Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis.

Using the powerful RDA-PCR-technique we could identify a novel Xenopus specific Sox-gene (xSox3) a transcription factor closely related to the sox sub-group B, which contains a HMG box. In normogenesis the xSox3 gene is expressed in the presumptive central nervous system. Furthermore a maternal component is also found in oocytes and in early cleavage stages in the animal hemisphere only. By whole-mount in situ hybridization the first zygotic transcription activities can be detected in the late blastula in the dorsal ectoderm and the dorsal and lateral part of the marginal zone. The expression reaches the highest level atthe late gastrula till the late neurula and fades after stage 30. The expression is restricted from gastrulation onwards to the presumptive brain area and the lens epithelium. Furthermore we could show that the gene is expressed in isolated Spemann organizer with adjacent neuroectoderm. The signal can be suppressed by suramin treatment, which inhibits neural development and causes a shift of dorsal to ventral mesoderm. The treatment of whole embryos with LiCl and UV results in an overexpression or an inhibition of the expression, respectively. In exogastrulae (pseudo-exogastrulae) the gene is expressed in the close vicinity to the endomesoderm only, but not in the distal most part of the ectoderm. This result indicates that it is unlikely that the gene can be activated by planar signals. The gene can also be activated in dissociated gastrula ectoderm without mesodermal or neural inducers. That means that the gene can be expressed in ectodermal cells in a cell autonomous manner.

Spatial and temporal transcription patterns of the forkhead related XFD-2/XFD-2' genes in Xenopus laevis embryos.

Two novel fork head related cDNA sequences, termed XFD-2 and XFD-2', have been isolated from a Xenopus laevis gastrula stage cDNA library. XFD-2 and XFD-2' proteins share 88% sequence identity; a comparison of their fork head domains yields 96% identity. Such close homology suggests that the two genes represent pseudo-allelic variants of a common ancestor and probably arose by the ancient tetraploidization event in this species. Both genes are activated at midblastula transition. Main transcriptional activity is found during blastula and gastrula stages of development; thereafter, there is a gradual decrease of transcripts until somite segregation stages. Whole mount in situ hybridisation of blastula stage embryos reveals that the genes are initially transcribed within the animal hemisphere. Subsequently, we observe their transcription in a circumferential mode along the marginal zone, i.e., within the forming mesoderm. During gastrulation, these cells enter the blastoporus at the ventral, lateral and dorsal sites. At the end of gastrula and during neural stages transcripts are localized within somitogenic mesoderm, notochord, lateral and ventral mesoderm, neural floor plate, spinal cords and in the developing brain.

Suramin prevents transcription of dorsal marker genes in Xenopus laevis embryos, isolated dorsal blastopore lips and activin A induced animal caps.

Suramin, a polyanionic compound which is known to interact with the receptors of growth factors inhibits the expression of dorsal marker genes in whole embryos and isolated dorsal blastopore lips. Suramin also prevents activin A induced dorsalization of animal cap explants from blastula stage embryos, but it simultaneously evokes a shift of the differentiation pattern from dorsal mesodermal structures (notochord, somites) to ventral mesodermal derivatives (mesothelium and erythroid precursor cells). The results are consistent with the assumption that the dorsal vegetal zone (Nieuwkoop center) primarily releases more general/ventral mesodermalization signals. They further suggest a dual role of activin A in early embryogenesis. While the maternal component may contribute to a more general/ventral type of induction, increasing concentrations of the zygotic component along with the activation of primary response genes may contribute to the dorsalization of the organizer.

Localization of a nervous system-specific class II beta-tubulin gene in Xenopus laevis embryos by whole-mount in situ hybridization.

A neural-specific beta-tubulin mRNA is expressed in the developing central nervous system shown by whole-mount in situ hybridization experiments. Of special interest is the fact that from the late blastula (stage 9; Nieuwkoop and Faber, 1967; Hausen and Ribesell, 1991) until the early neurula (stage 13) the signal can be found not only in the presumptive neural plate but also in the presumptive epidermis. Later in development (from stage 13) the specific mRNA becomes restricted to the presumptive brain and spinal cord area. The results are discussed in the context of predisposition and (pre)determination.

The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA- and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function.

The 5'-terminal region of the rat gene for the neuron-specific phosphoprotein, synapsin I, was isolated and sequenced. It comprises 1472 nucleotides (nt) of 5'-flanking sequence, 507 nt of the first exon, and 242 nt of the first intron. A single transcription start site was mapped by primer extension and S1 nuclease analysis. A sequence of 340 nt upstream from the transcription start site and the first exon are G+C-rich and enriched in CpG dinucleotides, resembling a CpG island. The 5'-flanking sequence lacks TATA and CAAT consensus elements but contains a consensus motif for the cAMP-responsive element. Furthermore, we notice two potential consensus motifs which are also found in corresponding positions in the genes for the nerve growth factor receptor and the 68-kDa neurofilament protein. The 5'-terminal region of the human synapsin I gene was also cloned and sequenced. A high degree of sequence conservation between rat and human is found in the upstream 340 nt that coincides precisely with the G+C-rich domain and includes the consensus elements, and throughout the first exon including the untranslated sequence. Sequence conservation is also observed further upstream and at the beginning of the first intron. In a transient chloramphenicol acetyltransferase expression assay, 5'-flanking sequences of the rat synapsin I gene function as strong promoters in neuroblastoma cells, but not in fibroblastoid cells. 225 nt of 5'-flanking sequence and 105 nt of 5'-untranslated sequence are sufficient for cell-type specific transcription in this assay.