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.     

小鼠早期胚胎发育期间TGF—β免疫组织化学定位

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1. Embryonic cells of early cleavage stages (2, 4 and 8 cells) and late morulae showed positive immunofluorescent reaction without any difference in staining intensity (Plate I, Figs. 1-4). 2. Marked staining of blastocysts in toto or sections with anti-TGF-beta-1 antibodies by either immunofluorescence or immunoperoxidase reaction was also observed. Inner cell mass (ICM) cells and trophoectoderm cells were both reacted, but more intense staining was found in primary endoderm cells differentiated from ICM cells adjacent to blastocoele (Plate II, Fig. 5). 3. Scattered granules stained strongly with immunoperoxidase reaction were present in embryonic ectoderm and visceral endoderm surrounding the forming mesoderm which was only slightly stained (Plate II, Fig. 6). 4. Intense immunoperoxidase staining was also present in mesoderm of visceral yolk sac of day 8 and day 10 embryos (Plate II, Fig. 7). 5. During the formation of somites, neural tube and limb bud, remarkable staining was found in mesenchyme, individual cells of somites, mucous layer of gut tubes, heart and limb buds (Plate III, Figs. 8-10). No significant staining was seen in neural cells per se except the inner surface of neural tube. The results of present studies indicate that abundant TGF-beta-1 is present in preimplantation mouse embryos including cleavage, morulae and blastocyst stages. In postimplantation embryos, TGF-beta-1 appears to play an important role in the differentiation of endoderm and mesoderm, particularly in the development of extraembryonic tissues, and in later morphogenetic and histogenetic events involving mainly mesoderm or mesenchyme cells.     

Gene transfer into cultured mammalian embryos by electroporation

To gain a better understanding of mammalian development at the molecular level, technology is needed that allows the transfer of exogenous genes into desired embryonic regions at defined stages of development. Our strategy has been to use electroporation (EP) of plasmid DNA following whole-embryo culture (WEC). In our gene transfer system, postimplantation rodent embryos are taken out of the uterus and a purified DNA solution of mammalian expression plasmid constructs is injected into the neural tube. A square-pulse current is delivered using an electroporator with an optimizer. Electroporated embryos are allowed to develop in the WEC system for 24--48 h. Within the targeted area, the proportion of transfected cells varied from 10% to approximately 100% depending on the test conditions (e.g., DNA concentration, voltage, duration of EP, and pulse number). The EP--WEC system has several advantages including rapid gene expression, minimal laboratory work, precisely targeted regions, and no risk for human beings. Application of the method is useful in improving our understanding of early neural development (E7--E12 in mice), e.g., alteration of gene function via ectopic expression, interference with dominant negative proteins, and fate mapping with marker genes. In addition, EP can complement genetic approaches such as the generation of knockout and transgenic mice.     

Fate map of the chicken neural plate at stage 4

A detailed fate map was obtained for the early chick neural plate (stages 3d/4). Numerous overlapping plug grafts were performed upon New-cultured chick embryos, using fixable carboxyfluorescein diacetate succinimidyl ester to label donor chick tissue. The specimens were harvested 24 hours after grafting and reached in most cases stages 9-11 (early neural tube). The label was detected immunocytochemically in wholemounts, and cross-sections were later obtained. The positions of the graft-derived cells were classified first into sets of purely neural, purely non-neural and mixed grafts. Comparisons between these sets established the neural plate boundary at stages 3d/4. Further analysis categorized graft contributions to anteroposterior and dorsoventral subdivisions of the early neural tube, including data on the floor plate and the eye field. The rostral boundary of the neural plate was contained within the earliest expression domain of the Ganf gene, and the overall shape of the neural plate was contrasted and discussed with regard to the expression patterns of the genes Plato, Sox2, Otx2 and Dlx5 (and others reported in the literature) at stages 3d/4.     

bFGF、NGF、EGF及其受体在人胚神经管早期发育中的表达

研究bFGF、NGF、EGF及其受体在人胚神经管早期发育中的表达。方法 应用免疫组织化学方法和图像分析。结果显示bFGF和NGF的表达时序不同,bFGF阳性细胞出现较早,在所检测在各个发育阶段均呈阳性表达,而NGF出现较晚,随着胚龄增加,免疫阳性着色逐渐增强,bFGF分布较NGF广泛,而EGF在所检测的各个发育阶段均呈阴性。flg、TrkA、EGFR表达时序和分布相似,三者在所检测的各个发育阶段均阳性。结果表明NGF和bFGF均通过其特异性受体介导,在胚胎神经管形成和分化的不同阶段发挥着重作用,EGF及其受体的作用有待进一步研究。     

Comparison of staging systems for the gastrulation and early neurulation period in rodents: a proposed new system.

