Blastomeres Of Embryos Research Articles

DNA Methylation Patterns of Metaphase Chromosomes in Human Preimplantation Embryos

We performed a stage-by-stage study of DNA methylation patterns in metaphase chromosomes from blastomeres of triploid and abnormal diploid human embryos. QFH-banded homologous parental chromosomes differ in their DNA methylation patterns at the metaphase of the 1st cleavage division. Chromosomes of both parental genomes are gradually demethylated at subsequent cleavages, undergoing hemimethylation in 2-cell embryos. At the 4-cell stage hypomethylated chromosomes initially appear and are further registered until the blastocyst stage. The proportion of hemimethylated and hypomethylated chromosomes varies between the blastomeres since the 4-cell stage with no preference for certain chromosomes to be hemi- or hypomethylated demonstrates random segregation of hypomethylated, undermethylated and methylated chromatids during cell cleavage. By the blastocyst stage the chromosomes acquire band- and, thus, chromosome-specific methylation patterns, with 5-methylcytosine-rich DNA preferentially accumulated in R- and T-bands and in the short arms of acrocentric chromosomes. Thus, demethylaton and remethylation of parental genomes of human embryos proceeds in the same manner from the 1st metaphase stage up to the blastocyst. These processes involve all chromosomes and all bands from each chromosome and lead to establishment of chromosome-specific DNA methylation patterns by the blastocyst stage with no differences between homologous chromosomes.

Traf2- and Nck-interacting Kinase Is Essential for Canonical Wnt Signaling in Xenopus Axis Formation

Wnt signaling pathways play important roles in various stages of developmental events and several aspects of adult homeostasis. Aberrant activation of Wnt signaling has also been associated with several types of cancer. We have recently identified Traf2- and Nck-interacting kinase (TNIK) as a novel activator of Wnt signaling through a comprehensive proteomic approach in human colorectal cancer cell lines. TNIK is an activating kinase for T-cell factor-4 (TCF4) and essential for the beta-catenin-TCF4 transactivation and colorectal cancer growth. Here, we report the essential role of TNIK in Wnt signaling during Xenopus development. We found that Xenopus TNIK (XTNIK) was expressed maternally and that the functional knockdown of XTNIK by catalytically inactive XTNIK (K54R) or antisense morpholino oligonucleotides resulted in significant malformations with a complete loss of head and axis structures. XTNIK enhanced beta-catenin-induced axis duplication and the expression of beta-catenin-TCF target genes, whereas knockdown of XTNIK inhibited it. XTNIK was recruited to the promoter region of beta-catenin-TCF target genes in a beta-catenin-dependent manner. These results demonstrate that XTNIK is an essential factor for the transcriptional activity of the beta-catenin-TCF complex and dorsal axis determination in Xenopus embryos.

Communication between nonadjacent blastomeres in early Xenopus embryos

Communication between nonadjacent blastomeres in early Xenopus embryos

Pregnancy and live birth following the transfer of vitrified–warmed blastocysts derived from zona- and corona-cell-free oocytes

This study reports two clinical pregnancies and one live birth following the transfer of vitrified blastocysts developed from oocytes with neither zona pellucida nor corona cells. Two zona-free oocytes obtained from two patients of advanced maternal age undergoing minimal stimulation were normally fertilized after intracytoplasmic sperm injection. In case 1, all four blastomeres of the zona-free embryo were loosely associated and inserted back into ruptured zona on day 2. Zona-free embryo from case 2 had tight contacts between blastomeres and was cultured without zona. Both embryos derived from zona-free oocytes progressed to blastocyst stage and were cryopreserved by vitrification. When patients came back for a cryopreserved embryo transfer, both vitrified blastocysts survived warming. In case 1, transfer of a warmed blastocyst with reconstructed zona resulted in a clinical pregnancy that ended in a spontaneous abortion at 22 weeks. In case 2, live birth was achieved with a normal healthy baby (male) weighing 2381 g at 40 weeks' gestation. This report emphasizes the importance of maintenance of normal cell arrangement on the subsequent embryonic development for a zona-free oocyte. Zona-free oocytes may provide a valuable source of embryos for infertility patients, especially for those with a limited number of oocytes.

