Cellular and cis-regulation of En-2 expression in the mandibular arch.

Investigations into early muscle development have focused primarily on somite derived cells. Cranial mesoderm does not undergo somitogenesis, and muscle formation in this region is less well understood. In the present study, we have focused upon the expression of engrailed in mandibular arch myoblasts. We demonstrate that En-2 is expressed in mandibular arch myoblasts of the mouse. The activity of the En-2 enhancer is maintained in several functionally related muscles that arise from the first arch. Through the use of reporter transgenics, we demonstrate that local cell-cell interactions are important in maintaining En-2 expression in the mandibular arch cells. En-2 enhancer activity in the first arch requires a combination of cis-acting sequences that includes a motif which is identical to one found in the Otx2 enhancer and which is sufficient to direct expression in the first arch. These data support the notion that cranial muscle development is regulated by local cell-cell interactions which distinguish distinct anatomical and functional muscle groups.     

Vertical regulation of En-2 expression and eye development by FGFs and BMPs

The formation of the midbrain region depends mainly on the activity of a signalling center located in the isthmus region, on the border between the prospective mesencephalon and metencephalon. FGF-8 has been proposed as a signalling molecule responsible for this specification because of its expression pattern and its ability to elicit duplication of the midbrain region when expressed ectopically in the neuroepithelium. Here we present evidence that members of the FGF family of growth factors when released in the cephalic mesenchyme are able to extend the expression of the mesencephalic marker En-2 to both the anterior and the posterior regions of its original landmark. This alteration in the expression pattern of En-2 is not accompanied by a significant alteration in the later development of the midbrain-cerebellar anlage, although the eye development is severely altered. Members of the bone morphogenetic protein family ectopically released from the mesenchyme down-regulate the expression of En-2 and also have an effect on the development of the eye. These results demonstrate that growth factor molecules produced in the mesenchyme (vertical signalling) participate in the correct establishment of the antero-posterior patterning of the cephalic nervous system during development.     

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.     

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.     

Histone genes from Xenopus laevis: molecular cloning and initial characterization

Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris. The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E. coli plasmid pCR1. A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA. A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII. The fragment was not cleaved by KpnI, AvaI, SalI and HindIII. Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA. The gene order was found to be H3, H4, H2A and H2B. The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence. Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.     

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.     

Molecular cloning, expression and in vitro functional characterization of Myb-related proteins in Xenopus.

Two cDNAs encoding Myb-related proteins have been cloned from Xenopus laevis and they have been termed Xmyb1 and Xmyb2. The Xmyb1 cDNA clone codes for an open reading frame of 733 amino acids and exhibits a high degree of similarity over the entire predicted protein sequence with the human B-Myb protein. Xmyb2 is a partial cDNA clone encoding three copies of amino-terminal tandem repeat elements typical for the Myb DNA-binding domain. The predicted protein sequence is most closely related to the human A-Myb gene product. In vitro translation of two deletion mutants of Xmyb1, truncated in the 3'-portion of the open reading frame, results in protein products which cross-react with polyvalent as well as monoclonal antibodies directed against the human c-Myb protein. The same two XMyb1 proteins, which both contain the complete set of aminoterminal repeats, specifically bind to the c-Myb-specific DNA binding sequence as evidenced by electrophoretic mobility shift analysis in vitro. RNA expression profiles of Xmyb1 and -2 are very different from each other; Xmyb1 is present throughout oogenesis and early Xenopus embryogenesis; in adult tissue it is primarily detected in blood. In contrast, Xmyb2 is expressed at only very low levels during oogenesis, not detectable in embryonic RNA preparations, and in adult tissue it is predominantly expressed in testis, with only a very low level seen in blood.     

Developmental expression of Shisa-2 in Xenopus laevis

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.     

Purification and molecular characterization of adenovirus type 2 DNA-binding protein.

An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure.     

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.