Development 101, 23-32 (1987) 23 Printed in Great Britain © The Company of Biologists Limited 1987 The development of animal cap cells in Xenopus: the effects of environment on the differentiation and the migration of grafted ectodermal cells E. A. JONES and H. R. WOODLAND MRC Animal Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Summary We have used blastocoel and vegetal pole grafts to stage 9 or earlier, or the host is stage 1(H or earlier, investigate the effect of environment on differen- the graft forms mesoderm, including striated muscle tiation and movement of animal pole cells of Xenopus. in the gut. This shows that muscle can develop in In the blastocoel of embryos earlier than stage 10, wholly the wrong environment, it suggests that the fragments of animal pole primarily form mesoderm. dorsal inductive signal from mesoderm is rather The cells are either integrated into normal host tissues general in the vegetal mass and suggests that dorsal or they organize a secondary posterior dorsal axis. If mesoderm development involves little subsequent either host or graft is later than stage 9 the graft adjustability. If the host is stage 11 or later, or the forms ectoderm and its cells all migrate into the host graft later than stage 9, the graft forms epidermis in ectoderm. Inner layer animal cells form sensorial the gut. This shows that the epidermal pathway of layer; outer cells move to the epidermis. Thus con- development is also insensitive to environment. siderable powers of appropriate movement are seen. In the vegetal pole no movement occurs. If the graft is Key words: Xenopus, animal cap cells, migration, graft. Introduction isolated animal hemispheres only form epidermis (Holtfreter & Hamburger, 1955; Asashima & Grunz, In this paper, we describe experiments using mono- 1983; Slack, 1984; Jones & Woodland, 1986). It is clonal antibodies to epidermis and muscle-specific believed that this mesoderm is formed by the in- epitopes to investigate the migration, development ducing action of cells in the presumptive endoderm and subsequent differentiation of animal cap cells of on competent ectoderm, the latter being reported to Xenopus embryos when they are transplanted into be able to respond to this induction up to gastrulation unusual positions in the embryo. (Dale, Smith & Slack, 1985). In experimental tissue In Xenopus, the ectoderm is primarily derived from combinations, at least, the presumptive ectoderm the pigmented half of the embryo (Keller, 1975; may also form pharyngeal endoderm (Sudarwati & Cooke & Webber, 1985; Dale & Slack, 1987) though Nieuwkoop, 1971). Thus, in atypical sites, presump- even vegetal pole cells of the 32-cell embryo give tive epidermis might be expected to form mesoderm rise to a little ectoderm at high frequency (Heas- and anterior gut, in addition to the epidermis or man, Wylie, Hausen & Smith, 1984). The ectoderm nervous system that it normally forms. eventually produces two main components, epider- When single cells are placed in the blastocoel of a mis and nervous system, a process involving a number host embryo their descendants appear in a variety of of steps of commitment, first to ectoderm rather than tissues and the cells concerned apparently conform to mesoderm and subsequently to either epidermis or the differentiated state of their surroundings (Wylie, nervous system. Recent fate mapping shows that the Smith, Snape & Heasman, 1985; Wylie, Snape, Heas- animal cap region also forms much of the mesoderm man & Smith, 1987; Snape, Wylie, Smith & Heas- (Cooke & Webber, 1985; Dale & Slack, 1987) though man, 1987). Do they differentiate in accordance with 24 E. A. Jones and H. R. Woodland their surroundings or do they settle on a differen- staining with quinacrine. They exhibit intensely fluorescent tiation pathway and then move to the appropriate chromatin granules, which are absent from X. laevis site? Indeed, how much are migratory abilities of cells (Fig. 1A; Thi^baud, 1983). responsible for maintaining and achieving the three Blastocoel-grafted embryos were made by inserting rho- damine-labelled or X. borealis donor ectoderm into a small germ layer structure of the embryo? In this paper, we slit at the animal pole of demembranated embryos. Pieces show that ectodermal cells have considerable ability of ectoderm were approximately one eighth of an animal to migrate to their appropriate location in an embryo, cap in size. They were cultured in MBS [88mM-NaCl; but that this location is not necessary for them to form lmM-KCl; 24mM-NaHCO3; 15mM-Tris-HCl; 0-33 mM- epidermis. Similarly, muscle can develop in com- Ca(NO3)2; lmM-MgSO4; lmM-NaHCO,; 2mM-sodium pletely unusual surroundings, although mesbderm phosphate pH7-4; and 0-1 mM-Na2EDTA (Gurdon, 1977)] cells probably also have migratory abilities around to heal and then transferred into 1/10 MBS to gastrulate. the general blastocoel region. The picture that Vegetal pole grafts were achieved by grafting similar explants into gaps teased