Blastomere Isolation Research Articles

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

Totipotency continuity from zygote to early blastomeres: a model under revision.

The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.

Decoupling brain from nerve cord development in the annelid Capitella teleta: Insights into the evolution of nervous systems

In the deuterostomes and ecdysozoans that have been studied (e.g. chordates and insects), neural fate specification relies on signaling from surrounding cells. However, very little is known about mechanisms of neural specification in the third major bilaterian clade, spiralians. Using blastomere isolation in the annelid Capitella teleta, a spiralian, we studied to what extent extrinsic versus intrinsic signals are involved in early neural specification of the brain and ventral nerve cord. For the first time in any bilaterian, we found that brain neural ectoderm is autonomously specified. This occurs in the daughters of first-quartet micromeres, which also generate anterior neural ectoderm in other spiralians. In contrast, isolation of the animal cap, including the 2d micromere, which makes the trunk ectoderm and ventral nerve cord, blocked ventral nerve cord formation. When the animal cap was isolated with the 2D macromere, the resulting partial larvae had a ventral nerve cord. These data suggest that extrinsic signals from second-quartet macromeres or their daughters, which form mesoderm and endoderm, are required for nerve cord specification in C. teleta and that the 2D macromere or its daughters are sufficient to provide the inductive signal. We propose that autonomous specification of anterior neural ectoderm evolved in spiralians in order to enable them to quickly respond to environmental cues encountered by swimming larvae in the water column. In contrast, a variety of signaling pathways could have been co-opted to conditionally specify the nerve cord. This flexibility of nerve cord development may be linked to the large diversity of trunk nervous systems present in Spiralia.

Specification of Larval Axes of Partial Embryos in the Temnopleurid Temnopleurus toreumaticus and the Strongylocentroid Hemicentrotus pulcherrimus.

Many sea urchins, including the strongylocentroid Hemicentrotus pulcherrimus, produce an amniotic cavity on the left for adult rudiment formation at the late larval stage. In contrast, temnopleurids form a cell mass at the early larval stage instead of an amniotic cavity. Although the mechanisms establishing left-right polarity of the amniotic cavity involve cell-cell interactions and signaling pathways, corresponding pathways for the cell mass are unknown. We analyzed the effects of blastomere isolation on the specification of larval axes in the temnopleurid Temnopleurus toreumaticus and compared them to those in H. pulcherrimus. Blastomere isolation at the two- or four-cell stages in T. toreumaticus disturbed the location of the cell mass and adult rudiment in approximately 10-20% of specimens. In contrast, isolation at the two-cell stage in H. pulcherrimus caused the left-right polarity to become random. When blastomeres isolated at the two-cell stage were cultured as pairs, approximately 20% of pairs had atypical polarity in both species. Following isolation at the four-cell stage, 71.4% of quartets produced larvae with atypical polarity in T. toreumaticus. Thus, cell-cell interaction between two daughter blastomeres after the second cleavage may be involved in the mechanism determining left-right polarity. Dye injection into a blastomere and subsequent observations indicated that the location of the boundary of the first cleavage showed similar patterns in both species. These observations suggest that species-specific mechanisms establish the larval axes and blastomeres at the two- and four-cell stages redistribute their cytoplasm, forming gradients that establish left-right polarity.

3D structured illumination microscopy of mammalian embryos and spermatozoa.

BackgroundSuper-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.ResultsHere we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.ConclusionsOur protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.

Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo

During early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanisms underlying developmental fate restriction are one of the major research subjects within developmental biology. In this article, this subject was addressed by combining blastomere isolation with microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord precursor cells and the other to nerve cord precursors. Approximately 2,200 each of notochord and nerve cord precursor cells were isolated, and their mRNA expression profiles were compared by microarray. This analysis identified 106 and 68 genes, respectively, that are differentially expressed in notochord and nerve cord precursor cells. These included not only genes for transcription factors and signaling molecules but also those with generalized functions observed in many types of cells. In addition, whole-mount in situ hybridization showed dynamic spatial expression profiles of these genes during segregation of the two fates: partitioning of transcripts present in the mother cells into either type of daughter cells, and initiation of preferential gene expression in either type of cells.