Governing cell lineage formation in cloned mouse embryos

Blastomeres of the pre-implantation mouse embryo form trophectoderm and inner cell mass via a process that requires the transcription factors Tead4, Cdx2, Oct4 and Nanog. In mouse morulae cloned by somatic cell nuclear transfer, we observed that the trophectoderm transcription factor Cdx2 is expressed very differently at the protein level compared to time- and stage-matched fertilized counterparts. Protein levels of Cdx2 in cloned embryos appear ‘erratic,’ i.e. are widely distributed, when plotted as histograms. In contrast to Cdx2, protein levels of the upstream factor Tead4 and of inner cell mass transcription factors Oct4 and Nanog are similar in cloned and fertilized embryos. These observations suggest that trophectoderm formation is initiated but not maintained correctly in cloned mouse morulae, which is consistent with cloned blastocysts' limited implantation and post-implantation success. Because a cell's ability to differentiate is greatly enhanced if it is surrounded by more cells differentiating the same way, a concept designated community effect by Gurdon, we reasoned that the insufficient cell numbers often observed in cloned embryos might lead to premature Cdx2 expression and differentiation of blastomeres into trophectoderm. Therefore, we created larger cloned embryos by aggregating them at the 4-cell stage. Homologous aggregation stimulates expression of multiple signaling pathways' components and results in cloned embryos with levels of Cdx2 similar to fertilized embryos. Most of the resultant morulae and blastocysts consist of cells of all three founders, indicating that aggregation increases stability of all of the individual components. We conclude that the induction of pluripotency in cloned embryos is more efficient than previously assumed, and we propose that a minimum cell number is necessary to stabilize pluripotency and inhibit premature expression of Cdx2 in cloned mouse embryos.

Development of Monozygotic Twin Mouse Embryos from the Time of Blastomere Separation at the Two-Cell Stage to Blastocyst1

The development of blastomeres separated from two-cell stage murine embryos has been compared. Blastomeres were removed from the zona pellucida (ZP) and cultured individually; the twin embryos were compared during their progression to blastocyst in terms of development rate, cell number, morphology, conformation at the four-cell stage, and CDX2 and POU5F1 (also known as OCT4) expression. In general, twin embryos, whether obtained from superovulated or normally bred dams, displayed comparable cell numbers as they advanced. They formed morulae and blastocysts more or less synchronously with each other and with control embryos, although possessing about half of the latter's cell number. Despite this apparent synchrony, the majority of twin blastocysts differed in terms of their relative complements of POU5F1+/CDX2- cells, which represent inner cell mass (ICM), and POU5F1+/CDX2+ cells, which identify trophectoderm (TE). Many, but not all, exhibited a disproportionately small ICM. By contrast, demiembryos retained within their ZP and created by randomly damaging one of the two blastomeres in two-cell stage embryos exhibited a more normal ratio of ICM to TE cells at blastocyst and significantly less variance in ICM cell number. One possible explanation is that ZP-free demiembryos only infrequently adopt the same conformation as their partners, including the favorable tetrahedral form, at the four-cell stage, suggesting that such embryos exhibit a high degree of plasticity with regard to the orientation of their first two cleavage planes and that a significant number likely deviate from paths that provide an optimal geometric progression to blastocyst. These data could explain the difficulty of creating monozygotic twins from two-cell stage embryos.