Because there is no standard developmental staging system for the early postimplantation period of rodent embryos, investigators must now choose between a variety of systems that differ significantly. We have reviewed many of these staging systems and have summarized the ambiguities within them and the inconsistencies among them. In order to compare systems, we first obtained a consensus of the order of developmental events from the literature, and then attempted to fit existing systems into this order taking into account inconsistencies in terminology and blurred borderlines between stages. We were able to do this for most systems but not all because some were too divergent. We found that inconsistencies in definition of some terms, such as "primitive streak stage" and those used to describe the early neurulation process (neural plate, neural groove, neural folds, and head fold) cause much confusion. In order to develop an unambiguous system which can be used by all investigators, we propose to modify Theiler's system, which is one of the most commonly used systems but is not defined precisely during the early postimplantation period. We suggest making subdivisions of the original stages as follows: 1) stage 8 into 8a and 8b, by the degree of extension of the proamniotic cavity into the extraembryonic region; 2) stage 10 into 10a and 10b, by the completion of amnion formation; 3) stage 11 into 11a, 11b, and 11c, by the appearance of neural folds and foregut pocket. After Stage 12, the number of somite pairs can be used to precisely stage embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic variability in human embryonic holoprosencephaly in the Kyoto Collection

BACKGROUND: Holoprosencephaly (HPE) is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Although numerous postnatal cases have been reported, early phases of its pathogenesis are not well understood. We examined over 200 cases of HPE human embryos both grossly and histologically, and studied their phenotypic variability and stage-specific characteristics. METHODS: Among over 44,000 human embryos in the Kyoto Collection of Human Embryos, 221 embryos have been diagnosed as HPE. Their developmental stages ranged from Carnegie stage (CS) 13 to CS 23. They were examined grossly and were also serially sectioned for detailed histological analysis. RESULTS: HPE embryos after CS 18 were classified into complete (true) cyclopia, synophthalmia (partially fused eyes in a single eye fissure), closely apposed separate eyes (possible forerunners of ethmocephaly and cebocephaly), and milder HPE with median cleft lip (premaxillary agenesis). At CS 13-17, when facial morphogenesis is not completed, HPE embryos had some facial characteristics that are specific to these stages and different from those in older HPE embryos. The midline structures of the brain, including the pituitary gland, were lacking or seriously hypoplastic in HPE embryos. Complete cyclopia was found in two cases after CS 18 but none at earlier stages. CONCLUSIONS: The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Neural cell adhesion molecule expression in Xenopus embryos

The spatiotemporal pattern of expression of the neural cell adhesion molecule NCAM was mapped immunohistochemically in embryos of the frog Xenopus, from blastula to early swimming stages, using a polyclonal antibody that recognizes Xenopus NCAM. The neural plate stage was the earliest at which NCAM could be detected. The initial sites of NCAM immunoreactivity were neural ectoderm, somitic mesoderm, and chordamesoderm. During formation of the neural tube, NCAM immunoreactivity became restricted to the neuroectoderm and its derivatives. During closure of the neural tube and for 2-4 hr thereafter, NCAM was expressed in a distinctive radial pattern in coronal sections of the neural tube. NCAM was observed in neural crest cells before migration and after formation of cranial and spinal ganglia. During the period of initial neurite outgrowth, NCAM became concentrated in the developing central nerve fiber pathways. NCAM was seen on peripheral nerves from the time of their initial outgrowth and it was strongly expressed at neuromuscular junctions during the period of their formation. These results show that NCAM is expressed after neural induction and functions during morphogenesis of the neural plate and tube, some neural crest derivatives, development of nerve fiber tracts, and formation of neuromuscular connections.     

DNA methylation,retroviruses, and embryogenesis

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or postimplantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfcctious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.     

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.     

The development of chick spinal cord in tissue culture. I. Fragment cultures from embryos of various developmental stages

Explants from neural tube and spinal cord of chick embryos at developmental stages 8 through 36 were cultured on collagen-coated cover glasses for 21 days. The cultures of neural tube at stages 10 to 14 contained many neuronal precursor cells which gave rise to mature neurons. This was verified by cumulative labeling of cultures with tritiated thymidine. Explants from spinal cords of stages 26 and 27 contained fewer precursor cells, and at stage 36, only 7% of mature neurons were labeled. Regardless of the stage of development at which explants were made (stages 8 through 36), all cultures had a similar appearance after 21 days, indicating that cells from explants taken from earlier developmental stages (before neurons were formed) "caught up" with the explants from later developmental stages, which already had formed neurons at the time of explantation.     

Homocysteine modifies development of neurulation and dorsal root ganglia in chick embryos

BACKGROUND: The formation of the neural tube (neurulation) involves two mechanisms: primary and secondary neurulation. In chicks, there is also an overlap zone, where both mechanisms work together. Homocysteine (Hcy) may have an important teratogenic role in neural tube defects (NTD) when folic acid levels are considered normal. Recently, Hcy capability to generate NTD and modify neural crest cell migration has been demonstrated in chick embryos. This study was aimed to evaluate the effects of Hcy on neurulation and the development of the dorsal root ganglia (DRG). METHODS: Chick embryos were treated with L-Hcy thiolactone 20 micromol to produce the highest rate of survival with embryos carrying neural tube defect (NTD) in the spine. Embryos at stages (st) 3-10 were treated and harvested at st 18-23. Only externally normal embryos or those carrying spinal NTD embryos were considered. RESULTS: Histological sections of Hcy-treated embryos showed: open spina bifida (39% of embryos), more than one tube forming the spinal cord (26%), disorganized spinal cord (26%), always affecting lumbosacral regions, probably in the overlap zone. Additionally, 32% of embryos had small and continuous DRG, associated with a slimmed roof plate. Three-dimensional reconstruction showed unsegmented DRG until the C8 ganglion level. There was a 75% reduction of C3 DRG cells in treated embryos in comparison to untreated ganglia. CONCLUSION: Hcy teratogenicity in avian embryos affected the neural tube in the overlap zone, secondary neurulation and the cervical DRG.     

