Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

High prevalence of defective human embryos at the early postimplantation period

Thirty-seven cases of human embryos at the early postimplantation period were procured after induced abortion and examined histologically. Their developmental stages ranged from Carnegie stages 6 to 11, and their standard ages ranged from 14 to 24 days after fertilization. Five cases (13.5%) were grossly abnormal, and seven (18.9%) were degenerating partially or in toto. Gross abnormalities included distorted embryonic disc, disorganized neural groove or tube, and neural tube dysraphism. The high prevalence rate of defective embryos at the early postimplantation period supports the clinical finding that a substantial proportion of human conceptions are eliminated from an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated pathological embryos is uncertain and remains to be determined.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

Critical period of rat development when sidedness of asymmetric body structures is determined

We recently reported that rat embryos cultured from the presomite stage in a medium containing the alpha-1 adrenergic agonist, phenylephrine, have a high incidence of situs inversus. In the present study, we have determined more precisely the critical period of development when situs inversus is induced. Rat embryos were harvested at 8 AM on Day 9 of gestation (plug day = Day 0), and divided into different stages of development, namely, early, mid, and late primitive streak stages and early, mid, and late neural plate stages. They then were cultured in rotating bottles to which phenylephrine, 0.5 mM, was added for various durations. After 49 hr of culture, embryos were examined for general morphology including sidedness of the bulboventricular loop, tail, and chorioallantoic placenta. Phenylephrine increased the incidence of situs inversus above control when administered throughout culture from either the early neural plate stage or before, and when administered for 4 hr or more from the early neural plate stage. This increase was significant even at the mid and late primitive streak stages when the control incidence was high. Our results suggest that sidedness of asymmetric body structures is determined during the early neural plate stage. This period is well before the 6-8-somite stage when morphological signs of body asymmetry first appear.     

The initial development of the human brain.

An account of the early development of the human brain has been prepared from the data available for the Carnegie Collection, as well as from published information from other sources. Although the site of the neural plate can be discerned at stage 7, the first visible indication of the nervous system is the neural groove in certain embryos of stage 8, in which the embryonic disc measures more than 1 mm and the notochordal process at least 0.3 mm. The progressive fusion of the neural folds during stage 10, and the closure of the rostral and caudal neuropores at stages 11 and 12, respectively, are detailed with further precision than hitherto. It is emphasized that the major subdivisions of the human brain do not begin as vesicles, but as enlargements of the open neural folds at stage 9. The relationships of the neuromeres to the otic region, the somites, and the neural crest are clarified and illustrated. The early appearance of the telencephalon medium (before cerebral vesicles have formed) is stressed, and the terminological implications for the subdivisions of the brain are discussed.     

SEM observations of the neural fold associated with neurulation in the rat

The topography of the ectoderm was examined by scanning electron microscopy during neurulation in rat embryos at stage 24 (somites 8-11). A zone of altered cell morphology was observed along the crest of neural folds. This zone was located between the presumptive neural tube and the surface ectoderm and exhibited numerous rounded cell blebs, immediately prior to fusion between the folds. It is suggested that the observed surface alterations may reflect a change in the properties of the altered cell which correlate with initial adhesion between the folds.     

"Micromere" formation and expression of endomesoderm regulatory genes during embryogenesis of the primitive echinoid Prionocidaris baculosa

Blastomere composition and expression profiles of wnt8 and hox11/13b orthologues were examined in the primitive indirect-developing echinoid Prionocidaris baculosa. We found that blastomere composition in the 16-cell-stage Prionocidaris embryos was different from that of the indirect-developing echinoids belonging to Euechinoidea, a derived group of the echinoids. The sizes of the blastomeres in the 16-cell-stage embryo varied, and no embryos formed a "micromere quartet," a group of four equal-sized micromeres. The smallest blastomere was usually located around the vegetal pole. We also found significant differences in early expression profiles of wnt8 orthologues of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of wnt8 orthologue of Prionocidaris was not detected at the 16-cell stage; it began at the 32-cell stage in the broad area containing the vegetal pole. However, in later stages, the expression profiles of hox11/13b and wnt8 orthologues of Prionocidaris were similar to that of euechinoid orthologues. The present study suggests that there are considerable differences between Prionocidaris and euechinoids in early developmental mechanisms in the vicinity of the vegetal pole.     

