![]() To obtain notochord and hindbrain for control transplants, the ventral endoderm and ectoderm were manually removed using a needle and forceps from anesthetized stage 35 tadpoles. 1A, was then excised with tungsten needles and fine scissors. The posterior-most region of the tail, approximately corresponding in size to the extent of the Xnot2-staining region shown in Fig. The ectoderm was removed using tungsten needles and the tailbud tissue transferred to 1× MBS for 10 minutes. The tip of the tail was removed from stage 35 embryos anesthetized in 0.01% Tricaine (Sigma) by excising the tailbud with iridectomy scissors and transferring to Ca 2+/Mg 2+-free OR2 medium ( Kay and Peng, 1991) in a 1% agarose dish for one hour. It is possible that Xnot ( von Dassow et al., 1993) and Xnot2 represent duplicated forms of the same gene. Because Xenopus is a pseudotetraploid organism, many genes are duplicated. The two probes would be expected to cross-hybridize, and expression patterns of the two genes appear identical at the level of resolution of wholemount in situ hybridization. (1993) shows that the predicted proteins are 90.1% identical overall and 96.6% identical in the homeodomains, with 91% of the amino acid changes being conservative ( Dayhoff, 1972). A comparison of the predicted amino acid sequence reported here with that reported by von Dassow et al. ![]() The homeodomain is most similar (62.7%) to the Drosophila empty spiracles homeodomain protein, making this a novel type of homeobox. The Xnot2 cDNA E-9 is 2097 bp, encodes a predicted 233 amino acid protein and contains a poly(A) tail. DNA sequences were analyzed using the University of Wisconsin Genetics Group ( Devereaux et al., 1984). The longest cDNA (E9) was subcloned and sequenced by the dideoxy method on both strands using T7 DNA polymerase (Pharmacia). Hybridization was at low stringency conditions in a solution containing 1.0 M NaCl/0.1 M Tris-HCl(pH 8.3)/6.6 mM EDTA/5× Denhardt’s/0.1% SDS/0.05%NaPP i/125 units per ml of heparin/1 mg per ml yeast RNA, and washed at high stringency (58☌) in 3 M TMAC/0.05 M Tris-HCl(pH 8.0)/0.2 mM EDTA ( Burglin et al., 1989). MATERIALS AND METHODSĪpproximately 1.5×10 5 plaques of an unamplified Xenopus egg cDNA library made as described ( Blumberg et al., 1992) were screened in duplicate on nitrocellulose filters with a 1024X degenerate mixture of 32P end-labelled oligonucleotides: corresponding to the sequence KIWFQ/KNRR of helix 3 of the homeodomain. We conclude that tail formation in Xenopus is a direct continuation of gastrulation movements and that the tip of the tail, which retains potent tail organizer activity, is a direct descendant of the dorsal blastopore lip. ![]() This was addressed by determining the fate map of the late blastopore lip and by transplantation studies. Having found that the tailbud consists of distinct cell populations, we then asked whether they were related by lineage to the different regions of the blastopore. The markers used were Xnot2, a homeobox gene that is expressed in the dorsal lip of the blastopore, and Brachyury (short-tail), a gene required for tail development and expressed in the entire blastoporal ring (see description below). ![]() In this study, we compare two gene markers that are expressed in distinct regions of the blastopore of the early gastrula and whose expression can be followed continuously as they become localized to distinct cell populations in the tailbud in the course of development. The question of whether or not the tailbud is homogeneous can now be directly addressed using appropriate molecular markers. Recently, the mechanism of tail development has received little attention, but there seems to be general agreement that the tailbud is a blastema ( Griffith et al., 1992). Pasteels (1943) favored a different view, in which the different tissues of the tail would derive from distinct cell populations. ![]() The concept of a tailbud blastema was proposed by Holmdahl (1925) who distinguished the ‘primary body development’ in which the three germ layers are formed by involution movements during gastrulation, from the ‘secondary body development’ in which an undifferentiated blastema directly gives rise to all tissues of the tail. An unresolved question is whether the tailbud is composed of truly undifferentiated pluripotential stem cells, i.e., a ‘blastema’, or whether it consists of several cell populations with differing cell fates despite its histologically homogenous appearance. The vertebrate tail develops from the tailbud, an apparently homogenous mass of cells at the posterior of the embryo. ![]()
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