Intersection clock reveals a rejuvenation event during human embryogenesis

Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.     

The effect of ethanol upon early development in mice and rats. I. In vivo effect upon preimplantation and early postimplantation stages

The preimplantation and early postimplantation effect of chronic alcohol consumption (at least a month before mating and during pregnancy until killing) and of acute ethanol intoxication during the preimplantation period (i.v. infection of ethanol) was studied on albino rats (Wistar) and albino mice (RAP). The main results were as follows: Chronic alcoholization. Rats: significant retardation of preimplantation development and in early postimplantation stages; a tendency of lowering of the mean litter size. Mice: significant increase of the number of preimplantation pathological forms; a tendency of lowering of the mean litter size. Pathological changes show, both in rats and mice, an obvious "litter effect". Acute ethanol intoxication. Rats: significant retardation in some litters, normal or even advanced development in others. This effect differs from the previously reported effect of acute ethanol intoxication during early postimplantation stages. The results obtained attest the prenatal noxious effect of chronic ethanol consumption in both species used and of acute ethanol intoxication during preimplantation development upon early postimplantation development in rats. Within the limits of extrapolation possibilities, they represent a risk signal for other species (including human).     

Morphometric analysis of the forebrain anomalies in the delayed Splotch mutant embryo

The early development of delayed Splotch mouse embryos was examined histologically using scanning electron microscopy and morphometric techniques. Embryos obtained from matings of mice heterozygous for the delayed Splotch gene exhibited a high incidence of lumbosacral (25%) or cephalic (7%) neural tube defects. The lumbosacral neural tube defects extended from the posterior neuropore region to the tip of the tailbud; cephalic neural tube closure defects were found in the hindbrain and midbrain regions. The frontal region of affected embryos was abnormal in that it was reduced in size, particularly in the developing midface. Histologically, the forebrain region of affected embryos appeared reduced, and the luminal surface of the neuroepithelium was often irregular and infolded. To quantify these alterations and to determine their contribution to the final form of the region, size and areal measurements were recorded and served as input for principal component and cluster analytic techniques. In affected embryos, significant reductions were found in lumen size, in neuroepithelial area but not thickness, and in overall area of forebrain but not hindbrain. Principal component analysis of data from unaffected embryos produced two factors, one containing hindbrain variables and the second forebrain variables; for the affected embryos, three factors were extracted. The first loaded on variables that measured the thickness and area of the neuroepithelium, the second on forebrain variables, and the third on hindbrain variables. Factor scores were then generated from a pooled analysis of normal and affected cases and were analyzed using cluster analysis. Three clusters were identified: one contained eight affected embryos with cephalic neural tube defects; another contained nine affected embryos with lumbosacral neural tube defects and five normal embryos; and the final cluster contained ten unaffected embryos. These results suggest a major role of the delayed Splotch gene on the neuroepithelium itself and support the suggested role of cerebrospinal fluid pressure in normal forebrain histogenesis.     

Characteristics of phenotypic expression of autosomal monosomies during pathological postimplantation human development

Autosomal monosomies represent a severe form of genomic disbalance which determines elimination of human embryos already at the preimplantation stages. As a rule, they occur very rarely in the materials of spontaneously aborted embryos and fetuses. Molecular-cytogenetic studies were carried out on the karyotype of cells of 60 spontaneous abortuses of I trimester of pregnancy with cell degeneration or absence of cell proliferation in the cultures, as a result of which the cells could not be studied using the standard metaphase analysis. The embryos were characterized by an unexpectedly high frequency of mosaic variants of monosomies for chromosomes 7, 15, 21, and 22, which amounted to 19% of all chromosome aberrations. Lethal forms of monosomies for human chromosomes 7 and 15 were described for the first time, since they are not found in spontaneous abortuses by standard cytogenetic methods. A hypothesis was proposed which accounts for the possibility of early postimplantation lethality of the embryos with mosaic forms of autosomal monosomies. The differences were found between the cells with monosomies for different autosomes in the mechanisms of origin, intertissue localization, and phenotypic effects. It was shown that monosomies for chromosomes 7, 15, 21, and 22 in a mosaic state with the normal cell line can be compatible with the early stages of postimplantation differentiation of the cytotrophoblast. Predominant compartmentalization of the cells with monosomies for chromosomes 21 and 22 in the extraembryonic mesoderm, a derivative of epiblast, can be a critical factor, which makes it impossible the normal morphogenesis of embryonic structures.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.