The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst

It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

126 DEVELOPMENT OF SINGLE BLASTOMERES DERIVED FROM PORCINE TWO-CELL EMBRYOS

Monozygotic mice and calves have been produced from demi-2-cell embryos (Wang K et al. 1997 J. Reprod. Dev. 43, 91-95; Tagawa M et al. 2008 Theriogenology 69, 574-82). These studies proved that single blastomeres of 2-cell embryos could still develop properly after being separated. However, such studies are very difficult to perform in pigs due to multiple pregnancies and relatively low success of embryo transfer. The production of genetically identical animals is very useful for animal husbandry, particularly to increase the number of progeny derived from genetically valuable parents. In the present study, we compared the developmental ability to the blastocysts and their quality in terms of total cell number and gene expression of blastomere pairs derived from 2-cell embryos (pair of blastomere for short). Evenly cleaved 2-cell embryos were collected during 24 to 30 h after IVF and IVM of follicular oocytes. They were split into pairs of single blastomeres by gentle pipetting after Pronase treatment for removal of zona pellucida. Blastomeres were then cultured separately in polydimethylsiloxane (PDMS) micro-wells. Embryos were cultured in IVC-PyrLac from Days 0 to 2 (Day 0 was defined as the day of IVF) and in IVC-Glu for next 4 days (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033-1041). At Day 6, single blastocysts were collected for total cell number or gene expression analysis. Eight replications were performed for each analysis. Three genes chosen for real-time PCR were high-mobility group box 1 (HMGB1); ATP synthase, H1 transporting, mitochondrial F1 (ATP5A1); and the small nuclear ribonuclear proteins (LSM2). These genes are considered as markers to identify embryos with high development competence (Withworth KM et al. 2005 Biol. Reprod. 72, 1437-1451). The expression levels were normalized to the housekeeping gene Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAG) using standard curve method. Assessment of 42 blastomere pairs at Day 6 showed that the percentages of the pairs developed to blastocysts (36.6 ± 5.3%) or degenerated (46.3 ± 10.3%) were significantly higher than those of the pairs in which one developed to blastocyst and the other degenerated (17.1 ± 7.8%, P < 0.05, 1-way ANOVA followed by Tukey test). Assessment of two blastocysts derived from one pair of blastomeres showed that one blastocyst (B#1) had better morphology than the other (B#2) in term of expansion and B#1s had significantly higher cell number (31.6 ± 2.9) than that of B#2s (19.1 ± 1.9; P < 0.05; 1-way ANOVA). Although the expression levels of ATP5A1, HMBG1, and LSM2 in B#1s (1.08 ± 0.37, 3.51 ± 1.01, and 2.63 ± 1.17, respectively) did not differ from those in B#2s (0.39 ± 0.13, 1.18 ± 0.38, and 1.68 ± 0.83, respectively), they tended to be higher in B#1s compared to B#2s (P < 0.1). In conclusion, blastomere pairs from the same origin had the same potential to develop to the blastocyst stage. However, the qualities of blastocysts in pairs were different.

The number of blastomeres in post-thawing embryos affects the rates of pregnancy and delivery in freeze-embryo-transfer cycles

To observe the differences in pregnancy rates (PRs), delivery rates, and abortion rates associated with frozen-embryo-transfer (FET)-based use of post-thawing embryos with different numbers of blastomeres. 959 FET cycles and 361 successful FET cycles performed between January 2007 and December 2007. Compare the PRs and abortion rates in post-thawing embryos with 8 blastomeres (8c), 7c, 6c, 5c, 4c,and 3c. 1. The total PRs of post-thawing 8c, 7c, 6c, 5c, 4c, and 3c embryos were 44.1%, 41.0%, 34.4%, 23.8%, 12.5%, and 0%, respectively (p < 0.05). 2. The abortion rates for the transferred embryos of the 8c, 7c, 6c, 5c, and 4c groups were 17.92%, 19.35%, 27.69%, 24%, 20%, respectively (p < 0.05). The number of blastomeres in the post-thawing embryos is an important factor influencing the occurrence of pregnancy in FET procedures; however, the criterion that post-thawing embryos with 50% intact blastomeres will lead to pregnancy may not be valid.

