Links-Rechts-Achsenentwicklung im Kaninchenembryo: Klonierung von Markergenen, Etablierung eines in vitro Kultursystems und Identifizierung des Wachstumsfaktors FGF8 als rechte Determinante

Abstract

Most of the fundamental mechanisms of embryonic axis formation are conserved between the various classes of vertebrates, for example the BMP-signaling system and its antagonists in the formation of the dorsal-ventral axis. A marked exception is the growth factor FGF8, which plays opposite roles in the transfer of an not yet identified asymmetric signal from the midline domain to the periphery during the formation of left-right asymmetry in chick and mouse. In the chicken embryo fgf8 is expressed asymmetrically on the right side of the primitive node and inhibits the transcription of factors of the left signaling cascade, nodal, Pitx2 and lefty2 on the right side. In mouse, fgf8 is transcribed symmetrically. A hypomorphic allel and missexpression experiments in cultured mouse embryos assign FGF8 an instructive function in the induction of the Nodal signaling cascade on the left side. This discrepancy could be attributed to the different expression patterns, evolutionary differences and the architecture of the early embryo, which forms a flat blastodisc in chick, in contrast to a cylindrical socalled egg-cylinder in mouse. Thus in the present work the function of FGF8 was analysed in the rabbit embryo, which as a mammal develops via a blastodisc. Cloning and expression analysis of marker genes showed a conserved asymmetric expression of the nodal cascade genes, whereas all genes, which are asymmetrically expressed in or at the chicken primitive node, for example fgf8 and shh, are always symmetrically active both in rabbit and mouse. Likewise in contrary to the chick, stable in vitro culture of rabbit embryos was only possible after the formation of the first pair of somites. Earlier cultures resulted in bilateral or missing expression of asymmetric marker genes. In order to check the suitability of the rabbit system Activin-soaked acrylic beads were introduced into the right side of cultured embryos. Activin, which functions like Nodal, induced bilateral expression of nodal and Pitx2. This experiment showed that the Nodal cascade is conserved in the rabbit and that the right side is competent to induce the Nodal cascade. Whereas, unlike in mouse, FGF8 did not show an inducing effect. In stage 1-3 somite embryos FGF8 had also no repressive function. But a clear correlation between the placement of a FGF8 bead and absence of nodal transcription was observed in earlier stages. An ectopically induced expression of the Nodal signaling cascade by BMP4 could also be efficiently repressed by the parallel administration of FGF8. An endogenous right-asymmetric role of FGF8 could be shown by repressing the FGF8 signaling cascade on the right side of early somatic stages by the inhibitor SU5402. This experiment resulted in bilateral expression of nodal and Pitx2. Gain- and loss of function experiments presented in this work assign FGF8 a right-asymmetric function in the rabbit embryo. They show that the different effects in chick, mouse and rabbit cannot be attributed to the asymmetric expression in the chick and also not to a reversal of FGF8 function in the course of evolution birds to mammals. In fact they suggest that the specific anatomy of the gastrula/neurula embryo plays a pivotal determinant in left-right axis formation.