Development of separated germ layers of rodent embryos on ectopic sites: a reappraisal.

The method of separation of germ layers of rodent embryos by treating the embryonic shields with proteolytic enzymes and by microsurgery with the subsequent transplantation to ectopic sites has helped to gain a more detailed insight into what is going on during gastrulation in mammals. The space under the kidney capsule of adult animals seems to be the most appropriate ectopic site for transplantation of early postimplantation rat embryos or separated germ layers. After transplantation the grafts develop into teratomas whose complex histological structure reflects the initial developmental capacities of the graft. At the pre-primitive streak and the early primitive streak stages the primitive ectoderm differentiates into tissue derivatives of all three definitive germ layers, often in complex organotypic combinations. This is indirect evidence that all cells of the embryonic body originate from the primitive embryonic ectoderm. Halves of the primitive ectoderm obtained by a longitudinal or transverse cut through the egg cylinder give the same result. At the head fold stage the capacity for differentiation of the ectoderm is restricted to ectodermal and mesodermal derivatives. One day before gastrulation the isolated primitive ectoderm is not able to differentiate as renal isograft. The mesoderm isolated at the head fold stage and at later stages when its segmentation occurs, differentiates almost exclusively into the brown adipose tissue. The embryonic endoderm differentiates only in combination with the mesoderm. After transplantation the embryonic ectoderm loses its epithelial organization and breaks up into a mass of mesenchyme-like cells in which epithelial structures subsequently appear and differentiate in a way reminiscent of the reaggregation of cells in mixed cell suspension in vitro.

Differentiation of the secondary elastic cartilage in the external ear of the rat.

The cartilage in the external ear of the rat belongs to the group of secondary cartilages and it has a unique structural organization. The chondrocytes are transformed into typical adipose cells, the proteoglycan cartilage matrix is reduced to thin capsules around the cells and the rest of the extracellular matrix is occupied by a network of coarse elastic fibers. It appears late in development (16-day fetus) and needs more than one month for final development. The differentiation proceeds in several steps which partly overlap: the appearance of collagen fibrils, elastin fibers, the proteoglycan matrix, and the adipose transformation of chondrocytes. The phenotype of this cartilage and the course of its differentiation are very stable, even in very atypical experimental environmental conditions. The only exceptions are explants in organ culture in vitro and perichondrial regenerates. In these conditions the development of elastic fibers is slow and poor while the production of the proteoglycan matrix is abundant. The resulting cartilage then displays structural characteristics of hyaline cartilage rather than those of the initial elastic one.

Morphogenetic behaviour of the rat embryonic ectoderm as a renal homograft.

Halves of transversely or longitudinally cut primary ectoderm of the pre-primitive streak and the early primitive streak rat embryonic shield developed after 15-30 days in renal homografts into benign teratomas composed of various adult tissues, often in perfect organ-specific associations. No clear difference exists in histological composition of grafted halves of the same embryonic ectoderm. The primary ectoderm of the pre-primitive streak rat embryonic shield grafted under the kidney capsule for 2 days displayed an atypical morphogenetic behaviour, characterized by diffuse breaking up of the original epithelial layer into mesenchyme. Some of these cells associated into cystic or tubular epithelial structures. The definitive ectoderm of the head-fold-stage rat embryo grown as renal homograft for 1-3 days gave rise to groups of mesenchymal cells. These migrated from the basal side of the ectoderm in a manner which mimicked either the formation of the embryonic mesoderm or the initial migration of neural crest cells. This latter morphogenetic activity was retained in the entire neural epithelium of the early somite embryo but was only seen in the caudal open portion of the neural groove at the 10- to 12-somite stage. The efficient histogenesis in grafts of dissected primary ectoderm and the atypical morphogenetic behaviour of grafted primary and definitive rat embryonic ectoderm were discussed in the light of current concepts on mosaic and regulative development, interactive events during embryogenesis and positioning and patterning of cells by controlled morphogenetic cell displacement.

Course of development of isolated rat embryonic ectoderm as renal homograft.

When the isolated head-fold stage rat embryonic ectoderm is grafted under the kidney capsule, it gives rise to a new mesenchyme with the capacity to differentiate into mesodermal tissues.

Technique of separation of germ layers in rat embryonic shields.

A microsurgical technique for the separation of germ layers in two- and three-layered rat embryonic shields is described.