Assessment of Sex Chromosomes of Human Embryos Arising from Monopronucleus Zygotes in in vitro Fertilization and Intracytoplasmic Sperm Injection Cycles of Chinese Women

Objectives: To examine abnormal fertilization phenomena in blastomeres of embryos generated from conventional IVF and ICSI displaying a single pronucleus at the zygote stage. Methods: 132 embryos from monopronuclear zygotes (1PN) generated from conventional IVF and ICSI were examined by FISH (fluorescent in situ hybridization) with X, Y dual color centromere probes. Results: In the embryos that were obtained from conventional IVF, the percentage of diploid, monoploid and mosaic sex chromosome were 54.35, 23.91 and 21.74%, respectively. In the embryos from 1PN zygotes derived from ICSI, the percentage of diploid, monoploid and mosaic sex chromosomes were 31.51, 31.51 and 36.99%, respectively. For monoploid embryos, the ratio of XO was significantly higher than that of YO in both conventional IVF and ICSI groups. Conclusion: The results demonstrated that the majority of embryos derived from 1PN zygotes generated with conventional IVF or ICSI have a high incidence of aneuploidy. Furthermore, the sex chromosome diploid ratio of embryos from 1PN zygotes derived from conventional IVF was significantly higher than those generated from ICSI procedure in Chinese women. Nevertheless, the high proportion of aneuploid embryos suggests that such embryos should not be utilized for embryo transfer.

Decreased cofilin1 expression is important for compaction during early mouse embryo development

Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarization by which cellular asymmetry is first established. During this process many molecules and organelles undergo polarized distribution, but the cytoskeletal basis for these distribution specifications remains to be explored. The present study focused on cofilin1, an actin-binding protein that depolymerizes actin filaments. We showed that cofilin1 expression decreased at the compaction stage, and that down-regulation of cofilin1 expression by siRNA microinjection accelerated compaction. Continuous observation using time-lapse video miscroscopy confirmed these findings. That is, the embryonic cells microinjected with anti-cofilin1 antibody exhibit earlier adherence properties compared to uninjected cells. Pronuclear microinjection of a site-directed mutated cofilin1 plasmid, in which cofilin1 is sustained in its active form produced embryos with blastomeres that did not adhere, suggesting that inactivation of cofilin1 is critical for cell flattening and adherence. Fluorescein-phalloidin staining indicated that decreased cofilin1 expression promoted the formation of the apical pole, which is a marker for polarity. Scanning electron microscopy results demonstrated the appearance of microvilli on the outer face of blastomeres in cofilin1 knockdown embryos. Our results suggest that cofilin1 plays an important role in cortical cytoplasmic organization during embryo compaction.

Differential cytotoxic effects of mono-(2-ethylhexyl) phthalate on blastomere-derived embryonic stem cells and differentiating neurons

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 μM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 μM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

BACKGROUNDRecently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres.METHODSFour 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs).RESULTSTen out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype.CONCLUSIONWe report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.

Particle tracking model of electrophoretic morphogen movement reveals stochastic dynamics of embryonic gradient

Some developmental events rely on an electrophoretic force to produce morphogenetic gradients. To quantitatively explore the dynamics of this process, we constructed a stochastic model of an early phase of left-right patterning: serotonin movement through the gap junction-coupled blastomeres of the Xenopus embryo. Particle-tracking simulations showed that a left-right gradient is formed rapidly, quickly reaching a final stable level. The voltage difference was critical for producing a morphogen gradient of the right steepness; gap junctional connectivity and morphogen mass determined the timing of the gradient. Endogenous electrophoresis drives approximately 50% of the particles across more than one cell width, and approximately 20% can travel across half the embryo. The stochastic behavior of the resulting gradients exhibited unexpected complexity among blastomeres' morphogen content, and showed how spatiotemporal variability within individual cells resulted in robust and consistent gradients across the embryonic left-right axis. Analysis of the distribution profile of gradient gain values made quantitative predictions about the conditions that result in the observed background level of laterality defects in unperturbed frog embryos. This work provides a general model that can be used to quantitatively analyze the unexpectedly complex dynamics of morphogens in a wide variety of systems.

Ernest Everett Just, Johannes Holtfreter, and the origin of certain concepts in embryo morphogenesis.

