Continuing organizer function during chick tail development

Vladimir Knezevic 0 1 Ronald De Santo 0 1 Susan Mackem 0 1 0 Laboratory of Pathology, NCI, NIH , Bethesda, M.D. 20892 , USA 1 V. Knezevic , R. De Santo and S. Mackem Development of the posterior body (lumbosacral region and tail) in vertebrates is delayed relative to gastrulation. In amniotes, it proceeds with the replacement of the regressed node and primitive streak by a caudal blastemalike mass of mesenchyme known as the tail bud. Despite apparent morphological dissimilarities, recent results suggest that tail development in amniotes is in essence a continuation of gastrulation, as is the case in Xenopus. However, this has been inferred primarily from the outcome of fate mapping studies demonstrating discrete, regionalized cell populations in the tail bud, like those present at gastrulation. Our analysis of the tail bud distribution of several molecular markers that are expressed in specific spatial domains during chick gastrulation confirms these results. Furthermore, we present evidence that gastrulation-like ingression movements from the surface continue in the early chick tail - During gastrulation, a uniform population of embryonic cells segregates to form definitive germ layers (definitive ectoderm, mesoderm and endoderm). While the mechanism by which this segregation is achieved may differ slightly from one species to another, the relationship and the fate of definitive germ layers is highly conserved among all vertebrates (Gilbert, 1991; Tam and Quinlan, 1996). An important feature of early gastrulation is the internalization of superficially located mesodermal and endodermal precursors. In amniotes this process occurs via movement of cells through the primitive streak. During this early phase of gastrulation, presumptive neuroectoderm and surface ectoderm precursors remain superficially located. Once formed, the germ layers will undergo a series of inductive interactions to establish and pattern the primary body axis. This induction is mediated by key signalling centers (or organizers) which have the defining properties of both contributing to and recruiting other cells into the forming embryonic axis. The properties of these signalling centers are also quite dynamic. The embryonic axis develops in a craniocaudal temporal sequence and there is considerable evidence in amphibians to support the idea first proposed by Mangold (1933), that the organizer contains distinct head, trunk and tail subdomains that function sequentially to regionalize different parts of the CNS with respect to their bud and that the established tail bud retains organizer activity. This tail organizer has the expected properties of being able to recruit uncommitted host cells into a new embryonic axis and induce host neural tissue with posteriorly regionalized gene expression when grafted to competent host cells that are otherwise destined to form only extra-embryonic tissue. Together, these results indicate that chick tail development is mechanistically continuous with gastrulation and that the developing tail in chick may serve as a useful experimental adjunct to investigate the molecular basis of inductive interactions operating during gastrulation, considering that residual tail organizing activity is still present at a surprisingly late stage. AP (anteroposterior) characteristics (reviewed by Lemaire and Kodjabachian, 1996). Expression patterns of different organizer-related genes in various vertebrate embryos are consistent with structural and functional heterogeneity within the organizer and recent functional studies also point to the presence of separable head and trunk organizer components in mammals, as well as amphibians and fish (reviewed by Tam and Behringer, 1997). Evidence for the existence and characteristics of a tail organizer is comparatively more nebulous in amniotes and, in fact, entirely different mechanisms have been previously proposed for development of the tail region (the lumbosacral region and caudad in amniotes; Criley, 1969; Holmdahl, 1925a,b, 1939; Lemire et al., 1975; Muller and ORahilly, 1987). In the tail bud, the primitive streak and Hensens node are replaced by a bulb-like structure consisting of a morphologically uniform mass of mesenchyme directly continuous with axial (neural tube, notochord, gut) and paraxial (segmental plate) structures formed during the earlier phases of gastrulation. This appearance has led to the proposal that structures in the tail are formed directly from a blastema without segregation of cells into germ layers (Holmdahl, 1925a,b, 1939; Hughes and Freeman, 1974; reviewed by Griffith et al., 1992). In amphibians, which lack such an apparent mesenchymal condensation, a variety of approaches have been used to demonstrate the continuity of developmental processes during gastrulation and tail formation, including analyses of the distribution of molecular markers (Gont et al., 1993); mapping studies with lineage tracers and homotypic grafts (Gont et al., 1993; Tucker and Slack, 1995a,b) and, most notably, the demonstration of functional organizer properties within the tail bud (Gont et al., 1993). However, in amniotes, evidence supporting continuity between gastrulation and tail formation has been more indirect and comes primarily from mapping studies using either lineage tracers in mouse (Wilson and Beddington, 1996) or homotypic grafts in chick (Catala et al., 1995, 1996). The size and accessibility of the chick embryo and availability of viral expression vectors provides a powerful system to experimentally manipulate gene expression during tail development. If tail development in amniotes is indeed comparable to gastrulation, then such studies would provide an alternate avenue to analyzing the function of genes critical for gastrulation. It was therefore of interest to examine tail development in the chick for various features characteristic of gastrulation. In this paper, we examined the regional distribution of several markers and investigated the movements of cells from the surface during tail bud formation, as well as testing for the presence of a functional tail organizer. Our results show not only that similar cell populations are continuously present through gastrulation and tail elongation, but that mesodermal precursors continue to internalize from the surface, recapitulating morphogenetic movements during gastrulation. We also demonstrate the presence of a functional organizer with posteriorizing features during tail elongation, strongly supporting the concept that formation of the caudal body axis is conceptually and mechanistically a continuation of gastrulation in amniotes. MATERIALS AND METHODS White Leghorn chick embryos or quail embryos (Truslow Farms) were incubated at 38.5C and staged as described by Hamburger and Hamilton (1951). Embryos were dissected in phosphate-buffered saline (PBS) and processed for experiments as described below. Whole-mount in situ hybridization of embryos Digoxigenin-UTP ribo (...truncated)