The chick embryo: hatching a model for contemporary biomedical research

Animal models play a crucial role in fundamental and medical research. Progress in the fields of drug discovery, regenerative medicine and cancer research among others are heavily dependent on in vivo models to validate in vitro observations, and develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical and technical issues that limit their usage. The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis and loss/gain of function experiments. It is also an established model for tissue/cell transplantation, and because of its lack of immune system in early development, the chick embryo is increasingly recognised as a model of choice for mammalian biology with new applications for stem cell and cancer research. Here, we review novel applications of the chick embryo model, and discuss future developments of this in vivo model for biomedical research.     

Induced mutation to monocotyledony in periwinkle,Catharanthus roseus, and suppression of mutant phenotype by kinetin

A recessive EMS-induced mutation inherited in Mendelian fashion caused monocotyledonous embryo formation and seed germination on high salt medium inCatharanthus roseus. Availability during embryo development of exogenously supplied cytokinin kinetin suppressed the mutant phenotype. These observations suggest that, inC. roseus, (i) insufficiency in endogenous kinetin may lead to monocotyledonous embryo patterning and (ii) dicotyledonous embryo formation requires a critical amount of kinetin in certain cells of early embryos.     

LATE OVULE AND EARLY EMBRYO DEVELOPMENT IN QUERCUS GAMBELII

The ovary of Quercus gambelii is tricarpellary and trilocular; each locule contains two anatropous, bitegmic ovules. The formation of a caecum from a chalazal extension of the embryo sac was observed. A central resistant column of cells, which exhibit some continuity with the vascular trace supplying the ovule, remains protruding into the embryo sac from the chalaza until the later stages of embryo development. Early embryogeny is characterized by a nearly vertical first division with both of the derivative cells contributing to the building of the embryo proper. Such observations suggest that the embryogeny of Quercus should be placed in the Piperad or Asterad type rather than the Onagrad type as has been the practice.     

Seed dormancy in Acer: The role of abscisic acid in the regulation of seed development in Acer platanoides L.

Germinability of isolated embryos from developing fruits of Acer platanoides was high at the earliest developmental stage assessed (90 dpa), but fell subsequently and at seed maturity was very low. These observations showed an inverse correlation with changes in endogenous free abscisic acid (ABA) levels in the embryo, which were low during early ontogeny, but reached maximum levels late in development (150–160 dpa). These observations suggest the possibility that dormancy may be induced during development as a result of ABA accumulation in the embryo, an argument strengthened by the obvious inhibitory effect of added ABA on the germinability of isolated embryos. The cotyledons appear to exert an inhibitory influence on embryo germinability that may result from their free ABA content although the embryonic axis itself possesses an innate dormancy that may reflect its own free ABA content. The increased germinability of isolated embryos resulting form added kinetin serves only to emphasise the complexity of the system and the dangers of simplistic interpretation.The correlation between germinability and ABA content is not complete, however, since much of the reduction in germinability had occurred before any appreciable increase in free ABA levels in the embryo was observed. Indeed the failure of the intact seed to respond to endogenous changes in embryonal ABA levels suggests that even though free ABA in the embryo may influence embryo germinability, it has little effect in the intact seed, where the presence of an intact testa may be a more important factor.The absence of a desiccation phase in the embryo during the late stages of development suggests that the large increases in endogenous free ABA did not cause dormancy by inhibiting water uptake, nor did they result from water stress in the embryonal tissues.     

EMBRYO SAC DEVELOPMENT IN THE CV. KS MALE-STERILE,FEMALE-STERILE LINE OF SOYBEAN (GLYCINE MAX)

Light microscopic observations were made on 22 ovules from fertile plants and 108 ovules from sterile plants of the cv. KS synaptic mutant, a highly male-sterile, female-sterile line of soybean [Glycine max (L.) Merr.] (2n = 2x = 40). Ovules of fertile siblings contained normal embryo sacs and embryos. Ovules from sterile plants contained various irregularities. The most consistent abnormality was the failure of the embryo sac to attain normal size. Small megasporocytes of irregular shape were seen; only one megasporocyte of normal shape and size was noted. No linear tetrads were found. However, two ovules contained nonlinear triads. A range from zero to 28 cells and nuclei, of various sizes, were identifiable in small megagametophytes and embryo sacs. Degeneration of these nuclei and cells was noted as early as the four-nucleate gametophyte stage. Other ovules contained degenerated nucellar centers without embryo sacs. Two ovules appeared to be normal. Late postpollination stages were marked by shrunken nucellus and integuments. The presence of pollen tube traces, endosperm, and aborting embryos in ovules of hand-pollinated flowers from sterile plants suggested that no incompatibility was involved. Degeneration of the gametophyte and embryo sac contents at many developmental stages indicated a wide array of effects, possibly resulting from meiotic irregularities similar to those seen in microsporogenesis of this mutant.     

