Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

A demonstration of cellular interactions during the formation of mesoderm and primordial germ cells in Pleurodeles waltlii

Animal, vegetal, dorsal and ventral blastomeres of eight-cell embryos of the urodele Pleurodeles waltlii were isolated and cultured for 15 days. The four animal blastomeres produced vesicles delimited by an irregularly shaped epidermis. In all other explants, the formation of mesodermal structures occurred, which can be interpreted as the result of inductive interaction, occurring during segmentation, between the ectodermal animal cap and vegetal yolk mass. Primordial germ cells (PGCs), which formed in 78% of cases when the presumptive ventral half to the embryo was cultured, occurred in only 48% of cases when the two ventral vegetal blastomeres were cultured alone. The absence of PGCs in the explants emanating from the four vegetal blastomeres is thought to have been due to inhibition of differentiation by notochord. This hypothesis has been confirmed by culture experiments in which the addition of presumptive chordomesoderm of young gastrulae prevented the differentiation of PGCs under conditions in which they are normally formed. These observations suggest that, in urodeles, PGCs do not arise from cells segregated as early as the eight-cell stage, but are the product of later inductive interaction between ectoderm and endoderm.     

A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos

The notochord is one of the defining features of chordates. The ascidian notochord is a rod like structure consisting of a single row of 40 cells. The anterior 32 ;primary' notochord cells arise from the A-line (anterior vegetal) blastomeres of the eight-cell stage embryo, whereas the posterior 8 ;secondary' notochord cells arise from the B-line (posterior vegetal) blastomeres of the eight-cell stage embryo. Specification of notochord precursors within these two lineages occurs in a spatially and temporally distinct manner. We show that specification of the secondary but not the primary notochord in Ciona intestinalis requires a relay mechanism involving two signalling pathways. First, we show evidence that acquisition of secondary notochord fate is dependent upon lateral Nodal signalling sources, situated in the adjacent b-line animal cells. Expression of the notochord specific gene Ci-Brachyury in the secondary notochord precursor was downregulated following selective inhibition of Nodal signal reception in B-line derivatives and also, strikingly, following selective inhibition of Nodal signal reception in A-line cell derivatives. Within the A-line, Nodal signals are required for localised expression of Delta2, which encodes a divergent form of Delta ligand. Using four distinct reagents to inhibit Delta2/Notch signals, we showed that Delta2 signalling from A-line cells, which activates the Notch/Su(H) pathway in adjacent B-line cells, is required for specification of the secondary notochord precursor. We propose a model whereby laterally produced Nodal acts to specify the secondary notochord precursor both directly in the B-line cells and via Delta2 induction in adjacent A-line cells.     

An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo

The major mesodermal tissues of ascidian larvae are muscle, notochord and mesenchyme. They are derived from the marginal zone surrounding the endoderm area in the vegetal hemisphere. Muscle fate is specified by localized ooplasmic determinants, whereas specification of notochord and mesenchyme requires inducing signals from endoderm at the 32-cell stage. In the present study, we demonstrated that all endoderm precursors were able to induce formation of notochord and mesenchyme cells in presumptive notochord and mesenchyme blastomeres, respectively, indicating that the type of tissue induced depends on differences in the responsiveness of the signal-receiving blastomeres. Basic fibroblast growth factor (bFGF), but not activin A, induced formation of mesenchyme cells as well as notochord cells. Treatment of mesenchyme-muscle precursors isolated from early 32-cell embryos with bFGF promoted mesenchyme fate and suppressed muscle fate, which is a default fate assigned by the posterior-vegetal cytoplasm (PVC) of the eggs. The sensitivity of the mesenchyme precursors to bFGF reached a maximum at the 32-cell stage, and the time required for effective induction of mesenchyme cells was only 10 minutes, features similar to those of notochord induction. These results support the idea that the distinct tissue types, notochord and mesenchyme, are induced by the same signaling molecule originating from endoderm precursors. We also demonstrated that the PVC causes the difference in the responsiveness of notochord and mesenchyme precursor blastomeres. Removal of the PVC resulted in loss of mesenchyme and in ectopic notochord formation. In contrast, transplantation of the PVC led to ectopic formation of mesenchyme cells and loss of notochord. Thus, in normal development, notochord is induced by an FGF-like signal in the anterior margin of the vegetal hemisphere, where PVC is absent, and mesenchyme is induced by an FGF-like signal in the posterior margin, where PVC is present. The whole picture of mesodermal patterning in ascidian embryos is now known. We also discuss the importance of FGF induced asymmetric divisions, of notochord and mesenchyme precursor blastomeres at the 64-cell stage.     

