The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin-gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhomben-cephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.

The embryonic ectoderm (epiblast) of the gastrulating mouse embryo is believed to be the sole precursor of all definitive tissues in the fetus. The evidence supporting this notion is provided by the extensive range of embryonic and adult tissues produced during the differentiation of the whole epiblast or fragments of it after transplanting to ectopic sites (Diwan & Stevens, 1976; Beddington, 1983; Svajger et al. 1986) and the multitude of tissue types colonized by the epiblast cells grafted orthotopically in the primitive-streak-stage embryo (Beddington, 1981, 1982). It is therefore conceivable that, in order to generate a embryonic body plan from the histologically homogeneous embryonic ectoderm (Reinius, 1965; Batten & Haar, 1979), an orderly allocation of cells to various tissue types in specific regions of the body needs to be accomplished during gastrulation. The process of pattern generation would be further facilitated if cells with diversified developmental fate are strategically located within the embryonic ectoderm so that tissues of different lineages but belonging to specific parts of the body are properly juxtaposed in preparation for ingression through the primitive streak.

An example of early regionalization of the embryonic ectoderm is the elevation of proliferative activity in a small group of cells in the anterior region of the embryonic ectoderm near the rostral end of the primitive streak of the gastrulating embryo (Snow, 1977). The developmental fate of the progeny of this mitotically active population is unknown though neuroectoderm has been suggested (Snow & Bennett, 1978). Morphological studies in the mouse embryo suggest that the embryonic ectoderm in the anterior region of the egg cylinder gives rise to major segments of the brain on the grounds that this part of the embryonic ectoderm is topographically related to the first three to four somitomeres normally underlying the forebrain to upper hindbrain (Tam & Meier, 1982; Meier & Tam, 1982; Jacobson & Tam, 1982). Orthotopic grafting of embryonic ectoderm cells to the most anterior region and the distal tip (the node, the head process or the archenteron -Theiler, 1972; Poelmann, 1981) of the primitive-streak-stage mouse embryo results in the colonization of, respectively, the head and trunk neurectoderm by the graft-derived cells but the precise segmental distribution of these cells in the cephalic neural tube is not known (Beddington, 1981). Details of the normal fate of cells in other anterior and lateral regions of the embryonic ectoderm are incomplete because previous mapping studies tended to focus mostly on specific groups of cells adjacent to and within the primitive streak of the gastrulating embryo (Beddington, 1981, 1982; Copp et al. 1986; Tam & Beddington, 1987). An in vitro study on the morphogenesis of isolated fragments of primitive-streak-stage embryo seems to suggest that, within the embryonic ectoderm, cell populations destined for specific brain parts are already spatially delineated (Snow, 1981). The present grafting study was carried out to examine the regionalisation of prospective ectodermal tissues in the anterior and lateral regions of the embryonic ectoderm. Special attention is given to (1) the segmental distribution of the epiblast-derived cells in the neural tube and (2) the location of cells destined for the surface ectoderm, the placodes and cephalic neural crest cells of the mouse embryo.

Recovery of embryos

Primitive-streak-stage embryos were obtained from a closebred colony of ICR strain mice. At 7·5 days p.c., embryos were dissected from the uterus in PB1 medium containing either 4 mg ml−1 bovine serum albumin (Miles Lab) or 10% fetal calf serum (FCS, Gibco). The parietal yolk sac was removed microsurgically with a pair of fine glass needles and the embryos were washed in several changes of fresh PB1 medium. Only late-primitive-streak-stage embryos (Fig. 1) showing expanded exocoelom and amniotic cavity, a complete amnion and clearly discernible embryonic ectoderm were used for labelling and grafting. Other features characteristic of embryos at this stage are the early allantoic rudiment and the anterior-posterior gradient of tissue opacity due to the presence of the spreading mesodermal wings.

Fig. 1.

Scanning electron micrograph of a bisected 7·5-day primitive-streak-stage embryo showing the exocoelom completely separated from the amniotic cavity by the amnion (am), al, allantoic rudiment; ch, chorion; ps, primitive streak; ect, embryonic ectoderm. Bar= 100 μm.

Fig. 1.

Scanning electron micrograph of a bisected 7·5-day primitive-streak-stage embryo showing the exocoelom completely separated from the amniotic cavity by the amnion (am), al, allantoic rudiment; ch, chorion; ps, primitive streak; ect, embryonic ectoderm. Bar= 100 μm.

In vitro culture of embryos

Primitive-streak-stage embryos were cultured in rotating (30 rev min’1) 50 ml glass bottle (Wheaton) containing 3-4 ml of culture medium. To culture embryos for 22-24 h until they reached the early-somite stage, a 1:1 (v/v) mixture of rat serum and Dulbecco’s modified Eagle’s medium (DMEM, Gibco) was used as the culture medium. For culturing embryos for 44-46h until the forelimb-bud stage, a mixture of mouse serum, rat serum and DMEM (1:2:1 by volume) was used and the embryos were transferred to fresh medium after 24h of culture (Hunter et al. 1988). The culture was gassed with 5% CO2, 5% O2 and 90% N2 during the first 24h of development to the early-somite stage and then with 5 % CO2 in air for further culture.

Labelling of embryonic ectoderm and preparation of grafts

Wheat germ agglutinin (WGA)-gold conjugate used for labelling the embryonic ectoderm was prepared as described by Tam & Beddington (1987). About 2–5 nl of the colloidal WGA-gold label was injected into the amniotic cavity of the primitive-streak-stage embryo, which was then cultured in rat serum-DMEM for 3–4 h. After culture, the embryo was transferred to PB1 medium and rinsed twice in the same medium. Fig. 2A shows the orientation of embryonic axes of the egg cylinder with the posterior aspect of the embryo indicated by the allantoic rudiment and the primitive streak and the proximal border by the amnion. Using a pair of fine glass needles, the egg cylinder was transected at the level of the amnion to remove the extraembryonic region. A longitudinal cut was made down the anterior midline (Fig. 2B) to unfold the embryonic portion of the egg cylinder. The dissected embryo was then left in PB1 medium on a warm stage (30–35°C) for about 5–10 min. Often this was sufficient to cause the spontaneous separation of the more turgid 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 primitive 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.

Fig. 2.