An embryological study ofPlatanthera bifolia (Orchidaceae)

Flowers ofPlatanthera bifolia were hand-pollinated and fixed in FPA₅₀ after 2, 5, 7, 14, and 21 days. Ovules, made transparent in Herr's clearing fluid, were investigated using confocal scanning laser microscopy. Pollination initiates the megasporogenesis. Two days after pollination dyads are frequent. Three days later most embryo sacs contain two nuclei. Seven days after pollination the embryo sacs are 4–8-nucleate and some are organized, and a week later all embryo sacs are organized and fertilization takes place. The embryo sac development follows thePolygonum type. Twenty-one days after pollination the egg nuclei have been fertilized and the embryo sacs contain 2- to many-celled embryos. A suspensor is formed during early stages of embryo development but degenerates later. Fertilization of the central nucleus does not lead to endosperm development.     

Cowpea embryo rescue. 1. Influence of culture media composition on plant recovery from isolated immature embryos

Factors responsible for successful rescue of immature embryos of cowpea [Vigna unguiculata (L.) Walp.] and V. vexillata (L.) and for in vitro embryo development were studied. A new basal medium for embryo development in vitro was formulated on the basis of the mineral composition of embryos. Sucrose, fructose and glucose were compared as carbohydrate sources. The highest frequency of embryos developing into plants was obtained with sucrose. Adding casein hydrolysate to the medium increased plant recovery by 30%. Among the plant growth factors used, cytokinins, zeatin, 6-benzylaminopurine and kinetin were the most effective in promoting embryo maturation and development. A method that can routinely ensure high plant recovery from cultured immature cowpea embryos is proposed. Received: 4 June 1996 / Revision received: 28 October 1996 / Accepted: 22 November 1996     

EMBRYOLOGICAL STUDY OF HERMINIUM MONORCHIS (ORCHIDACEAE) USING CONFOCAL SCANNING LASER MICROSCOPY

The embryology of Herminium monorchis (Orchidaceae) was studied using confocal scanning laser microscopy (CSLM), a new technique for embryological studies. This technique may contribute new information to plant embryology. Herminium monorchis has a monosporic embryo sac development. The mature embryo sac is 8-nucleate. Two integuments, both 2-layered, are formed, but only the inner takes part in formation of the micropyle. Double fertilization takes place. The primary endosperm nucleus does not divide, but remains alive at least at the 3-celled stage of embryo development. The three antipodals do not show any sign of degeneration at this stage.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.     

STRUCTURE OF THE SHOOT APEX IN LACTUCA SATIVA

Histological observations of the leaf lettuce ‘Black Seeded Simpson’ showed the dormant embryo to possess two visible leaves and a flkat to slightly depressed plumular apex. Observations conducted over a 12-day period of germination and growth showed the development of L₁ and L₂, emergence of L₃ and L₄, and periodic changes in size of the apex which were associated with leaf emergence. Thus the dormant embryo of Lactuca appears to be considerably more advanced in development than was previously believed. The shoot apex appeared flat to slightly depressed at all developmental stages studied.     

Reproductive Cycle and Developmental Processes During Embryogenesis of Clavularia hamra (Cnidaria,Octocorallia)

Clavularia hamra Gohar, 1948 is a common octocoral on the reefs of the Gulf of Eilat (northern Red Sea). Reproductive biology of C. hamra was studied for two years. Direct observations of spawning were conducted in situ and in aquaria. Cleavage of eggs and further embryo metamorphosis into mature planulae were examined by scanning electron microscopy and histological sections. Clavularia hamra is dioecious. Young oocytes appear annually in September, gradually grow in size and attain maturity within 11 months. The main spawning event of the population is highly synchronized, occurring on a single night each year between the last quarter and the new moon in mid summer. The released orange eggs are held together by mucus and remain attached amongst the polyps on the outside of the female colonies. Twelve hours after spawning, the young embryos have blastomeres of equal size with numerous microvilli on their external surface. Due to unequal cleavage, bizarre embryos are also formed. By 48 h a blastopore is visible, indicating that a gastrula is developing. Eight days after spawning mature planulae are observed. The external mode of embryo development on the surface of the parent colony reduces dispersal of the planulae. However, this reproductive feature enhances formation of locally dense populations of C. hamra, with distinct habitat preferences.     

