Development 107, 55-67 (1989) 55 Printed in Great Britain © The Company of Biologists Limited 1989 Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation PATRICK P. L. TAM Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong Summary The regionalisation of cell fate in the embryonic ecto- grafted to the anterior midline are found in the oral derm was studied by analyzing the distribution of graft- ectodermal lining, whereas cells from the archenteron derived cells in the chimaeric embryo following grafting are found in the notochord. With respect to mesodermal of wheat germ agglutinin-gold-labelled cells and cul- tissues, ectoderm at the archenteron and the distal- turing primitive-streak-stage mouse embryos. Embry- lateral region of the egg cylinder gives rise to rhomben- onic ectoderm in the anterior region of the egg cylinder cephalic somitomeres, and the embryonic ectoderm contributes to the neuroectoderm of the prosencephalon adjacent to the primitive streak contributes to the and mesencephalon. Cells in the distal lateral region give somitic mesoderm and the lateral mesoderm. Based rise to the neuroectoderm of the rhombencephalon and upon results of this and other grafting studies, a map of the spinal cord. Embryonic ectoderm at the archenteron prospective ectodermal tissues in the embryonic ecto- and adjacent to the middle region of the primitive streak derm of the full-streak-stage mouse embryo is con- contributes to the neuroepithelium of the spinal cord. structed. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes Key words: embryonic ectoderm, primitive-streak stage, and the cranial neural crest cells. Some labelled cells mouse embryo, microsurgical grafting, lectin-gold labelling. Introduction ectoderm is the elevation of proliferative activity in a small group of cells in the anterior region of the The embryonic ectoderm (epiblast) of the gastrulating embryonic ectoderm near the rostral end of the primi- mouse embryo is believed to be the sole precursor of all tive streak of the gastrulating embryo (Snow, 1977). definitive tissues in the fetus. The evidence supporting The developmental fate of the progeny of this mitoti- this notion is provided by the extensive range of cally active population is unknown though neuroecto- embryonic and adult tissues produced during the differ- derm has been suggested (Snow & Bennett, 1978). entiation of the whole epiblast or fragments of it after Morphological studies in the mouse embryo suggest transplanting to ectopic sites (Diwan & Stevens, 1976; that the embryonic ectoderm in the anterior region of Beddington, 1983; Svajger etal. 1986) and the multi- the egg cylinder gives rise to major segments of the tude of tissue types colonized by the epiblast cells brain on the grounds that this part of the embryonic grafted orthotopically in the primitive-streak-stage em- ectoderm is topographically related to the first three to bryo (Beddington, 1981, 1982). It is therefore conceiv- four somitomeres normally underlying the forebrain to able that, in order to generate a embryonic body plan upper hindbrain (Tarn & Meier, 1982; Meier & Tarn, from the histologically homogeneous embryonic ecto- 1982; Jacobson & Tarn, 1982). Orthotopic grafting of derm (Reinius, 1965; Batten & Haar, 1979), an orderly embryonic ectoderm cells to the most anterior region allocation of cells to various tissue types in specific and the distal tip (the node, the head process or regions of the body needs to be accomplished during the archenteron - Theiler, 1972; Poelmann, 1981) of the gastrulation. The process of pattern generation would primitive-streak-stage mouse embryo results in the be further facilitated if cells with diversified develop- colonization of, respectively, the head and trunk neur- mental fate are strategically located within the embry- ectoderm by the graft-derived cells but the precise onic ectoderm so that tissues of different lineages but segmental distribution of these cells in the cephalic belonging to specific parts of the body are properly neural tube is not known (Beddington, 1981). Details of juxtaposed in preparation for ingression through the the normal fate of cells in other anterior and lateral regions of the embryonic ectoderm are incomplete primitive streak. because previous mapping studies tended to focus An example of early regionalization of the embryonic 56 P. P. L. Tarn mostly on specific groups of cells adjacent to and within In vitro culture of embryos the primitive streak of the gastrulating embryo (Bedd- Primitive-streak-stage embryos were cultured in rotating ington, 1981, 1982; Copp etal. 1986; Tarn & Bedd- (30 rev min"') 50 ml glass bottle (Wheaton) containing 3-4 ml ington, 1987). An in vitro study on the morphogenesis of culture medium. To culture embryos for 22-24 h until they of isolated fragments of primitive-streak-stage embryo reached the early-somite stage, a 1:1 (v/v) mixture of rat seems to suggest that, within the embryonic ectoderm, serum and Dulbecco's modified Eagle's medium (DMEM, cell populations destined for specific brain parts are Gibco) was used as the culture medium. For culturing embryos for 44-46h until the forelimb-bud stage, a mixture of already spatially delineated (Snow, 1981). The present mouse serum, rat serum and DMEM (1:2:1 by volume) was grafting study was carried out to examine the regionali- used and the embryos were transferred to fresh medium after sation of prospective ectodermal tissues in the anterior 24 h of culture (Hunter et al. 1988). The culture was gassed and lateral regions of the embryonic ectoderm. Special with 5% CO2, 5% O2 and 90% N2 during the first 24 h of attention is given to (1) the segmental distribution of development to the early-somite stage and then with 5 % CO2 the epiblast-derived cells in the neural tube and (2) the in air for further culture. location of cells destined for the surface ectoderm, the placodes and cephalic neural crest cells of the mouse Labelling of embryonic ectoderm and preparation of embryo. grafts Wheat germ agglutinin (WGA)-gold conjugate used for Materials and methods labelling the embryonic ectoderm was prepared as described by Tarn & Beddington (1987). About 2-5 nl of the colloidal WGA-gold label was injected into the amniotic cavity of the Recovery of embryos primitive-streak-stage embryo, which was then cultured in rat Primitive-streak-stage embryos were obtained from a close- serum-DMEM for 3-4 h. After culture, the embryo was bred colony of ICR strain mice. At 7-5 days p.c., embryos transferred to PB1 medium and rinsed twice in the same were dissected from the uterus in PB1 medium containing medium. Fig. 2A shows the orientation of embryonic axes of either 4mgml~' bovine serum albumin (Miles Lab) or 10% the egg cylinder with the posterior aspect of the embryo fetal calf serum (FCS, Gibco). The parietal yolk sac was indicated by the allantoic rudiment and the primitive streak removed microsurgically with a pair of fine glass needles and and the proximal border by the amnion. Using a pair of fine the embryos were washed in several changes of fresh PB1 glass needles, the egg cylinder was transected at the level of medium. Only late-primitive-streak-stage embryos (Fig. 1) the amnion to remove the extraembryonic region. A longi- showing expanded exocoelom and amniotic cavity, a com- tudinal cut was made down the anterior midline (Fig. 2B) to plete amnion and clearly discernible embryonic ectoderm unfold the embryonic portion of the egg cylinder. The were used for labelling and grafting. Other features character- dissected embryo was then left in PB1 medium on a warm istic of embryos at this stage are the early allantoic rudiment stage (30-35°C) for about 5-10min. Often this was sufficient and the anterior-posterior gradient of tissue opacity due to to cause the spontaneous separation of the more turgid the presence of the spreading mesodermal wings. embryonic ectoderm from the loose mesoderm and the thin endodermal layer, which started to curl back from the free edges. Further separation of tissue layers was achieved by inserting a fine needle underneath the embryonic ectoderm, which was colored deep red by the gold label, to slice away the mesoderm and endoderm. A longitudinal cut was then made on each side of the primitive streak (Fig. 2B) to isolate two half-fragments of the embryonic ectoderm without the primi- tive streak (Fig. 2C). The embryonic ectoderm fragments were transferred to fresh PB1 medium and divided into four major fragments: two smaller anterior quadrants and two larger posterior quadrants (fragments A-D; Fig. 2C) from which smaller clumps of 20-30 cells were isolated for grafting. Usually 5 to 6 egg cylinders would provide enough tissues for grafting of 16-20 embryos. Grafting experiments The strategy was to graft WGA-gold-labelled cells isolated from the four major fragments of the embryonic ectoderm to different sites in the primitive-streak-stage mouse embryos. Donor cells were grafted to sites in the same quadrant from which they were isolated. Exact orthotopic grafting, which was technically much more complicated, was not attempted. The embryos were then cultured until they developed to the early-somite stage (22-24 h HI vitro) or to the stage of formation of forelimb bud and closure of anterior neuropore Fig. 1. Scanning electron micrograph of a bisected 7-5-day (stage 14 - Thieler, 1972; 44-46 h in vitro). The pattern of primitive-streak-stage embryo showing the exocoelom tissue colonization by the graft-derived cells was studied in the completely separated from the amniotic cavity by the chimaeric embryos following histological preparation and amnion (am), al, allantoic rudiment; ch, chorion; ps, silver enhancement of the colloidal gold particles in the primitive streak; ect, embryonic ectoderm.