Hildegard Tiedemann

Bone morphogenetic protein 4 (BMP-4), a member of the TGF-β family, in early embryos of Xenopus laevis: analysis of mesoderm inducing activity

We have screened a Xenopus ovary cDNA library using a synthetic oligonucleotide derived from that part of the inhibin beta A sequence, which is highly conserved within the TGF-beta family. Out of several clones yielding autoradiographic signals four turned out to represent Xenopus counterparts to the human bone morphogenetic protein 4 (BMP-4). Each two of the four sequences are nearly identical and probably account for different alleles whereas the two pairs showing 5% divergence may have arisen by genome duplication in this tetraploid species. The amino acid sequence of the Xenopus protein is 80% homologous to the human sequence showing no single exchange within the last 100 amino acids at the C-terminus. This region, which constitutes the main part of the mature, biologically active protein, also exhibits substantial homologies to other representatives of the TGF-beta family, especially to the Drosophila DPPC protein. Transfection of COS-1 cells with the Xenopus BMP-4 sequence under control of the CMV-promoter leads to the secretion of a protein which exhibits mesoderm inducing activity when tested with animal cap explants from Xenopus blastula stage embryos.

Induction of mesodermal tissues by acidic and basic heparin binding growth factors

The inducing activity of two heparin binding growth factors HBGF-1 (prostate epithelial cell growth factor; acidic pI) and HBGF-2 (fibroblast growth factor; basic pI) from bovine brain has been tested on totipotent ectoderm from early amphibian (Xenopus laevis, Ambystoma mexicanum) embryos. Both factors induced, at high concentrations, mostly compact spheres surrounded by a non-epidermal epithelium. When the concentration or time of incubation was reduced, large muscle inductions frequently organized as somites were formed besides endothelial vesicles, mesenchyme and smaller areas of intestine-like epithelium. Further reduction of the concentrations or the time of incubation led to an increase in size and number of endothelium-lined vesicles and of mesenchyme, whereas the induction of muscle decreased. At still lower concentrations the overall rate of inductions decreased. The relationship of the growth factors to the vegetalizing factor from chicken embryos, dilution of which shows a similar shift in induced organs, is discussed. The present and previous experiments suggest that different mesodermal and endodermal tissues are induced by secondary interactions in which additional factors are involved. The induced organs derive from dorsal as well as from ventral mesoderm.

Biological activity of vegetalizing and neuralizing inducing factors after binding to BAC-Cellulose and CNBr-Sepharose

SummaryCovalent binding to bromoacetyl-cellulose inactivates the vegetalizing factor. The bound factor is however still able to form a complex with an inhibitor for the factor. Covalent binding to CNBr-Sepharose likewise inactivates the vegetalizing factor. The neuralizing factor on the other hand is not inactivated when covalently bound to CNBr-Sepharose. When a crude fraction which contains the neuralizng factor as well as the vegetalizing factor is bound to CNBr-Sepharose the vegetalizing activity is greatly decreased whereas the neuralizing activity is not reduced. This suggests that the mechanisms of action of the two factors are quite different. Whereas the vegetalizing factor must be incorporated into the cells, the neuralizing factor interacts with the plasma membrane of competent ectoderm cells.

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity.

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.

A vegetalizing inducing factor isolation and chemical properties

A vegetalizing factor which induces the formation of endodermal and mesodermal organs in amphibian gastrula ectoderm was purified from chicken embryos. Preparative sodium dodecyl sulfate polyacrylamide electrophoresis and gel permeation chromatography on sephadex with different eluants were employed. In buffer containing 6 M urea the molecular weight of the factor was estimated to about 28 000-30 000. In buffer containing sodium dodecyl sulfate (SDS) the factor partially dissociates to smaller polypeptide chains. Because an equilibrium between molecules of different size is established, SDS-containing buffers are not suitable for preparative purposes. In 50%-70% formic acid the factor completely dissociates into smaller peptide chains (Mr about 13 000-15 000). Furthermore, very little absorption of the factor to the gel matrices or glass surfaces is observed in formic acid. The final purification can be achieved by high-performance gel permeation chromatography with glycerolpropyl-treated silica gel as column packing and 50% formic acid as eluant.

The vegetalizing factor A member of the evolutionarily highly conserved activin family

The mesoderm and endoderm inducing vegetalizing factor was partially sequenced after BrCN cleavage. A sequence which is highly conserved in activin A near the C‐terminal end was identified. This shows that the factor belongs to the activin family. The activins are not confined to embryos and gonads, but widely distributed in other tissues like calf kidney and calf liver. Functional aspects are discussed.

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

SummaryEctoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.