(A) Anterior (ANT)-posterior (POST) and proximal-distal embryonic axes of the 7·5-day egg cylinder. The primitive streak (PS) and allantois (AL) mark the posterior side of the egg cylinder and the proximal border is defined by the amnion attaching to the junction between the extraembryonic (EXT) and the embryonic (EMB) parts of the egg cylinder. (B) The position of cut I along the anterior midline of the egg cylinder and cuts II on the two sides of the primitive streak (PS) producing the fragments shown in fig. 2c. LAT, lateral aspect of the egg cylinder, ECT, embryonic ectoderm; AC, amniotic cavity. (C) The dissection of the half-embryo obtained in B to yield fragments A,B,C and D by cuts III and IV.

Fig. 2.

(A) Anterior (ANT)-posterior (POST) and proximal-distal embryonic axes of the 7·5-day egg cylinder. The primitive streak (PS) and allantois (AL) mark the posterior side of the egg cylinder and the proximal border is defined by the amnion attaching to the junction between the extraembryonic (EXT) and the embryonic (EMB) parts of the egg cylinder. (B) The position of cut I along the anterior midline of the egg cylinder and cuts II on the two sides of the primitive streak (PS) producing the fragments shown in fig. 2c. LAT, lateral aspect of the egg cylinder, ECT, embryonic ectoderm; AC, amniotic cavity. (C) The dissection of the half-embryo obtained in B to yield fragments A,B,C and D by cuts III and IV.

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 in vitro) or to the stage of formation of forelimb bud and closure of anterior neuropore (stage 14 -Thieler, 1972; 44–46 h in vitro). The pattern of tissue colonization by the graft-derived cells was studied in the chimaeric embryos following histological preparation and silver enhancement of the colloidal gold particles in the labelled cells. The manipulation of the egg cylinder and the grafting of labelled cells to the embryonic ectoderm followed that described by Beddington (1987). Briefly, grafting was done by microinjecting clumps of labelled cells to the embryonic ectoderm after pushing the injection needle through the primitive endoderm and the mesoderm of the egg cylinder. The graft was placed well within the ectodermal epithelium but it was inevitable that, because of the wound produced by the injection needle, some labelled cells might be lodged in a mesodermal or even endodermal position after grafting. The exact location of the donor cells was examined in 40 embryos which were fixed for histological study of WGA-gold-labelled tissues at 4-5 h after grafting.

Labelled donor cells were grafted to seven different sites of the embryonic ectoderm. Because of the variation in the size of the 7·5-day embryos, it was technically not feasible to obtain uniform results from different embryos by positioning the graft on the basis of absolute physical distance from any morphological landmark. Instead, the seven locations were so chosen that they represented the Cartesian points of an imaginary rectangular grid mapped on the lateral aspect of the embryo using the allantois, the amnion and the distal tip of the egg cylinder as the reference (Fig. 3). These regions were separated from one other by a distance of 80-100 //m along the orthogonal axes which could be discerned under a dissecting microscope at 40× magnification. The following grafts were made:

Fig. 3.

A schematic diagram showing the position in the embryonic ectoderm where microsurgical grafting of WGA-gold-labelled embryonic ectoderm cells were made. Three graftings were made to the midline position (ANT-MAR, MID-ANT and ARCH) and others to the lateral areas with two (PL-PS and ML-PS) adjacent to the primitive streak (PS) and two midway between the anterior and posterior midline of the embryo (P-LAT and D-LAT). PL-PS graft was made close to the allantoic rudiment (AL) and P-LATclose to amnion (AM). MID-ANT, D-LATand ML-PS was halfway down the proximal-distal distance in the embryonic part of the egg cylinder. ARCH was at tip of the egg cylinder.

Fig. 3.

A schematic diagram showing the position in the embryonic ectoderm where microsurgical grafting of WGA-gold-labelled embryonic ectoderm cells were made. Three graftings were made to the midline position (ANT-MAR, MID-ANT and ARCH) and others to the lateral areas with two (PL-PS and ML-PS) adjacent to the primitive streak (PS) and two midway between the anterior and posterior midline of the embryo (P-LAT and D-LAT). PL-PS graft was made close to the allantoic rudiment (AL) and P-LATclose to amnion (AM). MID-ANT, D-LATand ML-PS was halfway down the proximal-distal distance in the embryonic part of the egg cylinder. ARCH was at tip of the egg cylinder.

1 Labelled cells from proximal-anterior quadrant (fragment A -Fig. 2C) were grafted to

(1) the anterior midline position near the attachment of the amnion to the embryonic ectoderm (anterior-marginal: Ant-Mar).

11 Labelled cells from the distal-anterior quadrant (fragment B -Fig. 2C) were grafted to

(2) the region halfway down the anterior midline of the egg cylinder (mid-anterior: Mid-Ant).

III Labelled cells from the proximal-posterior quadrant (fragment C -Fig. 2C) were grafted to:

(3) the region lateral to the posterior end of the primitive streak (posterior-lateral primitive streak: PL-PS), and

(4) the proximal area near to the attachment of the amnion on the lateral flank of the egg cylinder (proximal-lateral: P-Lat).

IV Labelled cells from distal-posterior quadrant (fragment D -Fig. 2C) were grafted to:

(5) the region halfway down the posterior side of the egg cylinder and lateral to the middle portion of the primitive streak (mid-lateral primitive streak: MLPS),

(6) the area halfway down on the lateral flank of the egg cylinder (distal-lateral: D-Lat), and

(7) the distal tip of the egg cylinder, which corresponded to the site of the archenteron and the head process (archenteron: Arch).

Embryos were inspected under the dissection microscope immediately after grafting for the location of the graft. The labelled cells could easily be discerned by their red coloration. Embryos containing grafts in the mesodermal or endodermal germ layers were discarded.

Examination of embryos after culture

Embryos were harvested at 22–24 or 44–46 h of culture and examined for vitelline circulation and heart activity. The fetal membranes were then removed for a closer inspection of developmental features such as the appearance of neural folds and closure of cephalic neural tube, formation of somites and forelimb bud and axis rotation. Embryos were fixed with Carnoy fluid, embedded in paraffin wax and processed for light microscopy. To visualize the colloidal gold marker, histological sections were treated with a silver developer and counterstained with Fast Green (Tam & Beddington, 1987). The location of labelled cells in different embryonic tissues was identified in serial sections of the cultured embryos. The segmental position of the labelled cells in the neural tube, the surface ectoderm, the cranial mesenchyme, the somitic mesoderm and the lateral plate mesoderm was determined with respect to the primary brain parts and the branchial arches in the cephalic region and to the somite in the trunk region.

