Fates of the blastomeres of the 16-cell stage Xenopus embryo

The fate of each of the blastomeres in the 16-cell stage Xenopus embryo which had been carefully selected for stereotypic cleavages was determined by intracellularly marking a single blastomere with horseradish peroxidase and identifying the labeled progeny in the tailbud embryo by histochemistry. Each blastomere populated all three primary germ layers. The progeny of each blastomere were distributed characteristically both in phenotype and in location. For example, most organs were populated by the descendants of particular sets of blastomeres. Furthermore, within an organ the progeny of a single blastomere were restricted to defined spatial addresses. This study describes the fates of identified 16-cell stage blastomeres and demonstrates that they are distinct and predictable if embryos are preselected for stereotypic cleavages.     

Regulation of primary spinal neuron lineages after deletion of a major progenitor

Vertebrate embryos are able to reconstitute the body plan when early blastomeres are deleted, but it is not known whether this is accomplished by cells local to the lesion or by a readjustment of the entire pattern of the embryo. We distinguished between these two possibilities by studying which embryonic cells change primary spinal neuronal fates after deletion of a major spinal cord progenitor. After ablation of the V1.2 blastomere of the 16-cell Xenopus embryo, the spinal cord contained normal numbers of Rohon-Beard neurons and primary motoneurons, indicating that the remaining blastomeres numerically reconstituted these populations. Using lineage-tracing techniques we revealed a global response: 10 out of the 15 remaining blastomeres significantly changed the number of one or both neuronal types they produced. This widespread response indicates that position in the early embryo plays an important role in regulating the production of primary spinal neurons. However, not all cells are influenced solely by position; a vegetal cell transplanted into the position of the deleted V1.2 did not take on the neuronal fate of its new position. Thus, restitution of pattern relies on a combination of positional cues and intrinsic fate restrictions.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage

Cell lineages during embryogenesis of the ascidian Halocynthia roretzi were analyzed up until the stage where each blastomere was fated to be only a single tissue type (i.e., the tissue restricted stage) by intracellular injection of horseradish peroxidase using the iontophoretic injection method. Initially, the developmental fates of all blastomeres of the 64-cell stage embryo were examined, and thereafter, only the fates of daughter blastomeres of those blastomeres that were not tissue restricted at the 64-cell stage were traced. The developmental fates of blastomeres were highly invariant except for two candidates for "equivalence groups" (J. Kimble, J. Sulston, and J. White (1979). In "Cell Lineage, Stem Cells and Cell Determination," pp. 59-68. Elsevier, Amsterdam/New York), in which cellular interaction is suggested to be involved in the specification of the fates. The right and left a8.25 cells gave rise to the otolith and ocellus, and the right and left b8.17 cells gave rise to the spinal cord and endodermal strand in a complementary manner. No fixed relationship existed between the position of the blastomere and its derivative. Most restrictions of cell fates occurred early in cleavage. The numbers of blastomeres which generated a single type of tissue were 44 at the 64-cell stage and 94 at the 110-cell stage. Eight pairs of blastomeres had not yet become tissue restricted by the 110-cell stage. Almost complete lineages of epidermis, nervous system, muscle, mesenchyme, notochord, and endodermal tissues were described, and a fate map was constructed for the blastula. For certain tissues, the primordial cells occupied two different regions. Supplementary investigations of the lineage of muscle cells were also performed on embryos of another species, Ciona intestinalis.     

Pattern regulation in defect embryos of Xenopus laevis

Defect embryos of 24 series were prepared by removing increasing numbers of blastomeres from an 8-cell embryo of Xenopus laevis. They were cultured and their development was examined macroscopically when controls reached a tailbud stage or later. Results show that most of defect embryos of 12 series develop normally, and some of them become normal frogs. Each of these defect embryos contain at least two animal blastomeres, one dorsal, and one ventral blastomere of the vegetal hemisphere. This suggests that a set of these four blastomeres of the three types is essential for complete pattern regulation.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Fates of Animal-Dorsal Blastomeres of Eight-Cell Stage Xenopus Embryos Vary according to the Specific Patterns of the Third Cleavage Plane

The third cleavage plane in typical Xenopus embryos is horizontal. However, there are numbers of cases in which the third cleavage plane slants and yet the embryo develops normally. Pairs of animal-dorsal (AD) blastomeres of eight-cell stage Xenopus embryos with horizontal or oblique third cleavage plane were marked by intracellular injection of fluorescein dextran amine in order to locate their progeny. In neurulae, progeny of AD blastomeres was found mainly along the dorsal midline forming longitudinal clonal bands along the midline in the neural plate and the mesoderm underneath. AD blastomeres with oblique third cleavage plane further yielded the ventral endo-mesoderm in the head. On the other hand, they formed narrower clonal bands in the anterior ectoderm compared with AD blastomeres with horizontal third cleavage plane. Thus the fates of animal-dorsal brastomeres of eight-cell stage Xenopus embryos vary according to the specific patterns of the third cleavage plane. This indicates that the third cleavage in the Xenopus embryo does not affect the normal fate of each region of the embryo presumed at the eight-cell stage.     