Developmental Fates of Blastomeres of Eight-Cell-Stage Xenopus laevis Embryos

The developmental fates of animal, vegetal, dorsal, and ventral egg regions of Xenopus laevis embryos were examined. For this purpose, a tracer enzyme (horseradish peroxidase) was injected bilaterally into pairs of eight-cell-stage blastomeres and the clonal organization of marked cells in the early tail-bud embryos was examined. The epidermis over most of the body originated from animal-ventral micromeres, but that in the head originated from animal-dorsal blastomeres and that in the area surrounding the anus originated from vegetal-ventral blastomeres. The neural tube originated from animal-dorsal, vegetal-dorsal, and animal-ventral blastomeres. These results were consistent with those of previous studies. But in contrast to previous findings, results showed that the entire notochord is derived from animal-dorsal micromeres and that the somites originate from all four bilateral pairs of blastomeres in the eight-cell stage. These results are discussed in relation to the current maps of prospective fates based on results of vital-dye staining. Morphogenetic movements are also discussed on the basis of the clonal organization demonstrated in the present study.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of midstreak to neural plate stages of the mouse embryo was studied by microinjecting horseradish peroxidase (HRP) into single axial endoderm cells in situ, and tracing the labeled descendants to early somite stages in vitro. Axial endoderm cells along the anterior fifth of the late streak/neural plate stage embryo contributed descendants either to the yolk sac endoderm or to the anterior intestinal portal. Cells of the exposed head process contributed to the trunk endoderm and notochord; neighboring endoderm cells contributed to the dorsal foregut. Contributions to the ventral foregut came from endoderm at, and anterior to, the distal tip of the younger, midstreak embryo (in which the head process was not yet exposed). Endoderm over the primitive streak contributed to the postsomite endoderm. We argue from these results and those in the literature that during gastrulation the axial embryonic endoderm is of mixed lineage: (1) an anterior population of cells is derived from primitive endoderm and contributes to the yolk sac endoderm; (2) a population at, and anterior to, the distal tip of the midstreak embryo, extending more anteriorly at late streak/neural plate stages, is presumed to emerge from primitive ectoderm at the beginning of gastrulation and contributes to the foregut and anterior intestinal portal; (3) the axial portion of the head process that begins to incorporate into the ventral surface at the late streak stage contributes to notochord and trunk endoderm. Cells or their descendants that were destined to die within 24 hr were evident at the midstreak stage. There was a linear trend in the incidence of cell death among labeled cells at the late streak/neural plate stages, ranging from 27% caudal to the node to 57% in the anterior fifth of the embryo. The surviving axial endoderm cells divided sufficiently fast to double the population in 24 hr.     

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.     

Differentiation of the four animal and the four vegetal blastomeres of the eight-cell-stage ofTriturus alpestris

Summary The 4 animal and 4 vegetal blastomeres of the eight-cell-stage ofTriturus alpestris were isolated and cultured for up to 12 days. Because of the difficulty of obtaining intact animal and vegetal blastomeres of the same embryo, we either cut off the vegetal blastomeres or sucked off the animal blastomeres. The culture of early embryonic amphibian cells is improved by the use of 50% Leibovitz-medium with added fetal calf serum providing a stable pH and optimal osmotic pressure.Isolated animal blastomeres differentiated to irregularly shaped ciliated epidermis. 30% of the cases showed small amounts of myotomes, notochord and neuroid cells in addition to irregular epidermis. The vegetal blastomeres formed trunk and tail structures but only 6% of all cases formed nearly complete head structures in addition.From the results we conclude that the vegetal blastomeres as well as the animal blastomeres of the eight-cell-stage are already determined as to their future fate. The possibility of partial regulation and the influence of asymmetric or irregular cleavage on the further development of isolated blastomeres is discussed.     

Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube.     

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.     

Determinative properties of muscle lineages in ascidian embryos

Blastomeres removed from early cleavage stage ascidian embryos and reared to 'maturity' as partial embryos often elaborate tissue-specific features typical of their constituent cell lineages. We used this property to study recent corrections of the ascidian larval muscle lineage and to compare the ways in which different lineages give rise to muscle. Our evaluation of muscle differentiation was based on histochemical localization and quantitative radiometric measurement of a muscle-specific acetylcholinesterase activity, and the development of myofilaments and myofibrils as observed by electron microscopy. Although the posterior-vegetal blastomeres (B4.1 pair) of the 8-cell embryo have long been believed to be the sole precursors of larval muscle, recent studies using horseradish peroxidase to mark cell lineages have shown that small numbers of muscle cells originate from the anterior-vegetal (A4.1) and posterior-animal (b4.2) blastomeres of this stage. Fully differentiated muscle expression in isolated partial embryos of A4.1-derived cells requires an association with cells from other lineages whereas muscle from B4.1 blastomeres develops autonomously. Clear differences also occurred in the time acetylcholinesterase activity was first detected in partial embryos from these two sources. Isolated b4.2 cells failed to show any muscle development even in combination with anterior-animal cells (a4.2) and are presumably even more dependent on normal cell interactions and associations. Others have noted an additional distinction between the different sources of muscle: muscle cells from non-B4.1 lineages occur exclusively in the distal part of the tail, while the B4.1 descendants contribute those cells in the proximal and middle regions. During the course of ascidian larval evolution tail muscle probably had two origins: the primary lineage (B4.1) whose fate was set rigidly at early cleavage stages and secondarily evolved lineages which arose later by recruitment of cells from other tissues resulting in increased tail length. In contrast to the B4.1 lineage, muscle development in the secondary lineages is controlled less rigidly by processes that depend on cell interactions.     