Female meiosis in wild-type Arabidopsis thaliana and in two meiotic mutants

Female meiosis in Arabidopsis has been analysed cytogenetically using an adaptation of a technique previously applied to male meiosis. Meiotic progression was closely correlated with stages of floral development, including the length and morphology of the gynoecium. Meiosis in embryo sac mother cells (EMCs) occurs later in development than male meiosis, in gynoecia that range in size between 0.3 and 0.8 mm. The earliest stages in EMCs coincide with the second division to tetrad stages in pollen mother cells. However, the details of meiotic chromosome behaviour in EMCs correspond closely to the observations we have previously made in male meiosis. In addition, BrdU labelling coupled with an immunolocalisation detection system was used to mark the S phase in cells preceding their entry into prophase I. These techniques allow female meiotic stages of Arabidopsis to be analysed in detail, from the S-phase through to the tetrad stage, and are shown to be equally applicable to the analysis of female meiosis in meiotic mutants. Received: 3 April 2000 / Revision accepted: 2 August 2000     

Maturation of somatic embryos in conifers: Morphogenesis,physiology, biochemistry,and molecular biology

Summary In the past 15 years tremendons progress has been made towards the development of systems for the induction and development of somatic embryos of coniferous species. Since the first report in 1985, several species have been induced to produce somatic embryos. This has been rendered possible by the development of rational media and improvement of culture conditions, which have resulted in increased embryo quality and higher conversion frequency. Understanding the physiological and biochemical events occurring during in vivo embryogenesis has been fundamental in the design of new protocols for improving the somatic embryogenic process. Specifically, the inclusions of abscisic acid (ABA) and osmotic agents, such as polyethylene glycol (PEG), have been shown to be necessary for the functional development of somatic embryos. In the past few years, physiological and biochemical investigations have been useful in increasing our knowledge on the mode of action of ABA and PEG during embryo development. In comparison with the flowering plants, our understanding on the molecular mechanisms regulating the embryogenic process in coniferous species is still very limited. The application of new molecular techniques is therefore fundamental towards this end. The emphasis of this review is on recent information dealing with the maturation of conifer somatic embryos.     

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.     

The Structure of the IMaso-hypophysial Complex and the Mouth in Fossil and Extant Cyclostomes, with Remarks on Amphiaspiforms

The relationships between myxinoids and heterostracans are discussed on the basis of new observations on the structure of the prenasal sinus in an embryo of Myxine . A new interpretation of the feeding mechanism of heterostracans is proposed. Some remarks are made on the transformation of the prenasal sinus in more primitive heterostracans into a secondarily subdivided one in the amphiaspiforms.     

Localization of the proenzyme form of the vitellin-processing protease in Blattella germanica by affinity-purified antibodies

During Blattella germanica embryo development, the nutritive yolk protein vitellin is processed by a cysteine protease, which is activated proteolytically from a proprotease during acidification of yolk granules. A murine polyclonal antiserum was generated with the purified proprotease as the immunogen. The antiserum was made monospecific to proprotease by subtractive affinity chromatography using proprotease-free yolk proteins as ligand. The purified antibodies were employed to investigate the temporal and spatial expression of the proprotease during vitellogenesis and embryo development. Anti-proprotease-reactive peptides appeared in extracts of fat bodies and ovarian follicles of post-mating females, but not in fat bodies of males or the fat bodies or follicles of unmated females, suggesting that the proprotease is synthesized extraovarially. Use of the antibodies was extended to monitor the kinetics of proprotease disappearance during early embryo development. Arch. Insect Biochem. Physiol. 38:109–118, 1998. © 1998 Wiley-Liss, Inc.     

Morphology and ultrastructure of maternal seed tissues of soybean in relation to the import of photosynthate

Cytological observations were made on developing seeds of soybean (Glycine max (L.) Merr. “Amsoy 71”) using scanning and transmission electron microscopy and light microscopy. Attention was focused on the maternal tissues of the seed coat and embryo sac. An hypothesis of photosynthate import, unloading, and movement to the embryo is presented based on the results of these studies.