The initial location of grafted cells

Forty embryos receiving grafts at different sites of the embryonic ectoderm were examined at 4–5 h after grafting. Results in Table 1 show that about 68% (57–80%) of the experimental embryos have donor cells confined to the embryonic ectoderm (Fig. 4) and about 20 % have donor cells in both the ectoderm and the adjacent mesoderm. About 20-30 WGA-gold-labelled cells were found in these embryos. In about 12 % of cases, labelled cells were found only in the mesoderm and endoderm of the host embryo. Based upon this observation, 18 chimaeric embryos (of a total of 132) that showed tissue colonization by donor cells only in mesodermal and endodermal tissues and not in any ectodermal derivatives were excluded from this study, since they might represent artefacts of improper graftings.

Table 1.

The location of WGA-gold-labelled cells in embryos examined at 4–5 h after grafting

The location of WGA-gold-labelled cells in embryos examined at 4–5 h after grafting
The location of WGA-gold-labelled cells in embryos examined at 4–5 h after grafting
Fig. 4.

A primitive-streak-stage embryo examined 4h after grafting, showing the proper incorporation of WGA-gold-labelled cells (arrowheads) in the pseudostratified epithelium of the embryonic ectoderm, ect, embryonic ectoderm. Silver-enhanced and Fast Green. Bar = 20 μm.

Fig. 4.

A primitive-streak-stage embryo examined 4h after grafting, showing the proper incorporation of WGA-gold-labelled cells (arrowheads) in the pseudostratified epithelium of the embryonic ectoderm, ect, embryonic ectoderm. Silver-enhanced and Fast Green. Bar = 20 μm.

Embryonic development in vitro

After 22-24 h of culture, over 50% of experimental embryos developed an actively beating heart, and over 85 % of them formed early neural folds. About 4-8 pairs of somites were formed, which was similar to the number of somites found in the intact embryos cultured for the same duration (Table 2). When examined after 44-46h of culture, experimental embryos of the Ant-Mar, Mid-Ant, P-Lat and D-Lat groups developed to the same extent as the intact embryo with respect to axis rotation, cephalic neurulation, limb bud formation and somite number (Table 2). Embryos of Arch, ML-PS and PL-PS groups were retarded when compared to the intact embryo developing under similar culture conditions. Axis rotation and cephalic neural tube closure were delayed and fewer somites were formed (Table 2).

Table 2.

Development of embryos receiving grafts of labelled embryonic ectoderm at the egg cylinder stage and cultured for 22–24 or 44–46 h in vitro

Development of embryos receiving grafts of labelled embryonic ectoderm at the egg cylinder stage and cultured for 22–24 or 44–46 h in vitro
Development of embryos receiving grafts of labelled embryonic ectoderm at the egg cylinder stage and cultured for 22–24 or 44–46 h in vitro

Tissue distribution of labelled cells

The number of WGA-gold-labelled cells in different tissues of 36 chimaeric embryos was scored (Table 3). The total number of labelled cells varied from 28 to 154 per embryo. In embryos of the Ant-Mar, Mid-Ant, Arch, P-Lat and D-Lat groups, the majority of labelled cells was found in ectodermal derivatives such as the neural tube and epithelia of the oral cavity, body surface and cephalic placodes of chimaeric embryos. Colonization of the notochord by graft-derived cells occurred only in the Arch group embryos. More labelled cells were found in the paraxial and lateral mesoderm of the PL-PS, ML-PS and D-Lat embryos than in the other groups (P<0·01, Mann-Whitney test). About 12 % of the labelled population was found in the cranial mesenchyme and heart mesoderm of the Ant-Mar, Mid-Ant and Arch embryos, compared to 33% mesodermal colonization in embryos receiving graft in the lateral regions. Colonization of the endoderm was found in only 3 chimaeras, with about 10 % of the labelled cells (2–10 per embryo) present in the gut epithelium.

Table 3.

The distribution of the labelled cell population in the chimaeric embryos examined at 22–24 h after grafting of WGA-gold labelled embryonic ectoderm cells

The distribution of the labelled cell population in the chimaeric embryos examined at 22–24 h after grafting of WGA-gold labelled embryonic ectoderm cells
The distribution of the labelled cell population in the chimaeric embryos examined at 22–24 h after grafting of WGA-gold labelled embryonic ectoderm cells

Patterns of tissue colonization

Altogether 93 chimaeric embryos, including 15 from the cell counting study, were analysed in detail for the spatial distribution of labelled cells in various embryonic tissues (Tables 4–6).

Table 4.

Tissue colonization by WGA–gold-labelled cells in chimaeric embryos

Tissue colonization by WGA–gold-labelled cells in chimaeric embryos
Tissue colonization by WGA–gold-labelled cells in chimaeric embryos

(i) Neural tube

Colonization of the neural tube and placodes occurred more frequently in embryos receiving grafts along the anterior midline ectoderm (Ant-Mar, Mid-Ant and Arch) and the lateral ectoderm (P-Lat and D-Lat), in contrast to embryos with grafts made to embryonic ectoderm adjacent to the primitive streak (Table 4). The segmental distribution of labelled cells in the neural tube is summarized in Table 5. Most embryos receiving grafts at Ant-Mar ectoderm had labelled cells in the prosencephalon, mainly on the floor of the diencephalon near the invaginating Rathke’s pouch (Fig. 5), the lamina terminalis and the optic évagination. Some labelled cells were also found at the junction between the forebrain and the midbrain. Labelled cells of the Mid-Ant graft were found mostly in the mesencephalic floor and at the junction between the midbrain and adjacent brain segments. The rhombencephalon and the neural tube at the level of the first three (occipital) somites (Fig. 6) were colonized mainly by labelled cells grafted to the D-Lat ectoderm, with a minor contribution from the P-Lat and PL-PS ectoderm. Colonization of the neural tube at more caudal somitic levels was observed in embryos with Arch and ML-PS grafts.

Table 5.

