The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo

The HNF-3 alpha, beta and gamma genes constitute a family of transcription factors that are required for hepatocyte-specific gene expression of a number of genes, e.g. transthyretin, alpha-1 antitrypsin and tyrosine aminotransferase. These genes share a highly conserved DNA-binding domain first found in the Drosophila gene, forkhead, which is required for the normal patterning of the developing gut and central nervous system in Drosophila. In adult mouse tissues, transcripts from HNF-3 alpha and beta have been localised to the liver, intestine and lung, whereas HNF-3 gamma is found in the liver, intestine and testis. In light of the early developmental significance of forkhead in Drosophila, we have compared the patterns of expression of HNF-3 alpha, beta and gamma mRNAs during murine embryogenesis. We find that these genes are sequentially activated during development in the definitive endoderm. HNF-3 beta mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated. Subsequently, HNF-3 alpha is transcribed in the primitive endoderm in the region of the invaginating foregut and HNF-3 gamma appears upon hindgut differentiation. These genes have different anterior boundaries of mRNA expression in the developing endoderm and transcripts are found in all endoderm-derived structures that differentiate posterior to this boundary. Therefore, we propose that these genes define regionalization within the definitive endoderm. Furthermore, differential mRNA expression of HNF-3 alpha and beta is detected in cells of the ventral neural epithelium, chordamesoderm and notochord. In the neural epithelium, expression of HNF-3 alpha and beta mRNA becomes localised to cells of the floor plate. We propose that, in addition to their characterised requirement for liver-specific gene expression, HNF-3 alpha and beta are required for mesoderm and neural axis formation. We also conclude that HNF-3 beta is the true orthologue of the Drosophila forkhead gene.

Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm

Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

Two distinct patterns of chimaerism were found in conceptuses produced by injecting dissociated 4.5-day inner cell mass cells into genetically dissimilar blastocysts. Pattern 1: donor cells were found in the endoderm layer of the visceral yolk sac, but not in the adjacent mesoderm layer of this organ or in the foetus itself. Pattern 2: donor cells were found in the mesoderm layer of the visceral yolk sac and/or foetus, but never in the yolk-sac endoderm as well. Primitive endoderm cells of donor inner cell masses are responsible for the first pattern and primitive ectoderm cells for the second. These results, together with those of previous studies, suggest that the entire foetus, including its endodermal components, is formed from the primitive ectoderm, and that primitive endoderm forms only extra-embryonic endoderm of the conceptus.

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Investigation of the fate of 4.5 Day post-coitum mouse inner cell mass cells by blastocyst injection.

Les feuillets du cylindre-AEuf du rat ont ete disjoints par une solution composee de deux enzymes: 0,5% de trypsine et 2,5% de pancreatine. La separation a ete achevee par une aiguille de tungstene. Les feuillets ainsi detaches retiennent leur pouvoir de croissance et de differenciation comme homogreffes, sous la capsule renale.

Separation of germ layers in presomite rat emgryos.

Single germ layers (or combinations of two of them) were isolated from the primitive streak and the head-fold stage rat embryos and grown for 15 days under the kidney capsule of syngeneic adult animals. The resulting teratomas were examined histologically for the presence of mature tissues, with special emphasis on derivatives of the primitive gut.

Ectoderm isolated together with the initial mesodermal wings at the primitive streak stage gave rise to tissue derivatives of all three definitive germ layers. Derivatives of the primitive gut were regularly present in these grafts. At the head-fold stage, isolated ectoderm (including the region of the primitive streak) differentiated into ectodermal and mesodermal derivatives only.

Endoderm isolated at the primitive streak stage did not develop when grafted and was always completely resorbed. At the head-fold stage, however, definitive endoderm differentiated into derivatives of the primitive gut if grafted together with adjacent mesoderm.

These findings indirectly suggest the migration of prospective endodermal cells from the primitive ectoderm, and therefore a general analogy with the course of events during gastrulation in the chick blastoderm.

Investigation on the origin of the definitive endoderm in the rat embryo.

Two groups of embryonic shields were transferred under the kidney capsule, on days 7 ½ and 9 of normal pregnancy. The first group was without mesoderm and the second one with well-formed mesoderm. Random-bred and inbred Fischer rats were used. Killed and fixed 15 and 30 days after the operation, the grafts looked like teratomata with well-differentiated tissues. Differentiation was the same in embryos explanted at 7½ and 9 days, and from the two strains of rat. In order to study the growth potential, transfers within and between each strain of rat were carried out. A difference in weight was found 15 days after the operation among some series. This was not observed 30 days after the transfer. Although the grafts in random-bred rats were larger than in the inbred Fischer strain, factors other than the immunological influence were invoked for the explanation of the initial difference in growth. Long-term grafts were studied only in the inbred strain. Two, 4 and 6 months after the operation all well-differentiated tissues were still present. The amount of growth was very variable within each series, but no graft was found totally resorbed. Only in the largest graft, 6 months after the operation, large masses of cells were found which seemed to be undifferentiated.

Growth and differentiation of rat egg-cylinders under the kidney capsule

Die innere und die aussere Zellschicht 8 Tage alter Rattenkeimscheiben wurden enzymatisch voneinander getrennt und unter die Nierenkapsel erwachsener Ratten verpflanzt. Die isolierte innere Zellschicht (primitives Ektoderm) differenzierte sich nicht nur in ektodermale und mesodermale, sondern auch in typisch endodermale Gewebe (Darmepithel). Es wird daraus gefolgert, dass in diesem Entwicklungsstadium die innere bzw. aussere Zellschicht noch nicht als das definitive Ekto- bzw. Endoderm angesehen werden konnen.

Differentiation of endodermal tissues in homografts of primitive ectoderm from two-layered rat embryonic shields

Experimental results obtained many years ago revealed that during gastrulation (with the primitive streak and the mesoderm formation as distinct features) the early rodent embryo undergoes essential changes in its response to extrinsic teratogens (Russell & Russell, 1954; Wilson, 1954; Skreb, 1961; Skreb & Bijelic, 1962; Skreb & Frank, 1963). It has also been shown that the ultrastructural, histochemical and biosynthetic features of the embryo are subject to substantial changes during this period (Solter, Damjanov & Skreb, 1970, 1973; Dziadek & Adamson, 1978; Bode & Dziadek, 1979; Wartiovaara, Leivo & Vaheri, 1979; Jackson et al. 1981; Franke et al. 1982 a, b ). This suggests a restriction of developmental capacities (i.e. the loss of the capacity of regulation) in groups of embryonic cells at this developmental stage. According to the current concept, the initial cell population from which this restriction starts, resides within the embryonic ectoderm of the pregastrulation or preprimitive streak embryo (primitive or primary ectoderm).

B. Levak-Švajger

Papers

Separation of germ layers in presomite rat emgryos.

Investigation on the origin of the definitive endoderm in the rat embryo.

Growth and differentiation of rat egg-cylinders under the kidney capsule

Differentiation of endodermal tissues in homografts of primitive ectoderm from two-layered rat embryonic shields

Rat embryonic ectoderm as renal isograft.