Lucien Vakaet

Neural plate- and neural tube-forming potential of isolated epiblast areas in avian embryos

SummaryFormation, shaping, and bending of the neural plate and closure of the neural groove are complex processes resulting in formation of the neural tube. Two experiments were performed using avian embryos as model systems to examine these events. First, we transected blastoderms near the level of Hensens node to determine the potential of prenodal neural plate to form neural tube in isolation from primitive streak regression. Our results demonstrate that shaping and bending of the prenodal neural plate occur under these conditions, but neural groove closure is inhibited. Second, we isolated various areas of postnodal epiblasts to determine their potential to form neural plate. Our results suggest that the area of the postnodal epiblast that can form neural plate consists of paired tracts lying adjacent to the definitive primitive streak and extending caudally at least 1 mm from its cranial end.

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

SummaryA chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of 3H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of 3H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis

SummaryThe folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells.Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted.The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.

Fibronectin and its relation to the basal lamina and to the cell surface in the chicken blastoderm.

SummaryThe ultrastructural distribution of fibronectin immunoreactivity was investigated in the chicken embryo during late gastrulation. Sites of binding of anti-fibronectin antibodies were ascribed to the basal lamina and associated structures, and to the cell surface. The fibronectin-rich basal lamina was resolved into (1) a lamina densa, which appears as a continuous, dense sheet, (2) a lamina lucida, consisting of anchoring cords between lamina densa and epithelial cells, and (3) a lamina intima, closely juxtaposed to the cell surface. Cell-surface labelling was also observed in mesoblast cells, and along the dorsal side of the deep-layer cells. The ventral side of the latter cells was poorly stained in the endophyllic crescent, except in coated pits, and more regularly stained at the level of definitive endoblast. Some structures associated with the basal lamina reacted intensely with anti-fibronectin antibodies. These are (1) the interstitial bodies, which are aggregates of extracellular material, and (2) a kind of fibril or tubule, embedded in a fibronectin matrix and mainly found in the endophyllic crescent. Some intracellular labelling was found in most deep-layer cells, in few epiblast cells, never in mesoblast cells. These results extend previous studies on the localization of fibronectin, and correlate its presence and surface topology with its postulated role in migration of mesoblast cells on the basal lamina which, chemically, constitutes an appropriate substrate.

Time-lapse cinephotomicrography, videography, and videomicrography of the avian blastoderm.

Publisher Summary This chapter discusses the time-lapse cinephotomicrography, videography, and videomicrography of the avian blastoderm. Morphogenetic movements can be revealed by time-lapse cinephotomicrography or videography. Time-lapse cinephotomicrography or videography is now in widespread use in research and is an essential tool in the modern teaching of developmental biology. The goal of the research is to understand the mechanisms and dynamics of morphogenetic movements during gastrulation and neurulation. For the study described in the chapter, the setups for time-lapse videography and videomicrography were developed. Videography has superseded cinephotomicrography because sequences are copied faster, tape does not break as easy as film and does not scratch, video sequences can be observed forward and backward and as still images, and editing video sequences is less time-consuming. Video registrations have led to the new concept of mitotic pressure as a major mechanism of morphogenetic movements during gastrulation and neurulation. Video registration is a basic technique in the study of developmental biology as only video registration allows morphogenetic movements to be observed directly. It allows access to the fourth dimension: the evolution of shape in time, as morphogenesis may be called.

Microinjection in the chick blastoderm: An improved method to study the extracellular matrix in the living organism

A microinjection technique for the chick blastoderm is described. With a micropipette attached to a de Fonbrune micromanipulator, 25-45 nl of a reagent was injected into the entophyllic crescent of a chick blastoderm explanted in vitro according to New [7]. This procedure offers the advantage of eliminating the concentration variability which was observed after subblastodisc injection, and in contrast to the in ovo techniques, it allows one to stage the blastoderms properly. To check its applicability, testicular hyaluronidase was injected. On the basis of morphological and histochemical observations we ascertained that the experimental procedure itself did not interfere with the results. This method may provide a reliable experimental procedure with which to study the interactions between several macromolecules and the tissues during morphogenesis.

Connecting cords and morphogenetic movements in the quail blastoderm

SummaryConnecting cords are elongated telophase bridges persisting between separating daughter cells. We have studied them with Scanning Electron Microscopy in the upper cell layer of the quail blastoderm where a high mitotic activity accompanied by interkinetic nuclear migration coincides with morphogenetic movements. The predominant orientation of the connecting cords is parallel to the direction of the morphogenetic movements.

Mesoblast Anlage Fields in the Upper Layer of the Chicken Blastoderm at Stage 5V

The intraembryonic mesoblast of the chicken embryo originates from the upper layer by ingression through the primitive streak during gastrulation. There is no agreement about the disposition of the Anlage fields of the mesoblast, as appears from the reviews by Rudnick (1944) and Waddington (1952) and the maps of Rosenquist (1966) and Vakaet (1984).

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence

SummaryShortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1974) obtained by autoradiography after 3H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged “retarding” effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.

Mapping of Gastrulation Movements in Birds

Mapping gastrulation movements in Birds is theoretically impossible: a map is a still image and shows at best a snapshot of gastrulation. A map cannot show movements even when it is armed with arrows: to that end movies are needed. To enable the registration and reproduction of the movements within avian blastoderms during the first 24 hours of incubation, a cinephotomicrographic device was set up. The results of these observations are preceded by a demonstration of the procedure for preparing the germs. This procedure is derived from that of New (1955). Its main characteristic is that it allows normal development in vitro for a 24 hour period in laid blastoderms that are cultivated with their ventral side up, towards the observer.