Ernest E. Just (1883-1941) is best known for his discovery of the "wave of negativity" that sweeps of the sea urchin egg during fertilization, and his elucidation of what are known as the fast and slow blocks to polyspermy. Just's contemporary Johannes Holtfreter (1901-1992) is known for his pioneering work in amphibian morphogenesis, which helped to lay the foundation for modern vertebrate developmental biology. This paper, after briefly describing the life and scientific contributions of Just, argues that his work and ideas strongly influenced two of the concepts for which Holtfreter is best known: tissue affinity and autoneuralization (or autoinduction). Specifically, this paper argues that, first, Just's experiments demonstrating developmental stage-specific changes in the adhesiveness of the blastomeres of cleavage embryos helped lay the foundation for Holtfreter's concept of tissue affinity and, second, Just's notion of the intrinsic irritability of the egg cell, which is evident in experimental parthenogenesis, strongly informed Holtfreter's concept of the nonspecific induction of neural tissue formation in amphibian gastrula ectoderm explants, a phenomenon known as autoinduction. Acknowledgment of these contributions by Just in no way diminishes the importance of Holtfreter's groundbreaking work. It does, however, extend the impact of Just's work into the area of embryo morphogenesis. It connects Just to Holtfreter and positions his work as an antecedent to embryo research that continues to this day.

Use of picosecond infrared laser for micromanipulation of early mammalian embryos

A high repetition rate (80 MHz) picosecond pulse (approximately 2 psec) infrared laser was used for the inactivation (functional enucleation) of oocytes and two-cell mouse embryos and also for the fusion of blastomeres of two-cell mouse embryos. The laser inactivation of both blastomeres of two-cell mouse embryos by irradiation of nucleoli completely blocked further development of the embryo. The inactivation of one blastomere, however, did not affect the ability of the second intact blastomere to develop into a blastocyst after treatment. Laser inactivation of oocytes at Metaphase II (MII) stage and parthenogenetically activated pronuclear oocytes also completely blocked their ability for further development. Suitable doses of irradiation in cytoplasm region did not affect the ability of embryos and activated oocytes to development. The efficiency of laser induced fusion for blastomeres of two-cell embryos was 66.7% and all the tetraploid embryos developed successfully into blastocysts in culture. Our results demonstrate unique opportunities of the applications of a suitable infrared periodic pulse laser as a universal microsurgery tool for individual living cells.

Dynamics of cell volume in early mouse embryos subjected to hypotonic shock

Osmotic adaptation in a mouse embryo blastomere has been studied by direct measurement of the cell volume using microtomography (laser scanning microscopy followed by quantitative 3D reconstruction). Embryo cells subjected to hypotonic shock first swelled but then returned to the initial size. At the beginning of osmotic stress, the swelling obeyed the van’t Hoff equation with a water permeability coefficient of 0.4 µm min−1 atm−1. The regulatory volume decrease was not abolished by Na+/K+-ATPase inhibition.

CARM1 is Required in Embryonic Stem Cells to Maintain Pluripotency and Resist Differentiation

Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating pluripotency in mouse embryos. Blastomeres of four-cell embryos with high H3 methylation at these sites show unrestricted potential, whereas those with lower levels cannot support development when aggregated in chimeras of like cells. Increasing histone H3 methylation, through expression of coactivator-associated-protein-arginine-methyltransferase 1 (CARM1) in embryos, elevates expression of key pluripotency genes and directs cells to the pluripotent inner cell mass. We demonstrate CARM1 is also required for the self-renewal and pluripotency of embryonic stem (ES) cells. In ES cells, CARM1 depletion downregulates pluripotency genes leading to their differentiation. CARM1 associates with Oct4/Pou5f1 and Sox2 promoters that display detectable levels of R17/26 histone H3 methylation. In CARM1 overexpressing ES cells, histone H3 arginine methylation is also at the Nanog promoter to which CARM1 now associates. Such cells express Nanog at elevated levels and delay their response to differentiation signals. Thus, like in four-cell embryo blastomeres, histone H3 arginine methylation by CARM1 in ES cells allows epigenetic modulation of pluripotency.

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid Peronella japonica: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral-aboral (O-Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O-Ab axis.