The distribution of WGA–gold-labelled cells in the neural tube, placode and surface ectoderm of the chimaeric embryos

The distribution of WGA–gold-labelled cells in the neural tube, placode and surface ectoderm of the chimaeric embryos
The distribution of WGA–gold-labelled cells in the neural tube, placode and surface ectoderm of the chimaeric embryos
Fig. 5.

A sagittal section of the diencephalon (di) of an embryo cultured for 44 h after Ant-Mar grafting showing the presence of labelled cells in the neurohypophyseal diverticulum meeting the invaginating Rathke’s pouch (rp) on the roof of the oral cavity.

Fig. 5.

A sagittal section of the diencephalon (di) of an embryo cultured for 44 h after Ant-Mar grafting showing the presence of labelled cells in the neurohypophyseal diverticulum meeting the invaginating Rathke’s pouch (rp) on the roof of the oral cavity.

Fig. 6.

A sagittal section of an embryo cultured for 24 h after Mid-Ant grafting showing WGA–gold-labelled cells (arrowheads) in the rhombencephalon (rh) and in the mesenchyme adjacent to the truncus of the heart, fg, foregut portal; ht, heart tube.

Fig. 6.

A sagittal section of an embryo cultured for 24 h after Mid-Ant grafting showing WGA–gold-labelled cells (arrowheads) in the rhombencephalon (rh) and in the mesenchyme adjacent to the truncus of the heart, fg, foregut portal; ht, heart tube.

(ii) Surface ectoderm and placode

Colonization of the surface ectoderm was observed in 25–43 % of embryos receiving grafts in the lateral ectoderm and adjacent to the primitive streak (Table 4). The surface ectoderm over the branchial arches was colonized by cells grafted to lateral ectoderm but those over the hindbrain and somitic levels were derived from grafts in the lateral ectoderm as well as adjacent to the primitive streak (Table 5). Labelled cells of the P-Lat and PL-PS grafts were frequently found in the columnar placode epithelium on the lateral aspect side of the head folds of the early-somite embryo (Fig. 7). The labelled epithelium was located close to the root of the first two branchial arches and at the myelencephalic level of the chimaeric PL-PS and P-Lat embryos. Following further in vitro development, labelled cells were found in the trigeminal ganglia and the otic capsule of the advanced embryos, suggesting that the donor cells might have colonized the trigeminal and otic placodes after grafting to the PL-PS and P-Lat ectoderm.

Fig. 7.

A frontal section of an embryo cultured for 24 h after P-LAT grafting resulting in the colonization of placodal epithelium at the upper hindbrain level (arrowheads), fg, foregut; nt, neural tube.

Fig. 7.

A frontal section of an embryo cultured for 24 h after P-LAT grafting resulting in the colonization of placodal epithelium at the upper hindbrain level (arrowheads), fg, foregut; nt, neural tube.

(iii) Cranial mesenchyme and neural crest cells

Labelled cells were found in the cranial mesenchyme of all groups of experimental embryos especially in those with grafts in the Mid-Ant and lateral ectoderm (Table 4). When cells were grafted to Mid-Ant ecto derm, 46% of the embryos showed colonization of somitomeres II and III (Table 6). Labelled cells grafted to lateral areas (P-Lat and D-Lat) of the egg cylinder and to regions adjacent to the primitive streak (ML-PS and PL-PS) colonized predominantly the rhomben-cephalic somitomeres (Table 6). Only 3 examples of colonization of the cranial somitomeres were encountered in 42 embryos receiving grafts at Ant-Mar and Arch regions.

Table 6.

The distribution of WGA–gold-labelled cells in the cranial mesenchyme, somitic mesoderm and lateral plate mesoderm of the chimaeric embryos

The distribution of WGA–gold-labelled cells in the cranial mesenchyme, somitic mesoderm and lateral plate mesoderm of the chimaeric embryos
The distribution of WGA–gold-labelled cells in the cranial mesenchyme, somitic mesoderm and lateral plate mesoderm of the chimaeric embryos

In embryos receiving grafts at P-Lat and PL-PS regions, some labelled cells were found subjacent to the surface ectoderm on the lateral aspect of the cranial mesenchyme (Fig. 8). These were probably neural crest cells since they were localized along the migratory path where such cells are expected (Chan & Tam, 1988). Mesencephalic neural crest cells were derived from grafts in the P-Lat ectoderm and rhombencephalic neural crest cells came from grafts in the PL-PS ectoderm (Table 6).

Fig. 8.

A transverse section through the hindbrain level of an embryo cultured for 24 h after P-Lat grafting showing the presence of labelled cells (arrowheads) in the neuroepithelium (ne) and at the lateral subectodermal position along the putative migratory path for neural crest cells.

Fig. 8.

A transverse section through the hindbrain level of an embryo cultured for 24 h after P-Lat grafting showing the presence of labelled cells (arrowheads) in the neuroepithelium (ne) and at the lateral subectodermal position along the putative migratory path for neural crest cells.

In older embryos receiving P-Lat and PL-PS grafts, WGA–gold-labelled cells were found in cellular clusters at the base of the branchial arches, which are reminiscent of primordia of trigeminal and acoustico-facial ganglia. Although these cells were likely to be neural crest cells, a placodal contribution cannot be ruled out. In embryos with P-Lat and D-Lat grafts, labelled cells were sometimes found in the mesenchymal core of the branchial arches (Fig. 9) but again it was not possible in this study to identify their tissue of origin as either neural crest or cranial somitomere.

Fig. 9.

A section along the long axis of the first branchial arch (I) of an embryo cultured for 44 h after D-Lat grafting showing labelled cells (arrowheads) in the mesenchyme of the branchial arch, p, first pharnyngeal pouch; II, second branchial arch.

Fig. 9.

A section along the long axis of the first branchial arch (I) of an embryo cultured for 44 h after D-Lat grafting showing labelled cells (arrowheads) in the mesenchyme of the branchial arch, p, first pharnyngeal pouch; II, second branchial arch.

(iv) Somitic mesoderm and lateral plate mesoderm

Colonization of the somitic mesoderm (Fig. 10) was observed predominantly in embryos receiving grafts at Arch and ML-PS sites and to a lesser extent at D-Lat sites in the embryonic ectoderm (Table 4). Labelled cells grafted at the Arch and ML-PS ectoderm colonized the first 8-9 somites and the presomitic mesoderm. Labelled cells derived from the lateral ectoderm were allocated to the first three somites (Table 6).