Ectopic expression of a homeobox gene changes cell fate in Xenopus embryos in a position-specific manner.

We have injected XIHbox 6 mRNA together with the lineage tracer colloidal gold into individual dorso-anterior blastomeres of the 32-cell stage Xenopus embryo and analyzed their cell fate during embryogenesis. While the developing tadpoles appeared entirely normal, the fate of the progeny of the injected blastomere was altered. In the brain injected cells failed to differentiate terminally, as indicated by a loss of labeled cranial nerves. Differentiation of spinal nerves remained unaffected. Fate change in the CNS occurred at about the time of normal XIHbox 6 protein expression. In addition, progeny of injected blastomeres gained head epidermal fate and lost anterior notochord fate as a result of altered cell migrations during gastrulation. The results show that a homeodomain protein is capable of altering cell fate in a position-specific and cell-autonomous manner in Xenopus embryos. The experimental approach used here should be applicable to other molecules specifying cell fate.     

Elucidating the origins of the vascular system: a fate map of the vascular endothelial and red blood cell lineages in Xenopus laevis.

Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Slow intermixing of cells during Xenopus embryogenesis contributes to the consistency of the blastomere fate map

The relatively consistent fates of the blastomeres of the frog embryo could result from (i) predetermination of the blastomeres or (ii) reproducible morphogenetic cell movements. In some species, the mixing of the cells during development provides a test between these alternative hypotheses. If blastomeres are predetermined, then random intermixing of the descendants with neighboring cells could not alter their fate. To follow cell mixing during Xenopus development, fluorescent dextran lineage tracers were microinjected into identified blastomeres at the 16-cell stage. The labelled descendants of the injected blastomeres were followed over several stages of embryogenesis. After gastrulation, the labelled descendants formed relatively coherent groups in characteristic regions of the embryo. By larval stages, most of the labelled descendants were still located in characteristic regions. However, coherence was less pronounced and individual descendants were located in many regions of the embryo. Hence, cell mixing is a slow, but progressive, process throughout Xenopus development. This is in sharp contrast to the extensive mixing that occurs during the early development of other vertebrates, such as zebrafish and mice. The slow cell mixing in Xenopus development suggests a simple mechanism for the consistent fates of cleavage-stage blastomeres. The stereotyped cell movements of embryogenesis redistribute the largely coherent descendants to characteristic locations in the embryo. The small amount of mixing that does occur would result in variable locations of a small proportion of the descendants; this could contribute to the observed variability of the blastomere fate map. Because cell mixing during Xenopus development is insufficient to challenge possible lineage restrictions, additional experiments must be performed to establish when and if lineage restrictions occur.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme : II. The 16- and 32-cell stages

Cell lineages during development of ascidian embryos were analyzed by injecting horseradish peroxidase as a tracer enzyme into identified cells of the 16-cell and 32-cell stage embryos of Halocynthia roretzi. Most of the blastomeres of these embryos developed more kinds of tissues than have hitherto been reported, and therefore, the developmental fates of each blastomere are more complex. It has been thought that every blastomere of the 64-cell stage ascidian embryo gives rise to only one kind of tissues, but the finding that the several blastomeres at the 32-cell stage developed into at least three different kinds of tissues, clearly indicates that the stage at which the fates of every blastomere are determined to one tissue is later than the 64-cell stage. The results also clearly demonstrate that muscle cells are derived not only from B-line cells (B5.1, B5.2, B6.3, and B6.4) but also from A-line cells (A5.2 and A6.4) and b-line cells (b5.3 and b6.5). Based on the present analysis as well as other studies, complete cell lineages of muscle cells up to their terminal differentiation have been proposed. In addition, lineages of nervous system, notochord, and epidermis are also discussed.     

Fate map for the 32-cell stage of Xenopus laevis

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.     