Influence of early fate decisions at the two-cell stage on the derivation of mouse embryonic stem cell lines

The first event of differentiation in mammalian embryogenesis is the segregation of the inner cell mass and trophectoderm lineages in the blastocyst. Cellular and molecular events related to this process are still a controversial issue. During the years it was thought that first allocation of blastomeres before the blastocyst stage was done in the late eight-cell stage with the formation of inner and outer cells. Lately, many studies have pointed out that individual blastomeres at the four-cell stage differ in their developmental properties according to their position within the embryo. In this report, we wanted to elucidate whether these early decisions influence the production of mouse embryonic stem cell lines, so that a selective isolation of blastomeres at the four-cell stage to derive the lines could improve the efficiency of the derivation process. Results from blastomere tracking experiments support the idea of a different developmental potential of blastomeres within the four-cell stage embryo. However, we also show a high plasticity in the developmental pattern of blastomeres once isolated from the embryo, thus making all four-cell stage blastomeres equally competent to derive ESC lines.

Laser Killing of Blastomeres in Caenorhabditis elegans

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Within the embryo, contacts between cells often establish the polarization of neighboring cells. Blastomere isolation and recombination experiments have led to a wealth of understanding of the events in the four-cell C. elegans embryo. However, identifying individual blastomeres after isolation at stages past the four-cell stage is limited. In addition, removal of blastomeres from their native surroundings can interfere with many cell contacts besides the contacts of interest. An alternative approach for studying cell interactions within the C. elegans embryo is to use laser ablation of individual cells. Laser ablation can be used to kill one of two cells in contact with each other to understand what happens when a cell no longer signals to its neighbor. Additionally, killing a cell that is between two cells that will eventually contact each other can result in the corpse of the cell forming a steric barrier between the cells, preventing the contact. This protocol describes laser ablation of embryos mounted on an agar mount.

Tracing blastomere fate choices of early embryos in single cell culture

AbstractBlastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in &8805;50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro.

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

A “micromere model” of cell-cell interactions in sea urchin early embryos

It has been shown that isolation of sea urchin blastomeres before the post-division adhesion leads mainly to the formation of equal blastomeres at the stage of 4th cleavage division, whereas isolation after adhesion results in the formation of micromeres simultaneous with that in intact embryos. Similar results were obtained in five sea urchin species. It has been concluded that there exists a critical point in the cleavage process, when blastomeres exchange information that determines the further cleavage pattern. It has been shown with this “micromere model” that serotonin and its analogs influence the cleavage pattern of half-embryos. These data have served as a basis for the hypothesis of “protosynapse,” a bilaterally symmetric structure in which the blastomeres are not only source and target of the signal but also a passive obstacle to leakage of the signal substance from the interblastomere cleft to the milieu. Such a structure may also specify the primary asymmetry of the blastomeres. The micromere model may be useful in specific pharmacological screening.

Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors

Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P < 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

A spectroscopic assessment of cellulose and the molecular mechanisms of cellulose biosynthesis in the ascidian Ciona intestinalis

Tunicates are the only animal group known to synthesize cellulose. The current hypothesis is that a horizontally acquired cellulose synthase gene of bacterial origin might have contributed to the establishment of this unique trait. Cellulose biosynthesis in tunicates thus provides an opportunity to understand how a foreign gene was assimilated into a new genome to establish a new trait. Because little is known of the molecular mechanisms underlying cellulose biosynthesis, we set up a practical assessment of cellulose in the ascidian tunicate Ciona intestinalis. We first demonstrated and characterized cellulose in the tunic of adult specimens by chemical purification and by subsequent scanning electron microscopic observation and X-ray diffractometry. Next, we showed that Fourier transform infrared spectroscopic microscopy (FTIR microscopy) can be used to assess cellulose in the small tunic of individual larval specimens without chemical purification. Using FTIR microscopy together with a blastomere isolation technique, we demonstrated that cellulose biosynthesis occurred cell-autonomously in the animal hemisphere of an embryo where the future epidermis, the known site of cellulose biosynthesis, will arise. We combined FTIR microscopy with morpholino antisense oligonucleotide-mediated gene knockdown technology to generate a reverse genetic system to identify genes involved in cellulose biosynthesis. FTIR microscopy was thus able, in combination with current research resources, to contribute to cellular and molecular investigations of animal cellulose biosynthesis.