between vegetal pole cells or into emerges is first that the structure of the embryo is the holes left after removing whole vegetal pole blasto- probably maintained by sophisticated migratory abili- meres. All grafted embryos were healed in MBS. They ties in its constituent cells. Second, it seems that once were either maintained in this medium to produce exogas- certain major choices in differentiation pathways are trulae or transferred to 1/10 MBS to gastrulate normally. made, cells differentiate autonomously. Fixation, embedding, sectioning and staining with anti- bodies or simpler chemicals were as described by Jones & We have recently isolated two monoclonal anti- Woodland (1986). Fig. 1B,C shows the normal staining bodies that react specifically with the epidermis and pattern of the epidermal and muscle-specific antibodies on striated muscle of the amphibian embryo. The epider- stage-46 X. laevis embryos. mal marker reacts with all of the surface epidermal cells of the neurula, even though these cells may be as Results different as the cement gland and ciliated cells (Jones, 1985; Jones & Woodland, 1986). This antigen, which Migration and differentiation of epidermal cells in first appears in the stage-12^ late gastrula, is a major ectodermal sandwiches secreted molecule with a protein component, present In a normal embryo, the cells that form epidermis, in all superficial cells of the early neurula, except the the outermost layer of which binds 2F7.C7, bound the future central nervous system. It can be used as a embryo. The same is very largely true when a morula marker of the appearance of the epidermal pheno- or blastula explant of animal cap cells is cultured in type, even when cells do not gain the morphological saline, although in this case there is also a scattering characteristics of epidermis. For example, the marker of somewhat more lightly stained cells within the solid subsequently appears when cell division is blocked at ball of 'atypical epidermis' which forms (Jones & the mid-blastula stage, even though the cells become Woodland, 1986). Can highly pigmented ectodermal multicellular and disorganized (Jones & Woodland, cells differentiate into the strongly positive pheno- 1986). The muscle-specific marker is a monoclonal type in an internal position or is it essential that they antibody (5A3.B4) raised by immunizing Balb/c mice migrate to the cell surface before differentiating in with a homogenate of adult Xenopus muscle. It stains this way? striated muscle from stage 20 onwards and reacts with To find if this was so, we made a sandwich of two no other tissue type (Fig. 1). We also used a further animal caps from stage-9 blastulae and placed a muscle-specific antibody (Kinter & Brockes, 1985). smaller piece of animal cap from embryos of various All the antibodies used in this study stain X. laevis and stages in the centre (Fig. 2A). Implants were taken X. borealis in an identical way. from embryos between stage 3 and stage 9 (8-cell to late blastula) and placed in stage-9 tissue. In every case, greater than 20 in total, a high proportion of the implant, and all of its heavily pigmented cells, bound Methods 2F7.C7 strongly. However, the great majority did so without moving to the surface (Fig. 2B-D). An Embryo culture, manipulations and histology outside position is thus not necessary to form epider- Embryos were cultured and explants made as described by mis and migration to the surface of the explant does Jones & Woodland (1986). Ectodermal sandwich exper- not occur. However, the overall environment of the iments were made with ectodermal explants from X. explants is still ectodermal. borealis sandwiched between complete animal caps derived from two X. laevis stage-9 blastula and incubated. They Migration of ectodermal cells in blastocoel grafts were fixed when embryos synchronous with the implant had Since ectodermal cells do not migrate in the wholly reached stage 19. X. borealis cells were recognized by ectodermal environment of an ectodermal sandwich, Development of animal cap cells in Xenopus 25 we have tested the ability of ectodermal cells to migrate after grafting into different regions of the whole embryo. Initially, ectoderm was grafted into the blastocoel. Classically this operation was used as a test of the ability of the dorsal mesoderm to induce a secondary CNS, that is as a modification of the original Spemann and Mangold graft (Spemann & Mangold, 1924). As pointed out by Slack (1983), this kind of experiment introduces the graft into variable situations, with complex results, at least in terms of the overall tissue organization of the embryo. How- ever, we have grafted animal cap and not dorsal mesoderm, and primarily ask three very simple ques- tions: do the grafted cells migrate and, if so, what kind of tissue do the grafted cells enter and what differentiated phenotype do they display? Similar approaches have been used in Xenopus with single ectodermal and endodermal cells (Heasman et al.