Fig. 10.

A sagittal section through the trunk of an embryo cultured for 44 h after ML-PS graft showing the colonization of somites by WGA–gold-labelled cells (arrowheads), se, surface ectoderm; scl, sclerotome; dm, dermamyotome. Bars= 100μ. Silver-enhanced and Fast Green.

Fig. 10.

A sagittal section through the trunk of an embryo cultured for 44 h after ML-PS graft showing the colonization of somites by WGA–gold-labelled cells (arrowheads), se, surface ectoderm; scl, sclerotome; dm, dermamyotome. Bars= 100μ. Silver-enhanced and Fast Green.

Colonization of the lateral plate mesoderm was observed primarily after grafts were made to the D-Lat embryonic ectoderm and to the ectoderm adjacent to the primitive streak (Table 4). The graft-derived cells colonized both the somatopleure and the splanchno-pleure at the level of the lower hindbrain to first 4 somites (Table 6).

(v) Other embryonic tissues

Extensive colonization of the notochord was found in embryos receiving grafts at the archenteron (Table 4). In 5 out of the 14 cases, labelled cells were distributed in the notochord over a length equivalent to 5-6 somites. Donor cells colonized the ectodermal lining of the stomadeum, the buccopharyngeal membrane and the foregut endoderm of embryos receiving Ant-Mar grafts (Table 4). Two of the 13 Mid-Ant chimaeras also contained labelled cells in the oral ectoderm, but not in the foregut endoderm. Colonization of the endoderm of mid- and hind-gut occurred in 6 cases following grafting to Arch and ML-PS sites (Table 4). Labelled cells were found in the epimyocardium and pericardium of 4 embryos in the Ant-Mar and P-Lat groups and in 1 D-Lat embryo (Table 4).

The developmental fate of cells located in the anterior and lateral regions of the embryonic ectoderm of intact late-primitive-streak-stage embryos has been examined. Experimental evidence has been obtained for regionalisation in the deployment of embryonic ectoderm cells to specific segments of the neural tube and other ectodermal tissues during gastrulation. The study of the mesodermal fate of embryonic ectoderm was, however, fraught with technical problems and proved less conclusive.

Technical consideration

In the present study, labelled cells were isolated from four major quadrants of the embryonic ectoderm and were grafted back to various sites in their quadrant of origin. Grafting of cells to exactly orthotopic sites was not attempted because of technical difficulties. There is, however, no significant variation in the pattern of tissue colonization among chimaeric embryos of the same grafting groups. Indeed, it has been shown that when embryonic ectoderm is grafted to heterotopic sites, the cells may change their normal fate to conform with the pattern of tissue differentiation of the new locations in the embryonic ectoderm (Beddington, 1982). The one exception is the anterior marginal ectoderm which is predisposed to produce ectodermal derivatives. Since in the present study, donor cells were always grafted to quasi-orthotopic sites, the differentiation of the graft-derived cells is likely to reflect the normal fate of cells at the selected sites in the embryonic ectoderm.

One major drawback of the microsurgical grafting technique is the possibility that, despite great caution taken to place the graft within the embryonic ectoderm, some grafted ectodermal tissue may accidentally be grafted to the subjacent germ layers. The passage of the injection micropipette through the tissue layers might also create artefactual channels through which grafted cells in the ectoderm could readily migrate to other germ layers. A survey of 40 embryos examined at 4–5 h after grafting did reveal that, in about 20% of cases, grafted cells were distributed to both the ectoderm and mesoderm which might result in the colonization of tissues derived from both germ layers in these chimaeric embryos. In another 12% of cases, the grafted cells were not found in the embryonic ectoderm and therefore colonization by graft-derived cells would probably be confined to mesodermal and endodermal tissues. In both cases, it would be difficult to draw any conclusion about the normal fate of embryonic ectodermal cells with respect to mesodermal and endodermal development. In the present study, chimaeric embryos that showed no colonization of ectodermal derivatives by WGA–gold-labelled cells were excluded on the assumption that they were the result of misintegration of grafts. Only embryos containing labelled cells in the ectodermal tissues were studied, with specific emphasis on external tissues such as the neural tube and the surface ectoderm. The results for internal (mesodermal and endodermal) tissues must be viewed with caution since they might be an artefact of grafting procedure.

The ectodermal derivatives

Results of the present grafting experiments demonstrate clearly that cells in the anterior regions of the embryonic ectoderm give rise to the neuroectoderm of the prosencephalon and mesencephalon. Cells destined for the rhombencephalon come from the ectoderm in the distal-lateral regions flanking the rostral end of the primitive streak. The neuroectoderm of the spinal cord is derived from the ectoderm overlying the archenteron (alias the node region) and lateral to the anterior and the middle regions of the primitive streak (sites 4 and 5, Fig. 11). The various segments of the brain and the trunk neural tube are represented in the correct cranio-caudal order along the anterior-posterior axis of the embryonic ectoderm of the primitive-streak-stage embryo. Labelled cells grafted to the midline of the embryo colonize the floor plate and the basal plate whereas cells grafted to more lateral positions end up predominantly on the lateral wall of the neural tube. It seems therefore that not only the craniocaudal pattern but also the dorsoventral orientation of the neural tube is established within the embryonic ectoderm at this stage of gastrulation.

Fig. 11.

The normal fate of cells in the embryonic ectoderm of the primitive-streak-stage mouse embryo studied by orthotopic graftings of labelled cells (Data from the present study: grafts 1–4 and 6–8; Tam & Beddington, 1987: grafts 5 and 9–11; Beddington, 1981, 1982 and Copp et al. 1986: grafts 1,6 and 11). The dotted lines mark the boundary of the various brain parts and the spinal cord. For results obtained in the present study, only data showing colonization of the tissue in over 20% of the chimaeras were used for constructing this map. Neural tube (NTube): Pros, prosencephalon; Di∼Me, diencephalic-mesencephalic junction; Me, mesencephalon; Me—Rh, mesencephalic-rhombencephalic junction; Rh, rhombencephalon; SpC, spinal cord. Surface ectoderm (SEct): CrSEct, head surface ectoderm; TrSEct, trunk surface ectoderm; Pl, trigeminal placode or otic placode/vesicle. Ectomesenchyme (Ect∼Mes): BAr, branchial arch mesenchyme; NCC, presumptive neural crest cells. Paraxial mesoderm (PxMeso): Sme, cranial somitomeres I-VII; Som, somites and presomitic mesoderm. Other mesodermal tissue (MiscMes): LPM, lateral plate mesoderm including somatopleure and splanchnopleure; TB, caudal mesenchyme; ExtEmb, extraembryonic mesoderm of the amnion, yolk sac and allantois. Gastrointestinal tract (GITract): OrEct, ectodermal lining of the oral cavity and buccopharyngeal membrane; MGN, midgut endoderm; HGN, hindgut endoderm. Others: Noto, notochord; PS, primitive streak; PGC, primordial germ cells.