The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo

The 40 notochord cells of the ascidian tadpole invariably arise from two different lineages: the primary (A-line) and the secondary (B-line) lineages. It has been shown that the primary notochord cells are induced by presumptive endoderm blastomeres between the 24-cell and the 64-cell stage. Signaling through the fibroblast growth factor (FGF) pathway is required for this induction. We have investigated the role of the bone morphogenetic protein (BMP) pathway in ascidian notochord formation. HrBMPb (the ascidian BMP2/4 homologue) is expressed in the anterior endoderm at the 44-cell stage before the completion of notochord induction. The BMP antagonist Hrchordin is expressed in a complementary manner in all surrounding blastomeres and appears to be a positive target of the BMP pathway. Unexpectedly, chordin overexpression reduced formation of both primary and secondary notochord. Conversely, primary notochord precursors isolated prior to induction formed notochord in presence of BMP-4 protein. While bFGF protein had a similar activity, notochord precursors showed a different time window of competence to respond to BMP-4 and bFGF. Our data are consistent with bFGF acting from the 24-cell stage, while BMP-4 acts during the 44-cell stage. However, active FGF signaling was also required for induction by BMP-4. In the secondary lineage, notochord specification also required two inducing signals: an FGF signal from anterior and posterior endoderm from the 24-cell stage and a BMP signal from anterior endoderm during the 44-cell stage.     

Distinct Neuronal Lineages of the Ascidian Embryo Revealed by Expression of a Sodium Channel Gene

The ascidian larva contains tubular neural tissue, one of the prominent anatomical features of the chordates. The cell-cleavage pattern and cell maps of the nervous system have been described in the ascidian larva in great detail. Cell types in the neural tube, however, have not yet been defined due to the lack of a suitable molecular marker. In the present work, we identified neuronal cells in the caudal neural tube of theHalocynthiaembryo by utilizing a voltage-gated Na⁺channel gene, TuNa I, as a molecular marker. Microinjection of a lineage tracer revealed that TuNa I-positive neurons in the brain and in the trunk epidermis are derived from the a-line of the eight-cell embryo, which includes cell fates to epidermal and neural tissue. On the other hand, TuNa I-positive cells in the more caudal part of the neural tissue were not stained by microinjection into the a-line. These neurons are derived from the A-line, which contains fates of notochord and muscle, but not of epidermis. Electron microscopic observation confirmed that A-line-derived neurons consist of motor neurons innervating the dorsal and ventral muscle cells. Isolated A-line blastomeres have active membrane excitability distinct from those of the a-line-derived neuronal cells after culture under cleavage arrest, suggesting that the A-line gives rise to a neuronal cell distinct from that of the a-lineage. TuNa I expression in the a-line requires signals from another cell lineage, whereas that in the A-line occurs without tight cell contact. Thus, there are at least two distinct neuronal lineages with distinct cellular behaviors in the ascidian larva: the a-line gives rise to numerous neuronal cells, including sensory cells, controlled by a mechanism similar to vertebrate neural induction, whereas A-line cells give rise to motor neurons and ependymal cells in the caudal neural tube that develop in close association with the notochord or muscle lineage, but not with the epidermal lineage.     

Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos

An extracellular signaling molecule acts on several types of cells, evoking characteristic and different responses depending on intrinsic factors in the signal-receiving cells. In ascidian embryos, notochord and mesenchyme are induced in the anterior and posterior margins, respectively, of the vegetal hemisphere by the same FGF signal emanating from endoderm precursors. The difference in the responsiveness depends on the inheritance of the posterior-vegetal egg cytoplasm. We show that macho-1, first identified as a localized muscle determinant, is also required for mesenchyme induction, and that it plays a role in making the cell response differ between notochord and mesenchyme induction. A zygotic event involving snail expression downstream of maternal macho-1 mediates the suppression of notochord induction in mesenchyme precursors.     

Variability of morphometric indices of cleaving embryos in two anuran amphibians under modified magnetic conditions

We studied the effects of modified magnetic fields, such as reduced geomagnetic field or only its horizontal component, strengthened vertical component, or periodic alteration of horizontal component polarity, on two-, four-, and eight-cell embryos of Bufo viridis and Rana macrocnemis. Modified magnetic conditions did not affect the first or second cleavage furrow geometry. The strengthened vertical component of geomagnetic field alone decreased the frequency of two-cell embryos with the second furrow, which was not closed on the animal pole. Modified magnetic conditions more distinctly affected the vertical sizes of animal and vegetal blastomeres of eight-cell toad embryos due, apparently, to displacement of the third cleavage plane towards the animal or vegetal pole. The response of frog embryos to modified magnetic conditions was much less pronounced.     

Role of the FGF and MEK signaling pathway in the ascidian embryo

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.     

Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)