Oncogenic Met receptor induces ectopic structures in Xenopus embryos.

When aberrantly expressed or activated, the Met receptor tyrosine kinase is involved in tumor invasiveness and metastasis. In this study, we have used the Xenopus embryonic system to define the role of various Met proximal-binding partners and downstream signaling pathways in regulating an induced morphogenetic event. We show that expression of an oncogenic derivative of the Met receptor (Tpr-Met) induces ectopic morphogenetic structures during Xenopus embryogenesis. Using variant forms of Tpr-Met that are engineered to recruit a specific signaling molecule of choice, we demonstrate that the sole recruitment of either the Grb2 or the Shc adaptor protein is sufficient to induce ectopic structures and anterior reduction, while the recruitment of PI-3Kinase (PI-3K) is necessary but not sufficient for this effect. In contrast, the recruitment of PLCgamma can initiate the induction, but fails to maintain or elongate supernumerary structures. Finally, evidence indicates that the Ras/Raf/MAPK pathway is necessary, but not sufficient to induce these structures. This study also emphasizes the importance of examining signaling molecules in the regulatory context that is provided by receptor/effector interactions when assessing a role in cell growth and differentiation.

Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo.

The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription-polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4-injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.

Immunodetection of Xenopus bone morphogenetic protein-1 in adult and embryonic cells.

In order to analyze biochemical properties of Xenopus bone morphogenetic protein-1 (XBMP-1), rabbit antiserum (alpha-B1) was raised against a synthetic peptide (P1) corresponding to a hydrophilic N-terminal region. XBMP-1B (Xtld) synthesized in the reticulocyte lysate was successfully immunoprecipitated by this antiserum. This precipitation was completely blocked when P1 was added to the reaction, indicating that alpha-B1 recognized XBMP-1B specifically. In Western blot analysis, two distinct sizes of protein (107 and 34 kD) were detected in hind limbs in metamorphosing animals. Both proteins were detected in various adult tissues such as lung, liver, kidney, heart, muscle, intestine, brain, and testis. The mixing of the liver and muscle extracts, and the following detection of immunoreactive proteins suggested that the 34 kD band was a proteolytic product of the 107 kD protein. In the embryonic extracts from the unfertilized egg (stage 0) to swimming tadpoles (stage 40), a 63 kD protein was detected in addition to the 107 kD protein. We also showed that the 107 kD protein was much more expressed in the animal half of the unfertilized eggs than in the vegetal half, but that it was ubiquitously expressed in the gastrula embryos. We suggest that the 63 and 107 kD proteins correspond to full-length proteins encoded by XBMP-1A and XBMP-1B genes, and these proteins are expressed in embryo and in various adult tissues.

GATA-1 inhibits the formation of notochord and neural tissue in Xenopus embryo.

The expression of GATA-1, which encodes for a hemopoietic transcription factor, initiates at gastrula stage in the Xenopus embryo (1). In order to examine a possible function of GATA-1 in dorso-ventral patterning of mesoderm and ectoderm derivatives, the synthesized RNA of GATA-1 was overexpressed in embryonic cells to assess its biological effects. In the embryos injected with GATA-1 RNA in the dorsal marginal zone at 4-cell stage, dorsal epidermis did not cover the vegetal cells so that the gastrulation was not completed. The same dose of GATA-1 RNA injected into ventral marginal zone did not influence the development, and GATA-2 RNA transcribed from the same vector had little effect, suggesting that this phenomenon is physiologically important. The morphological and immunohistochemical studies revealed that notochord and neural tissue were mostly eliminated in the embryos or the dorsal marginal zone explants after injection of GATA-1 RNA. GATA-1 also inhibited neurogenesis in animal cap explants, which was induced by the injection with noggin RNA. Northern blot analysis using dorsal marginal zone explants showed, however, that only a slight amount of alpha-globin message was induced, and cardiac alpha-actin message was retained. Therefore, GATA-1 did not convert completely the dorsal phenotype to the ventral one. Furthermore, the injection of GATA-1 RNA didnot alter the expression of early dorsal and ventral markers at the onset of gastrulation. These results suggest that GATA-1 is an potential inhibitor of the dorsalization and the neurogenesis, but it affects on the specification of dorsal tissues in relatively later steps.