Fig. 11.

The normal fate of cells in the embryonic ectoderm of the primitive-streak-stage mouse embryo studied by orthotopic graftings of labelled cells (Data from the present study: grafts 1–4 and 6–8; Tam & Beddington, 1987: grafts 5 and 9–11; Beddington, 1981, 1982 and Copp et al. 1986: grafts 1,6 and 11). The dotted lines mark the boundary of the various brain parts and the spinal cord. For results obtained in the present study, only data showing colonization of the tissue in over 20% of the chimaeras were used for constructing this map. Neural tube (NTube): Pros, prosencephalon; Di∼Me, diencephalic-mesencephalic junction; Me, mesencephalon; Me—Rh, mesencephalic-rhombencephalic junction; Rh, rhombencephalon; SpC, spinal cord. Surface ectoderm (SEct): CrSEct, head surface ectoderm; TrSEct, trunk surface ectoderm; Pl, trigeminal placode or otic placode/vesicle. Ectomesenchyme (Ect∼Mes): BAr, branchial arch mesenchyme; NCC, presumptive neural crest cells. Paraxial mesoderm (PxMeso): Sme, cranial somitomeres I-VII; Som, somites and presomitic mesoderm. Other mesodermal tissue (MiscMes): LPM, lateral plate mesoderm including somatopleure and splanchnopleure; TB, caudal mesenchyme; ExtEmb, extraembryonic mesoderm of the amnion, yolk sac and allantois. Gastrointestinal tract (GITract): OrEct, ectodermal lining of the oral cavity and buccopharyngeal membrane; MGN, midgut endoderm; HGN, hindgut endoderm. Others: Noto, notochord; PS, primitive streak; PGC, primordial germ cells.

Other ectodermal tissues besides the neuroectoderm are also derived from cells of the embryonic ectoderm. Cells in the more proximal areas of the lateral embryonic ectoderm contribute to the cranial surface ectoderm, the epidermal placode and the neural crest cells. A transplantation study in the mid-to late-primitive-streak-stage chick embryo shows that a crescent-shaped zone in the anterior and lateral aspects of the epiblast is destined for neural crest cells and peripheral to this zone are those cells destined for the epidermis (Rosen-quist, 1981). If such a neural crest cell zone could be taken to demarcate the boundary of the neural jirimor-dium then the neural plate of the mouse embryo is occupying a major portion of the anterior and lateral regions of the embryonic ectoderm leaving a small area in the proximal-lateral embryonic ectoderm for the non-neural ectodermal tissues. Fig. 11 shows the subdivision of the neural primordium into domains for the brain segments and the spinal cord, based on the distribution of graft-derived cells in major neural tube segments and particularly at junctions between segments. The forebrain and the midbrain occupy a relatively small area making up about one third of the anterior embryonic ectoderm. The hindbrain covers the distal area of the anterior embryonic ectoderm and most of the lateral region. The trunk neural tube occupies the node areas and extends posteriorly and proximally to embryonic ectoderm lateral to the primitive streak. Taking into account the cup-shaped configuration of the primitive-streak-stage mouse embryo, there is a remarkable similarity in the spatial pattern of brain segments in the embryonic ectoderm when compared to that in the epiblast of the stage 4-5 chick embryo. In the chick, the neural primordium is mapped in a series of wedges stretching from the prenodal to the postnodal-areas in the epiblast adjacent to the cranial end of the primitive streak (Nicolet, 1971; Rosenquist, 1981; Packard, 1986).

Labelled cells derived from grafts in the anterior-marginal embryonic ectoderm also colonize the ectodermal lining of the oral cavity, the buccopharyngeal membrane and the Rathke’s pouch, in addition to lamina terminalis and the diencephalic (neurohypophyseal) diverticulum of the forebrain. It is interesting to draw a comparison with the prosencephalic plate of the early-somite-stage avian embryo. Using chick-quail chimaeras, the anterior neural primordium of the avian embryo is found to give rise to the typical neural structures such as the telencephalon and diencephalon and also to non-neural tissue including the lining of the nasal and oral cavities, the prosencephalic neural crest cells and the hypophysis (Couly & Le Douarin, 1985, 1987; and the morphological studies by Takor Takor & Pearse, 1975).

The relative size of different parts of the neural tube as mapped onto the embryonic ectoderm of the primitive-streak-stage embryo is not in proportion to their ultimate size at subsequent stages of development. Although the forebrain and midbrain occupy a small portion of the anterior embryonic ectoderm, by the early-somite stage they become the most prominent brain segments and constitute over half the tissue volume of the head folds (Morriss-Kay, 1981; Jacobson & Tam, 1982). This is probably the result of differential tissue growth in the neural tube. Indeed, it has been shown in the rat embryo that the most active tissue growth is encountered in the developing forebrain, which may account for the rostral and lateral expansion of this part of the brain during neurulation (Tuckett & Morriss-Kay, 1985). Concomitant to the morphogenesis of the neural tube, there is an enormous expansion in the area of the proximal-lateral embryonic ectoderm leading to the separation of the hindbrain and the spinal cord which are originally juxtaposed in the embryonic ectoderm of the primitive-streak-stage embryo. Analysis of the movement of cells in the proximal regions of the embryonic ectoderm has revealed a relocation of cells converging towards the primitive streak during gastrulation (Lawson et al. 1987) similar to that de scribed in the chick epiblast (Vakaet, 1984). Such cell movement is probably linked to the anisotropic growth of the egg cylinder in the posterior and distal direction (Tam & Meier, 1982) and could be achieved by the active proliferation of cells in the lateral and distal embryonic ectoderm (Snow, 1977; Poelmann, 1980). The expansion in tissue areas can also be achieved by the changes in cell size and shape as exemplified by the attenuation of the epithelium of the surface ectoderm accompanying neurulation in the rat embryo (Morriss-Kay, 1981).