Formation of mesodermal pattern by secondary inducing interactions

SummaryA highly purified vegetalizing factor induces endoderm preferentially in amphibian gastrula ectoderm. After combination of this factor with less pure fractions, a high percentage of trunks and tails with notochord and somites are induced. The induction of these mesodermal tissues depends on secondary factors which may act on plasma membrane receptors of the target cells. The secondary factors are probably proteins as they are inactivated by trypsin or cellulose-bound proteinase K. They are not inactivated by thioglycolic acid.The implication of these findings for tissue determination and differentiation in normal development in relation to the anlageplan for endoderm and mesodermal tissues is discussed.

Inducing activity of subcellular fractions from amphibian embryos

SummaryThe homogenate from unfertilized eggs, gastrulae, neurulae and hatched embryos ofXenopus laevis was fractionated by differential centrifugation and subsequent repeated centrifugation on discontinuous sucrose gradients. A high archencephalic-neural inducing activity was found in RNP particles, which were released from the high-speed (“microsomal”) sediment by treatment with EDTA, and in a fraction of heterogeneous small vesicles. The highest archencephalic inducing activity was observed in RNP particles from unfertilized eggs and from gastrulae. RNP particles isolated from hatched embryos had a lower inducing activity. The neuralizing factor can be extracted from the small vesicles with pyrophosphate buffer at pH 8.6, but it is not solubilized with a non-ionic detergent (Triton X 100). The high-speed supernatant from the gastrula homogenate contains soluble neuralizing factor, whereas the supernatant from egg homogenate has a low inducing activity. The plasma membrane fraction (isolated from gastrulae) also has only a low inducing activity. The possible significance of the subcellular distribution of neuralizing factors for the transmission of neuralizing inducer from the mesoderm to competent gastrula ectoderm and the processing of signals which are generated on the plasma membrane of induced cells is discussed.

Über die Induktionsfähigkeit von Microsomen- und Zellkernfraktionen aus Embryonen und Leber von Hühnern☆

Zusammenfassung Das starke Induktionsvermogen 9 Tage alter Huhnerembryonen ist an die Strukturbestandteile der Zellen gebunden. Um zu bestimmen, welche Zellstrukturen die Induktionsstoffe enthalten, wurden Microsomen-, Ribosomen- und Zellkernfraktionen isoliert und ausgetestet. Ausser Huhnerembryonen diente auch Huhnerleber als Ausgangsmaterial (Abb. 1). 1. 1. Die Microsomen aus Huhnerembryonen induzieren in der prasumptiven Bauchepidermis von Triturus alpestris sowohl deuterencephal als such spinocaudal. Aus der Microsomenfraktion nach Behandlung mit Desoxycholsaure abzentrifugierte Ribosomen induzieren stark deuterencephal und daneben schwach archencephal. Die deuterencephalen Induktionen enthielten nicht nur die neuralen Hinterkopforgane Rautenhirn und Gehorblasen, sondern haufig auch zur Hinterkopfregion gehorende mesodermale Organe wie Kopfmuskulatur, kleine Chordastucke und Knorpel. Organe des spinocaudalen Komplexes fehlten jedoch. Der Uberstand, welcher nach dem Abzentrifugieren der Ribosomen verbleibt, induziert ebenfalls stark, jedoch vorwiegend spinocaudal (Tabelle 1a; Abb. 5 und 6). 2. 2. Fraktionen aus Huhnerleber verhalten sich ebenso wie Fraktionen aus 9 Tg. alten Huhnerembryonen (Tabelle 1b). 3. 3. Die Sedimentationsanalyse mit der Ultrazentrifuge zeigte, dass die Ribosomen ein heterogenes Gemisch aus mehreren Komponenten darstellen. Die Ribosomenfraktion aus Huhnerembryonen weist zahlreichere und zum Teil langsamer wandernde Komponenten auf als die entsprechende Fraktion aus adulter Leber (Abb. 3). 4. 4. Durch Behandlung mit Pyrophosphat-Desoxycholsaure (pH 8,5), mit starker alkalischem Pyrophosphat (pH 9,5) oder mit Athylendiamintetraessigsaure konnen die Ribosomen in kleinere Bruchstucke gespalten werden, deren Induktionswirkung bestehen bleibt. 5. 5. Die Zellkernfraktionen weisen eine geringere, vorwiegend deuterencephale Induktionsleistung auf. Die Induktionen enthielten ausser Hinterhirn und Gehorblasen nur etwas Mesenchym und Knorpel, jedoch keine Muskulatur oder Chorda (Abb. 9). Ob diese Induktionsstoffe tatsachlich aus den Kernen oder aber aus schwer abzutrennenden Verunreinigen stammen, ist noch nicht geklart.