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.     

Developmental variability during early embryonic development of zebra fish, Danio rerio

Early vertebrate embryos pass through a period of remarkable morphological similarity. Possible causes for such similarity of early embryos include modularity, developmental constraints, stabilizing selection, canalization, and exhausted genetic variability. Supposedly, each process creates different patterns of variation and covariation of embryonic traits. We study the patterns of variation of the embryonic phenotype to test ideas about possible evolutionary mechanisms shaping the early embryonic development. We use the zebra fish, Danio rerio, as a model organism and apply repeated measures of individual embryos to study temporal changes of phenotypic variability during development. In particular, we are looking at the embryonic development from 12 hours post fertilization until 27 hours post fertilization. During this time period, the development of individual embryos is documented at hourly intervals. We measured maximum diameter of the eye, length of embryo, number of somites, inclination of somites, and the yolk size (as a maternal effect). The coefficient of variation (CV) was used as a measure of variability that was independent of size. We used a principal component analysis for analysis of morphological integration. The experimental setup kept environment x genotype interactions constant. Nongenetic parental contributions had no significant effects on interindividual variability. Thus all observed phenotypic variation was based on additive genetic variance and error variance. The average CV declined from 14% to 7.7%. The decline of the CV was in particular expressed during 15-19 h post fertilization and occurred in association with multiple correlations among embryonic traits and a relatively high degree of morphological integration. We suggest that internal constraints determine the patterns of variability during early embryonic development of zebra fish.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

The allocation of cells in the presomitic mesoderm during somite segmentation in the mouse embryo

Orthotopic grafts of wheat germ agglutinin-colloidal gold conjugate (WGA-gold) labelled cells were used to demonstrate differences in the segmental fate of cells in the presomitic mesoderm of the early-somite-stage mouse embryos developing in vitro. Labelled cells in the anterior region of the presomitic mesoderm colonized the first three somites formed after grafting, while those grafted to the middle region of this tissue were found mostly in the 4th-7th newly formed somites. Labelled cells grafted to the posterior region were incorporated into somites whose somitomeres were not yet present in the presomitic mesoderm at the time of grafting. There was therefore an apparent posterior displacement of the grafted cells in the presomitic mesoderm. Colonization of somites by WGA-gold labelled cells was usually limited to two to three consecutive somites in the chimaera. The distribution of cells derived from a single graft to two somites was most likely due to the segregation of the labelled population when cells were allocated to adjacent meristic units during somite formation. Further spreading of the labelled cells to several somites in some cases was probably the result of a more extensive mixing of mesodermal cells among the somitomeres prior to somite segmentation.     

In vitro development of the postimplantation embryos of laboratory rodents in human blood serum

Rat and mouse embryos at the stage of the first somites formation (1-5 pairs) cultivated in human blood serum demonstrated its embryolethal and teratogenic effect. The embryos taken at a later stage (11-18 pairs of somites) developed normally and could be compared with the development of the rat embryos in homologous blood serum. There was no difference in the development when the embryos were cultivated either in male or female blood serum. The stage of embryogenesis 11-18 pairs of somites is recommended for in vitro experimental revealing in the human serum of embryotoxic factors induced by certain external influences.     

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.     

The effects of 5-bromodeoxyuridine (BrdU) on morphogenesis of the early chick embryo

Summary The effects of BrdU (3×10^–4 M) on morphogenesis of the chick embryo explanted at the definitive streak stage and cultured for 24 hours were studied. Compared to controls treated embryos often showed (1) an open neural tube and (2) less numerous somites. Heart development was not significantly affected by BrdU. The damage caused by BrdU was not permanent, i.e., the embryos retained the ability to undergo fairly normal morphogenesis when, after 4–5 hours of BrdU treatment, they were subcultured on a medium with excess thymidine.This study was supported by a grant from the Rutgers University Research Council No. 07-2189.     