Mesodermal derivatives

Results of tissue colonization by donor cells in this study suggested that precursor tissues for the paraxial mesoderm and the lateral plate mesoderm are largely confined to the distal-lateral and posterior regions of the embryonic ectoderm close to the primitive streak. Colonization of the first three cranial somitomeres occurs in 1 out of 20 Ant-Mar and 6 out of 13 Ant-Mid chimaeric embryos. A minor contribution to ‘loose head mesoderm’ and even heart mesoderm by grafts of anterior ectoderm has previously been reported (Beddington, 1981, 1982). As previously discussed, it is doubtful whether this actually reflects the normal fate of cells in this regions of the embryonic ectoderm. The translation of the anterior and lateral ectoderm into the cephalic neural tube necessitates a forward displacement of a coherent epithelial tissue and a concomitant expansion of the posterior ectoderm adjacent to the streak to generate the spinal cord and the paraxial mesoderm. If the colonization of the rhombencephalic mesoderm by Ant-Mid graft is real then the generation of such mesodermal tissues from an anterior sites in the epithelial sheet would pose a difficult mechanistic problem. A specific group of prospective mesodermal cells will have to move towards the streak in a direction opposite to that of tissue sheet expansion and then to reverse their course after invagination to reach their final segmental position. Alternatively, the colonization of the mesoderm by labelled cells could readily be explained by a misplaced graft or a local delamination of the grafted cells from the embryonic ectoderm.

More extensive colonization of the paraxial mesoderm begins with the 4th cranial somitomeres and extended to all subsequent somites in embryos receiving grafts in the lateral and posterior embryonic ectoderm adjacent to the primitive streak. Previous grafting experiments carried out for cells in the primitive streak and adjacent to the streak (Fig. 11: sites 5, 9—11; Beddington, 1981; Tam & Beddington, 1987) have demonstrated a significant contribution to the paraxial mesoderm and lateral mesoderm during gastrulation of the mouse embryo. That colonization by graft-derived cells began with the rhombencephalic somitomeres also agrees with the morphological finding that the first three somitomeres are already established in the mesoderm at the mid-to late-primitive-streak stage (Tam & Meier, 1982) and with the result of grafting studies that newly recruited paraxial mesoderm from the embryonic ectoderm and the primitive streak is allocated to more caudal somitomeres (Tam & Beddington, 1986, 1987). Similar to the situation of the ectodermal derivatives, there is again striking homology in the mesodermal fate of cells in the mouse embryonic ectoderm and that of the chick epiblast at the primitive streak stage of development. In both cases, the paraxial mesoderm is located lateral to the anterior region of the primitive streak and posterior to the Hensen’s node. The lateral plate mesoderm is found adjacent to and within the middle region of the primitive streak, whereas the extraembryonic mesoderm is associated with the posterior part of the streak (Nicolet, 1971; Meier & Jacobson, 1982; Packard, 1986; Copp et al. 1986; Tam & Beddington, 1987).

Regionalisation of the embryonic ectoderm

Sufficient information is now available to build a map of prospective ectodermal and mesodermal tissues for the embryonic ectoderm of the primitive-streak-stage mouse embryo. Fig. 11 shows the grafts of embryonic ectoderm that have been investigated: sites 1-4 and 6-8 from the present study and sites 5 and 9-11 from study by Tam & Beddington (1987). Additional information for sites 1, 6 and 11 is provided by similar grafting studies by Beddington (1981, 1982) and Copp et al. (1986). The neural tube occupies most of the anterior and lateral embryonic ectoderm. The proximal areas of the embryonic ectoderm contain the cells destined for the surface ectoderm in the head and oral regions, the epidermal placodes and the neural crest cells. Paraxial mesoderm (rhombencephalic somitomeres and somites) is mapped to the embryonic ectoderm in the distal-lateral regions and adjacent to the primitive streak. Within the primitive streak, distinctive regional diversity of tissue fate is observed: the anterior region is associated with the trunk neural tube and paraxial mesoderm, the middle region with the lateral plate mesoderm and caudal mesoderm and the posterior region with the extraembryonic mesoderm and primordial germ cells (Copp et al. 1986; Tam & Beddington, 1987). The present fate map, which is based upon the result of grafting experiments, is very similar to the map constructed by Snow (1981) using an entirely different experimental approach: the prospective fate of the embryonic ectoderm is deduced from the types of embryonic tissues formed in embryo fragments containing a full complement of germ layers. Although a spatial pattern of tissue diversification has been demonstrated within the embryonic ectoderm by these studies, it does not imply that the cells are precommitted to specific lineages prior to gastrulation. Although the consensus of studies using heterotopic grafts and ectopic teratomas indicates that there is little regional restriction in the developmental potency of embryonic ectoderm cells (Beddington, 1983; Svajger et al. 1986), definitive information on cell potency has to be obtained by a proper clonal analysis of individual cells of the embryonic ectoderm (Lawson et al. 1987).