Correlations between cell fate and the distribution of proteins that are synthesized before the midblastula transition in Xenopus

Summary The proteins synthesized before the 512-cell stage by Xenopus blastomeres with different fates were compared by one dimensional PAGE. Blastomeres that contributed more progeny to antero-dorsal axial structures produced proportionately more of two proteins of 225000 and 245000 daltons. Additionally, these proteins were reversibly increased in ventralized embryos and were decreased in dorsalized embryos. These observations indicate that some proteins that are synthesized during cleavage stages are expressed to different degrees in different regions of the embryo, that their expression can be correlated to cell fate in the normal embryo, and that their expression is altered quantitatively in dorsalized and ventralized embryos. The inverse relationship between the production of these proteins and the potential to produce dorsal structures in the normal and in dorsalized/ventralized embryos is consistent with a model in which cell fate is influenced by a gradient of particular proteins.Supported by NIH grants HD 06619 (SLK) and GM 33932 (MLK).     

Onset of competence to respond to activin A in isolated eight-cell stage Xenopus animal blastomeres

Single animal hemisphere blastomeres isolated from the eight-cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32-cell stage) of normal development. The competence gradually increased during the morula stages.     

16细胞期爪蟾胚胎背方与腹方裂球的发育能力

在两栖类爪蟾胚胎发育中,由受精引起的皮层转动造成了受精卵的背腹极性。为了研究受精卵细胞质的不均一分布对胚胎体轴形成的影响,我们进行了16细胞期动物极背、腹方裂球的外植和异位移植实验。16细胞期的动物极背方裂球在外植和移植到腹方位置后都表现出背方特征,如外植块培养到原肠中期时伸长,背方裂球在移植到腹方后引发第二体轴等;而16细胞期动物极腹方裂球移植到背方后其发育命运则遵循背方裂球的命运,参与背方结构的形成。我们认为在16细胞期,动物极背、腹方的裂球由于包含着不同的卵质,因而在发育能力上已经具有背、腹的差异。     

Dorsal Blastomeres in the Equatorial Region of the 32-Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32-cell Xenopus embryos, their roles in head formation in normal development and the organizer-inducing capabilities of the dorsal-most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis . When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32-cell stage and comes into existence not under the inductive influence of the dorsal-most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32-cell embryo.     

Binary specification of nerve cord and notochord cell fates in ascidian embryos

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.     

Fates of the blastomeres of the 32-cell-stage Xenopus embryo

A detailed fate map of all of the progeny derived from each of the blastomeres of the 32-cell-stage South African clawed frog embryo (Xenopus laevis), which were selected for stereotypic cleavages, is presented. Individual blastomeres were injected with horseradish peroxidase and all of their descendants in the late tailbud embryo (stages 32 to 34) were identified after histochemical processing of serial tissue sections and whole-mount preparations. The progeny of each blastomere were distributed characteristically, both in phenotype and location. Most organs were populated largely by the descendants of particular sets of blastomeres, the progeny of each often being restricted to defined spatial addresses. Thus, the descendants of any one blastomere were distinct and predictable when embryos were preselected for stereotypic cleavages. However, variations among embryos were common and the frequencies with which one may expect organs to contain progeny from any particular blastomere are reported. The differences in the fates of the 16-cell-stage blastomeres and their 32-cell-stage daughter blastomeres are outlined and can be grouped into three general categories. The two daughter cells may give rise to equal numbers of cells in a particular organ, one daughter cell may give rise to many more of the cells in an organ derived from the mother blastomere, or one daughter cell may give rise to all of the progeny in an organ derived from the mother blastomere. Thus, cell fates are segregated during cleavage stages in both symmetric and asymmetric manners, and the lineages exhibit a diversification mode (G. S. Stent, 1985, Philos. Trans R. Soc. London Ser. B 312, 3-19) of cell division.     

The role of tensile fields and contact cell polarization in the morphogenesis of amphibian axial rudiments

Summary The role of stretching-generated tensile stresses upon the organization of axial rudiments have been studied. Pieces of the dorsal wall ofXenopus laevis andRana temporaria embryos at the late gastrula stage were rotated through 90°, transplanted into the field of neurulation tensions of another embryo and replaced by ventral tissues already insensitive to inductive influences. The axial rudiments which developed from rotated and transplanted dorsal tissues oped from rotated and transplanted dorsal tissues almost completely reorientated according to the tensile patterns in adjacent host tissues. Some of the donor cells changed their presumptive fates in accordance with their new positions in the host tensile field. Transplanted ventral tissues were involved in the morphogenetic movements specific for the dorsal regions and imitated some typical dorsal structures. In the regions without pronounced tensions the structure of transplanted axial rudiments was chaotic. It is suggested that the organization of the axial structures is established and maintained by tensile fields created by uniformly polarized cells. Cell polarization can be transmitted by contact from host to donor tissues. The specificity of this propagating process and its morphogenetical role is discussed.