Extirpation experiments on embryonic rudiments of the carapace of Chelydra serpentina

Somites, along with adjacent neural tube and overlying ectoderm, were extirpated unilaterally from embryos of Chelydra serpentina. Mesoderm of three somites was removed from various levels. The operations included the last formed somite and were done on embryos with 12 to 22 pairs of somites. In practice it was found that ventromedial portions of the somites were not included in the extirpation. The animals were preserved before pigmentation became heavy. The cartilaginous skeleton was stained selectively. The extirpations resulted in depletions of ribs consonant with relating the second rib to the fourteenth somite. The somites behaved as mosaics; they did not reconstitute each other nor did they regenerate after partial extirpation. The rudiments for the ribs were separable from the rudiments of the vertebrae, the sclerotomes, and were found to arise from a more lateral portion of the somite. The scutes are ectodermal derivatives, which are held to be dependent upon underlying somitic mesoderm for their differentiation. The extirpations resulted in abnormalities and depletions of scutes.     

The development of the hemopoietic system: colony-forming splenic units in mouse embryogenesis

Localization and time of appearance as well as dynamics of quantitative changes of splenic colony-forming units (CFU-S) in mouse (C57BL/6 X CBA)F1 embryos were studied. Cells taken from the whole embryo (day 8), yolk sac and embryo per se (day 9), and also liver (day 10) were injected into the lethally irradiated syngenic mice. 7-8 days after the injection the spleens were fixed and the number of macrocolonies was counted. Statistically significant number of CFU-S was detected starting from day 10 of development, first in the embryo (30-33 somites), then in yolk sac and blood (37-38 somites) and liver (after the 40 somites stage). Rapid increase of CFU-S number during days 11-12 (two-fold increase in about 4.6 hours) suggest that not only active proliferation of CFU-S but also maturation of CFU-S precursors take place.     

The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

A study of the inductive capacity of post-nodal primitive-streak pieces after treatment with follicle-stimulating hormone

Summary Chick embryos at the primitive-streak stage were treatedin vitro with 0.002 and 0.008% follicle-stimulating hormone (FSH)(NIH-FSH-S1) for 24 hours. Post-nodal primitive-streak pieces 0.8 mm behind the node level were grafted into host chick embryos at the primitive-streak stage to assess the capacity of the grafted pieces to produce inductions.Control grafts from donors maintained under identical conditions were unable to cause induction; all of them were resorbed into the host embryo. The post-nodal pieces treated with FSH acquired capacity to induce neural tissue. The grafts seemed to induce foregut formation also. FSH appeared to have supported differentiation of the grafts into somites and mesenchyme.     

Effects of acute exposures to elevated temperatures on rat embryo growth and development in vitro

Day 10 rat embryos (8-12 somites) cultured in vitro were exposed to elevated temperatures (41-43 degrees C) for varying lengths of time. A 15-minute exposure to a temperature of 43 degrees C (109.4 degrees F) was sufficient to produce malformed embryos when observed on day 11. Longer exposures at this temperature produce higher incidences of malformed embryos and also more severely affected embryos. Temperatures of 42 degrees C (107.6 degrees F) or 41 degrees C (105.8 degrees F) also produced malformed embryos, but the required length of exposure was increased compared to 43 degrees C. The minimal length of exposure at 42 degrees C was 60 minutes, while at 41 degrees C it was increased to 4 hours. The central nervous system was particularly sensitive to increased temperatures, and embryos exposed to "teratogenic doses" of hyperthermia exhibited primarily microcephaly and microphthalmia. In addition, histological analyses revealed that at 4.5 hours after a 30-minute exposure to 43 degrees C, necrotic debris was prevalent in the neuroepithelium, less prevalent in the surrounding mesenchyme and surface ectoderm, and absent in the tissues of the heart.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.     

