Faculty Bibliography

Using antibodies against homeoprotein XIHbox 1 from Xenopus laevis, we have detected a new embryonic protein with a much larger molecular weight, 115 kDa. Antibodies fractionated according to their affinity for 3 different domains of the XIHbox 1 protein were used to show that this new protein is related to the C-terminal region of XIHbox 1 protein, downstream from the homeodomain. By immunohistochemistry, the protein was shown to be localized in nuclei of embryonic cells. On SDS-polyacrylamide gels, the 115 kDa protein appears as a set of closely spaced bands whose pattern varies with the stage of development and with the parental origin of the embryos. The protein could be extracted from embryos in a multiprotein complex of approximately 600 kDa. In contrast, the 18 and 27 kDa proteins predicted from the sequence of cloned cDNA to be transcribed and translated from the XIHbox 1 gene could not be detected, suggesting that they are rare or unstable in embryos. These data suggest that the new protein is involved in the development of Xenopus embryos, with a function possibly related to that of the homeoprotein XIHbox 1.

Like all known LIM class homeobox genes, Xlim-1 encodes a protein with two tandemly repeated cysteine-rich LIM domains upstream of the homeodomain. In Xenopus laevis, Xlim-1 is specifically expressed in the Spemann organizer, whose major functions include neural induction and dorsalization of ventral mesoderm. From RNA injection experiments we conclude here that: (1) the LIM domains behave as negative regulatory domains; (2) LIM domain mutants of Xlim-1 elicited neural differentiation in animal explants; (3) mutant, and to a lesser extent wild-type, Xlim-1 enhanced muscle formation after coinjection with Xbra; (4) both of these activities are mediated by extracellular signals as seen in combined explant experiments; (5) Xlim-1 mutants activated goosecoid (gsc) expression in animal explants, but not expression of noggin or follistatin; (6) mutant Xlim-1 elicited formation of partial secondary axes, and cooperated with gsc in notochord formation. Thus Xlim-1 has latent activities, implicating it in organizer functions.

A fate map of the progeny derived from all blastomeres of the 32-cell stage embryo of the leopard frog Rana pipiens has been generated. Embryos presenting regular cleavages were injected into two pairs of blastomeres with fluorescein- and Texas red-lysine dextran. By stage 21, embryos were sectioned and the tissue distribution of labeled clones was determined. The results of 93 clones were pooled to give a fate map which represents the derivation of each tissue from the different blastomeres of the 32-cell embryo. The results show that all blastomeres give rise to multiple tissues and all tissues are derived from at least two, and usually more, pairs of blastomeres. Although there is a general tendency for ectoderm to derive from the animal hemisphere, endoderm from the vegetal hemisphere, and mesoderm from the equatorial region, the boundaries between germ layers are not sharply defined at the 32-cell stage but rather appear as a series of overlapping zones. The fate map of R. pipiens is quite similar to that of Xenopus laevis but differs in some details that are discussed. As in all vertebrates, the R. pipiens fate map is not fully deterministic but nevertheless has predictive value in that tissues are populated by the progeny of the same blastomeres in different embryos.

In an attempt to document at the molecular level the behavior of mesodermal cells in Keller explant preparation, we have analyzed the time course of expression of four molecular markers of mesoderm gsc, Xbra, Xnot and XLIM-1. Our findings demonstrate that, (i) all mesodermal markers tested were expressed in the explants, but patterning of the mesoderm appeared incomplete; (ii) during convergence and extension of the explants, mesodermal cells did not invade the ectodermal tissue at any time tested, supporting the view that mesoderm establishes exclusively planar contacts with the ectoderm in this preparation; (iii) planar contacts were not sufficient to promote the neural expression of XLIM-1 protein in these explants.

Cadherins, a family of Ca-dependent adhesion molecules, have been proposed to act as regulators of morphogenetic processes and to be major effectors in the maintenance of tissue integrity. In this study, we have compared the effects of the expression of two truncated cadherins during early neurogenesis in Xenopus laevis. mRNA encoding deleted forms of XB- and N-cadherin lacking most of the extracellular domain were injected into the four animal dorsal blastomeres of 32-cell stage Xenopus embryos. These truncated cadherins altered the cohesion of cells derived from the injected blastomeres and induced morphogenetic defects in the anterior neural tissue to which they chiefly contributed. Truncated XB-cadherin was more efficient than N-cadherin in inducing these perturbations. Moreover, the coexpression of both truncated cadherins had additive perturbation effects on neural development. The two truncated cadherins can interact with the three known catenins, but with distinct affinities. These results suggest that the adhesive signal mediated by cadherins can be perturbed by overexpressing their cytoplasmic domains by competing with different affinity with catenins and/or a common anchor structure. Therefore, the correct regulation of cadherin function through the cytoplasmic domain appears to be a crucial step in the formation of the neural tissue.

Epithelial-mesenchymal interactions are of major importance during development to direct correct differentiation and morphogenesis of embryonic tissues. One subset of lateral mesoderm-derived mesenchymal cells will form the smooth muscle (SM) layer of the primary epithelial lining of hollow internal organs. It has been previously reported that the differentiation of SM cells in Xenopus laevis can be followed by the expression of alpha-SM actin. It was also shown that basic fibroblast growth factor (bFGF) had the ability to induce this actin isoform in isolated blastula animal caps. In this paper, by injection at the two-cell stage of mRNA encoding a truncated form of the FGF receptor which can act as a dominant negative inhibitor, we have analyzed the role of the FGF signaling pathway in the formation of the SM lineage. We have observed that mutated embryos presented significant delay in the differentiation of the SM cells compared to control embryos, demonstrating the importance of this signaling pathway for the formation of the lateral mesoderm-derived SM cells. Moreover, a correlation could be established between this delay and the dramatic defects observed in the morphogenesis of the intestine with which mesenchyme-derived SM cells are normally associated. This phenotype was efficiently rescued by coinjection of the wild-type FGF receptor. Our data suggest that the differentiation of SM cells at the correct time could be an essential event for the proper morphogenesis of the endoderm-derived digestive tract.

The neural plate in the amphibian embryo is induced in the ectoderm by signals from the dorsal mesoderm. In the extensively studied species Xenopus laevis, such signals are believed to proceed along two alternate pathways, defined as vertical and planar induction. We have studied the relative importance of these pathways in Rana pipiens. In the embryo of this frog, dorsal mesoderm involution can be diverted from its normal course by injection of peptides that inhibit interaction of fibronectin with its receptor. In such embryos, dorsal mesoderm failed to migrate across the blastocoel roof but moved bilaterally along the equator, leading to the formation of two notochords. Neural tissue differentiation occurred in close association with each notochord, but no neural tissue formed along the dorsal midline as might have been predicted by a predominantly planar induction model. While in X. laevis planar induction has been reported to be a major pathway in neuralizing the ecoderm, the results presented here indicate that vertical induction predominates in initiating neural development in R. pipiens embryos.