary axis, resulting in a twinned embryo and thus, the dorsal lip of blastopore is named ``the organizer"[1]. Due to the functional importance of the organizer, intense efforts have been placed on deciphering its molecular properties and in past decades dozens of organizer genes have been identified(reviewed by Heasman J[2]).

    The organizer is progressively induced during blastula stage by maternal inducing signals. It's generally accepted that mesoderm-inducing signals and dorsal determinants are both necessary for the formation of the organizer[2]. The most promising candidates of mesoderm-inducing signals are factors encoded by genes of the members of transforming growth factor-beta (activin, Vg1, BMP-4, nodal related genes etc.) and fibroblast growth factor super families[3],[4]. Increasing evidence also revealed that components of Wnt signaling pathway have been implicated in dorsalization of mesoderm[2]. These two kinds of maternal signals orchestrated in the vegetal dorsal territories of blastula, resulting in the establishment of the organizer. However, how these maternal inherited factors induce the formation of the organizer, in terms of initiating the expression of organizer spesific genes, remains unclear.

    NOGGIN, a secreted factor, is expressed in the organizer region during gastrulation and has nearly all characterestic properties of the organzier, such as in patterning body axis, dorsalizing ventral mesodrm and converting prospective ectoderm to neural ectoderm. It can also induce an imcomplete additional body axis when mis-expressed in ventral marginal zone[5]. Therefore, to analyze the regulation of noggin expression will help greatly in understanding the establishment of the organizer. To this aim, we cloned a 1.5 kb noggin 5' upstream DNA by screening a partial Xenopus genomic library. Reporter gene assays indicated that this genomic sequence was able to direct reporter gene to express in vivo and respond to activin and Wnt signalings. Furthermore, a 229 bp fragment has been identified to be essential for the basal promoter activity by serial deletion analysis.

    Xenopus genomic DNA were digested with EcoRI and fractionated by 0.7 % agarose gel, then recovered according to molecular weight ladder (1-2, 5-6kb, and over than 6 kb. A pair of PCR primers specific to noggin 5' untranslated region (UTR) was used to probe the genomic fragments. Since the positive signal was detected in the 2-3 kb genomic fragments with the highest abundance(see Fig 1A), a special library containing the EcoRI digested 2-3 kb genomic fragments was constructed in pBluescript II SK- vector for further screening. By using the probe amplified from the 5' UTR of noggin cDNA, one positive clone was screened out from about 200,000 transformants. Sequencing result indicated that the positive insert was 2066 base pair covering 516 base pair 5' UTR of noggin cDNA and 1550 bp 5' flanking region. The positive clone was therefore named pSK-N2066. PCR primers specific to noggin 5' cDNA are as follows.

    Functional beta-galactosidase gene was cloned from pCH110 (phamarcia) with BamHI/HindIII (blunted) into pSK-N2066 with BamHI/SmaI. This reporter construct was named 2066-LacZ.

    A 2066 bp long genomic sequence was obtained from a special fractionated genomic library by a noggin 5' UTR specific probe (see details in Materials and Methods). Sequencing indicated that this genomic sequence covered 516 bp of 5' UTR and 1550 bp of 5' flanking region.

    The promoter activity of this genomic fragment was examined by injecting reporter construct 2066-LacZ at 2-cell stage in marginal zone of one cell. In reporter construct 2066-LacZ, the functional betagalactosidase gene was inserted immediately downstream to 2066 bp of genomic fragment. X-Gal staining through gastrula to taibud stages embryos demonstraed that the 2066 bp of noggin genomic fragment was indeed able to direct reporter gene to express in vivo, i.e. the noggin genomic fragment we cloned could respond to in vivo signals. Four injected embryos at late neurula stge were shown in Fig 2, demonstrating the reporter gene was expressed primarily in dorsal territories. 1.5kb noggin5'regulatory
region is able to respone to mesoderm induction signal activin and signals