Temporal/spatial expression and efflux activity of ABC transporter, P-glycoprotein/Abcb1 isoforms and Bcrp/Abcg2 during early murine development

ABC transporters pump out from cells a large number of endo- and xenobiotics including signal molecules and toxins; they are molecular markers of stem/progenitor cells as well. Here, we present the study of temporal/spatial patterns of Abcb1 isoforms and Abcg2 transporter expression and efflux activity in pre- and early postimplantation murine embryos. We found in 2-cell embryos abcb1a, abcb1b and abcg2 mRNAs which were believed to be maternally inherited. The expression of abcb1b and abcg2 genes was found in blastocysts and in 7 days postcoitum (dpc) embryos, while in 9dpc embryos beside of abcb1b/abcg2, the abcb1a gene was expressed. The abcb2 mRNA was detectable neither in pre- nor in postimplantation embryos. Moreover, we analysed temporal/spatial patterns of rhodamine 123/Hoechst 33342 efflux, which mirrors the ABC transporter phenotype, from individual cells of pre- and postimplantation murine embryos. The blastomeres of 2-, 4- and 8-cell embryos had efflux-inactive phenotype. Single, efflux-active cells emerged first in the morulae and their number increased in blastocyst inner cell mass. In 6 and 7 dpc embryos, all embryonic cells hold the efflux-active phenotype. Proximal embryonic endoderm of 6-8 dpc embryos contained two sub-domains: one consisted of efflux-active cells and another one of efflux-inactive cells reflecting polarity of an embryo. Between 7 and 8 dpc, at the onset of organogenesis, the vehement surge of efflux-inactive embryonic cells occurred, and their number increased in 9 dpc embryos, which consequently contained few efflux-active cells.     

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of fibroblast growth factors (FGF-2, FGF-4) on the development of parthenogenetic mouse embryos

We studied the effects of two growth factors, FGF-2 and FGF-4, on development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1. Parthenogenetic embryos were treated with FGF-2 or FGF-4 in vitro at the morula stage and, after they reached the blastocyst stage, transplanted into the uteri of pseudopregnant females. FGF-2 and FGF-4 did not affect the number of blastocysts formed in vitro or implantation into the uterus. However, FGF-2 and FGF-4 at optimal doses decreased the mortality rate of parthenogenetic embryos at the early postimplantation stages and increased twofold the number of embryos that developed in utero to the somite stages: 42 and 36%, respectively, versus 20% in the control. The results obtained suggest that the treatment of parthenogenetic mouse embryos with FGF-2 or FGF-4 modulate the effects of genomic imprinting and prolong the development of parthenogenetic embryos at the postimplantation stages.     

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics

In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8–16 cell stage): I and II – early (day 2) and late (day 3) 5–8 cell, III and IV – early (day 3) and late (day 4) 8–16 cell, and V and VI – early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5–8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8–16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block.     

Differences in the effects of treatment of uncompacted and compacted mouse embryos with phorbol esters on pre- and postimplantation development

Abstract. Differences are described in the effects of treatment of preimplantation mouse embryos with low levels (0.01–1 n M ) of phorbol myristate acetate (PMA), during three different periods of a 48-h culture from the 2-cell stage, on pre- and postimplantation development. Treatment of embryos with PMA for 48 h (first group) or 24 h (second group) from the 2-cell stage caused premature cavitation (prior to the 16-cell stage) and it also reduced the size and alkaline phosphatase (ALPase) activity of inner cell masses (ICMs), as well as the numbers of cells in blastocysts, in a dose-dependent manner. Treatment of early morulae with PMA for 24 h (third group) did not have the abovementioned effects on embryos but inhibited the formation and subsequent enlargement of the blastocoel. The blastocysts that were allowed to develop in the three treatment groups were examined for postimplantation development. Implantation was unaffected in all groups. The survival rate after implantation was low in the first and second groups but relatively high in the third group. The results indicate that an embryo exposed to PMA for 24 h from the 2-cell stage forms a premature blastocoel, and, in such an embryo, quantitative and qualitative differentiation into the ICM is blocked but qualitative differentiation into trophectoderm is uninhibited. Consequently, the embryo can implant but does not survive for a long time. When embryos were exposed to PMA for 24 h from the early morula stage, the formation and enlargement of the blastocoel were inhibited even though the treatment had a minimal effect on other developmental events. It is suggested that the effects of PMA on early mouse development are specific to each period at which the drug is applied.     

Studies on the mechanisms of neurulation in the chick: interrelationship of contractile proteins, microfilaments, and the shape of neuroepithelial cells

Electron microscopy and indirect immunofluorescence were employed to correlate the distribution patterns of major contractile proteins (actin and myosin) with 1) the organizational state of microfilaments, 2) the apical cell surface topography, 3) the shape of the neuroepithelial cells, and 4) the degree of bending of the neuroepithelium during neurulation in chick embryos at Hamburger and Hamilton stages 5-10 of development. Both actin and myosin are present at these developmental stages and colocalize in the neural plate as well as in later phases of neurulation. During elevation of neural folds, actin- and myosin-specific fluorescence is always most intense in regions where the greatest degree of bending of the neuroepithelium takes place [e.g., the midline of the V-shaped neuroepithelium (early neural fold stage) and the midlateral walls of the "C"-shaped neuroepithelium (mid-neural-fold stage)]. This intense fluorescence coincides with 1) a particularly dense packing of microfilaments and 2) highly constricted cell apices. After neural folds make contact, there is an overall reduction in both the intensity of apical fluorescence and the thickness of apical microfilament bundles, especially in the roof and floor of the neural tube. The remaining fluorescence in the contact area is apparently related to cellular movements during fusion of neural folds.