A revised fate map for amphioxus and the evolution of axial patterning in chordates

The chordates include vertebrates plus two groups of invertebrates (the cephalochordates and tunicates). Previous embryonic fate maps of the cephalochordate amphioxus (Branchiostoma) were influenced by preconceptions that early development in amphioxus and ascidian tunicates should be fundamentally the same and that the early amphioxus embryo, like that of amphibians, should have ventral mesoderm. Although detailed cell lineage tracing in amphioxus has not been done because of limited availability of the embryos and because cleavage is radial and holoblastic with the blastomeres nearly equal in size and not tightly adherent until the mid-blastula stage, a compilation of data from gene expression and function, blastomere isolation and dye labeling allows a more realistic fate map to be drawn. The revised fate map is substantially different from that of ascidians. It shows (1) that the anterior pole of the amphioxus embryo is offset dorsally from the animal pole only by about 20°, (2) that the ectoderm/mesendoderm boundary (the future rim of the blastopore) is at the equator of the blastula, which approximately coincides with the 3rd cleavage plane, and (3) that there is no ventral mesoderm during the gastrula stage. Involution or ingression of cells over the blastopore lip is negligible, and the blastopore, which is posterior, closes centripetally as if by a purse string. During the gastrula stage, the animal pole shifts ventrally, coming to lie about 20° ventral to the anterior tip of the late gastrula/early neurula. Comparisons of the embryos of amphioxus and vertebrates indicate that in spite of large differences in the mechanics of cleavage and gastrulation, anterior/posterior and dorsal/ventral patterning occur by homologous genetic mechanisms. Therefore, the small, nonyolky embryo of amphioxus is probably a reasonable approximation of the basal chordate embryo before the evolution of determinate cleavage in the tunicates and the evolution large amounts of yolk in basal vertebrates.

Regionalization of the Tail-tip Epidermis Requires Inductive Influence from Vegetal Cells and FGF Signaling in the Development of an Ascidian, Halocynthia roretzi

The epidermis of an ascidian larva derived from animal-hemisphere cells is regionalized along the anterior-posterior (AP) axis through inductive signals emanating from vegetal-hemisphere cells in early stages of the development. Previously, we showed by blastomere isolation and ablation experiments that the contact between the animal and vegetal hemispheres until the 32-cell stage is necessary for the proper AP patterning of the epidermis in the tailbud-stage embryo. We here addressed the patterning mechanism of the posteriormost epidermis using a tail-tip epidermis marker, HrTT-1. Employing blastomere isolation and ablation experiments along with knockdown of a master regulator gene for posterior mesoderm, we have demonstrated that presence of the posterior vegetal cells after the 32-cell stage is necessary for the expression of HrTT-1. To explore the timing and nature of the influence of the posterior vegetal cells, we treated the embryos with FGF signaling inhibitors at various developmental stages and found that HrTT-1 expression was lost from embryos treated with the inhibitors from stages earlier than the late neurula stage, just prior to the onset of HrTT-1 expression but not after the initial tailbud stage, at which the expression of HrTT-1 had started. In embryos lacking HrTT-1 expression, the expression domain of Hrcad, which would otherwise be localized anterior to that of HrTT-1, expanded to the tail-tip. These results suggest that FGF signaling from the neurula to initial tailbud stages is necessary for the initiation but not maintenance of HrTT-1 expression in the tail-tip epidermis. The contact with posterior vegetal cells until and after the 32-cell stage may be required for FGF signaling to occur in the posterior tail, which in turn regionalizes the tail-tip epidermal territory.

Isolation and characterization of embryonic stem cell‐like cells from in vitro‐produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.

Microarray analysis of localization of maternal transcripts in eggs and early embryos of the ascidian, Ciona intestinalis

The establishment of body axes and specification of early embryonic cells depend on maternally supplied transcripts and/or proteins, several of which are localized at specific regions of fertilized eggs and early embryos. The ascidian is known to exhibit a mosaic mode of development, and this mode is largely dependent on localized maternal factors. Using blastomere isolation, microarray and whole-mount in situ hybridization, the present study of Ciona intestinalis demonstrates that maternal transcripts of a total of 17 genes are localized at the posterior-most region of fertilized eggs and early embryos. Ten of them are newly identified in the present study, while the remaining seven genes have already been characterized in previous studies. In addition, maternal transcripts of two genes, in addition to 14 genes encoded by the mitochondrial genome, showed a mitochondria-like distribution. Despite the present comprehensive approach, we could not identify maternal transcripts that are clearly localized to the animal-pole side, the vegetal-pole side, the anterior-side or other specific regions of the early embryo. Therefore, we concluded that the posterior-most localization and mitochondria-like distribution appear to be major specialized patterns of maternal transcripts in early Ciona embryos.

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage.

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.