Batten
,
B. E.
&
Haar
,
J. L.
(
1979
).
Fine structural differentiation of germ layers in the mouse at the time of mesoderm formation
.
Anat. Rec
.
194
,
125
142
.
Beddington
,
R. S. P.
(
1981
).
An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo
.
J. Embryol. exp. Morph
.
64
,
87
104
.
Beddington
,
R. S. P.
(
1982
).
An autoradiographic analysis of tissue potency in different regions of the embryonic ectoderm during gastrulation in the mouse
.
J. Embryol. exp. Morph
.
69
,
265
285
.
Beddington
,
R. S. P.
(
1983
).
Histogenetic and neoplastic potential of different regions of the embryonic egg cylinder
.
J. Embryol. exp. Morph
.
75
,
189
204
.
Beddington
,
R. S. P.
(
1987
).
Isolation, culture and manipulation of post-implantation mouse embryos
.
In Mammalian Development. A Practical Approach
(ed.
M.
Monk
),
IRL Press
,
Oxford
, pp.
43
70
.
Chan
,
W. Y.
&
Tam
,
P. P. L.
(
1988
).
A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin-gold conjugate as the cell marker
.
Development
102
,
427
442
.
Copp
,
A. J.
,
Roberts
,
H. M.
&
Polani
,
P. E.
(
1986
).
Chimaerism of primordial germ cells in the early postimplantation mouse embryo following microsurgical grafting of posterior primitive streak cells in vitro
.
J. Embryol. exp. Morph
.
95
,
95
115
.
Couly
,
G. F.
&
Le Douarin
,
N. M.
(
1985
).
Mapping of the early neural primordium in quail-chick chimaeras. I. Developmental relationships between placodes, facial ectoderm and prosencephalon
.
Devi Biol
.
110
,
422
439
.
Couly
,
G. F.
&
Le Douarin
,
N. M.
(
1987
).
Mapping of the early neural primordium in quail-chick chimaeras. II. The prosencephalic neural plate and neural fold: implications for the genesis of cephalic human congenital abnormalities
.
Devi Biol
.
120
,
198
214
.
Diwan
,
S. B.
&
Stevens
,
L. C.
(
1976
).
Development of teratomas from ectoderm of mouse egg cylinders
.
J. natn. Cancer Inst
.
57
,
937
942
.
Hunter
,
E. S.
,
Balkan
,
W.
&
Sadler
,
T. W.
(
1988
).
Improved growth and development of presomitic mouse embryos in whole embryo culture
.
J. exp. Zool
.
245
,
264
269
.
Jacobson
,
A. G.
&
Tam
,
P. P. L.
(
1982
).
Cephalic neurulation in the mouse embryo analyzed by SEM and morphometry
.
Anat. Rec
.
203
,
375
396
.
Lawson
,
K. A.
,
Meneses
,
J. J.
&
Pederson
,
R. A.
(
1987
).
Germ layer formation in the mouse embryo studied with an intracellular lineage tracer
.
Abstract of Bath Symposium on Craniofacial Development, British Society of Developmental Biology
.
Meier
,
S.
&
Jacobson
,
A. G.
(
1982
).
Experimental studies of the origin and expression of metameric pattern in the chick embryo
.
J. exp. Zool
.
219
,
217
232
.
Meier
,
S.
&
Tam
,
P. P. L.
(
1982
).
Metameric pattern development in the embryonic axis of the mouse. I. Differentiation of the cranial segments
.
Differentiation
21
,
95
108
.
Morriss-Kay
,
G. M.
(
1981
).
Growth and development of pattern in the cranial neural epithelium of rat embryos during gastrulation
.
J. Embryol. exp. Morph
.
65
Supplement
,
225
241
.
Nicolet
,
G.
(
1971
).
Avian gastrulation
.
Adv. Morphog
.
9
,
231
262
.
Packard
,
D. S.
, Jr
(
1986
).
The epiblast origin of avian somite cells
.
In Somites in Developing Embryos
, (ed.
R.
Bellairs
,
D. A.
Ede
&
J. W.
Lash
),
New York
:
Plenum Press
, pp.
37
45
.
Poelmann
,
R. E.
(
1980
).
Differential mitosis and degeneration patterns in relation to the alterations in the shape of the embryonic ectoderm of early post-implantation mouse embryos
.
J. Embryol. exp. Morph
.
55
,
33
-
51
.
Poelmann
,
R. E.
(
1981
).
The head process and the formation of the definitive endoderm in the mouse embryo
.
Anat. Embryol
.
162
,
41
49
.
Reinius
,
S.
(
1965
).
Morphology of the mesoderm, from the time of implantation to mesoderm formation
.
Z. Zellforsch. mikrosk. Anat
.
68
,
711
723
.
Rosenquist
,
G. C.
(
1981
).
Epiblast origin and early migration of neural crest cells in the chick embryo
.
Devi Biol
.
87
,
201
211
.
Snow
,
M. H. L.
(
1977
).
Gastrulation in the mouse: growth and regionalisation of the epiblast
.
J. Embryol. exp. Morph
.
42
,
293
303
.
Snow
,
M. H. L.
(
1981
).
Autonomous development of parts isolated from primitive-streak-stage mouse embryo. Is development clonal?
J. Embryol. exp. Morph
.
65
Supplement
,
269
287
.
Snow
,
M. H. L.
&
Bennett
,
D.
(
1978
).
Gastrulation in the mouse: assessment of cell populations in the epiblast of tw,8/tw18 embryos
.
J. Embryol. exp. Morph
.
47
,
39
52
.
Svajger
,
A.
,
Levak-Svajger
,
B.
&
Skreb
,
N.
(
1986
).
Rat embryonic ectoderm as renal isograft
.
J. Embryol. exp. Morph
.
94
,
1
27
.
Takor Takor
,
T.
&
Pearse
,
A. G. E.
(
1975
).
Neuroectodermal origin of avian hypothalamo-hypophyseal complex: the role of the ventral neural ridges
.
J. Embryol. exp. Morph
.
34
,
311
325
.
Tam
,
P. P. L.
&
Beddington
,
R. S. P.
(
1986
).
The metameric organization of the presomitic mesoderm and somite specification in the mouse embryo
.
In Somites in Developing Embryos
, (ed.
R.
Bellairs
,
D. A.
Ede
&
J. W.
Lash
),
New York
:
Plenum Press
, pp.
17
36
.
Tam
,
P. P. L.
&
Beddington
,
R. S. P.
(
1987
).
The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis
.
Development
99
,
109
126
.
Tam
,
P. P. L.
&
Meier
,
S.
(
1982
).
The establishment of a somitomeric pattern in the mesoderm of the gasatrulating mouse embryo
.
Am. J. Anat
.
164
,
209
225
.
Theiler
,
K.
(
1972
).
The House Mouse
.
Springer-Verlag
,
Berlin
.
Tuckett
,
F.
&
Morriss-Kay
,
G. M.
(
1985
).
The kinetic behaviour of the cranial neural epithelium during neurulation in the rat embryo
.
J. Embryol. exp. Morph
.
85
,
111
119
.
Vakaet
,
L.
(
1984
).
Early development of birds
.
In Chimaeras in Development
, (ed.
N. M.
Le Douarin
&
A.
McLaren
).
Academic Press
,
London
, pp.
71
88
.