真水狼蛛胚胎发育过程中形态和主要化学物质含量的变化

采用常规石蜡切片和液体石蜡透明卵壳的方法,系统地观察了真水狼蛛的胚胎早期,体节期,胚胎速转期和幼虫期4个发育阶段的形态特征和发育过程,并测定了胚胎发育过程中卵的内主要化学物质含量的变化,在28度时,真水狼蛛的卵从产卵到孵化共需144小时,其中胚胎早期42小时,体节期33小时,胚胎逆转期27小时,在胚胎逆转期后进入前幼虫期,前幼虫期42小时,真水狼蛛的胚胎逆转现象很明显,表明真水狼蛛的进化程度较高,在胚胎发育过程中,卵的含水量,含脂量和卵重在胎发育24hr后开始下降,卵的蛋白质含量在48小时后也开始下降,含糖量下降不明显。     

Neural crest origin of cardiac ganglion cells in the chick embryo: Identification and extirpation

Using interspecific grafting of neural crest between quail and chick embryos, it was determined that the cardiac ganglia originate from the cranial region (somites 1–2) of the vagal neural crest (somites 1–7). Neuronal uptake of [³H]choline was used as an index of neuronal development in the chick atrium. Normal uptake was found to be quite high between Days 8 and 14 of incubation. Following extirpation of neural crest over somites 1 to 3 at stages 8 to 10, neuronal uptake in 8-day chick atrium was decreased by 25–60% depending on the stage at which the lesion was performed. It is thought that the residual uptake represents preganglionic terminals from the dorsal motor nucleus of the vagus. Embryos with extirpations of neural crest over somites 1–3 performed at stage 9 showed the greatest decrease of neuronal choline uptake and did not live beyond 11 days of incubation. However, hearts from embryos with partial lesions (performed at stage 10) were treated on incubation Days 12 and 15 for demonstration of acetylcholinesterase in the subepicardial plexus. These hearts showed much less extensive neural plexus with sparse, small cardiac ganglia.     

The influence of axial structures on chick somite formation

The influence of the axial structures on somite formation was investigated by culturing, on a nutritive agar substrate, segmental plates from chick embryos having 8 to 20 pairs of somites. In the first set of experiments, segmental plate was explanted together with adjacent notochord and approximately the lateral halves of the neural tube and node region. These explants formed 18 to 20 somites within 30 hr. In a second series of experiments, the notochord and neural tube were included as before, but further regression movements in the explants were prevented by removing the node region. These explants formed only 11.9 ± 1.1 somites. Finally, explants of segmental plate that included no neural tube, notochord, or node region were made. These explants had formed 10.7 ± 1.1 somites 14 to 17 hr later. When such explants were cultured for periods longer than 17 hr, there was a marked tendency for the more posterior somites to disperse and for all of the somites to develop a peculiar “hollow” morphology. It was concluded from these results that during the period of development when chick embryos possess 8 to 20 pairs of somites, the segmental plate mesoderm (1) represents about 12 prospective somites, (2) may segment into its full complement of somites without further contact with the axial structures, but (3) requires continued intimate contact with the axial structures for normal somite morphologic differentiation and stability.     

Expression of Notch signaling pathway genes in mouse embryos lacking beta4galactosyltransferase-1

A requirement for beta4galactosyltransferase-1 (beta4GalT-1) activity in the modulation of Notch signaling by the glycosyltransferase Fringe was previously identified in a mammalian co-culture assay. Notch signaling is necessary for the formation of somites in mammals. We therefore investigated the expression of eleven Notch pathway and somitogenic genes in E9.5 mouse embryos lacking beta4GalT-1. Four of these genes were altered in expression pattern or expression level. The Notch target genes Hes5 and Mesp2 were affected to some degree in all mutant embryos. The Notch ligand genes Dll1 and Dll3 were reduced or altered in expression in a significant proportion of mutants. While there were no differences in the number or morphology of somites in E9.5 B4galt1 null embryos, the number of lumbar vertebrae in mutant embryos differed from control littermates (P < or = 0.01). The subtlety of the in vivo phenotype may be due to redundancy since several B4galt genes related to B4galt1 are expressed during embryogenesis.