Timing of the potential of micromere-descendants in echinoid embryos to induce endoderm differentiation of mesomere-descendants

It has been reported that the micromeres of echinoid embryos have the potential to induce an archenteron in animal cap mesomeres recombined at the 16- or 32-cell stage. In the present study, experiments were performed to determine the exact period when the micromeres transmit their inductive signal to respecify the cell fate of mesomeres as endo-mesoderm. An animal cap was recombined with a quartet of micromeres, or micromere-descendants cultured in isolation, to form a recombinant embryo. The micromere-descendants were completely removed at various developmental stages, resulting in an embryo composed only of mesomere-descendants that had been under the inductive influence of micromeres for a limited period. The resulting embryos were cultured and examined for their potential to differentiate endoderm. The results indicated that the signal effective for inducing an archenteron in mesomere-descendants emanated from the micromere-descendants at the early blastula stage around hatching onward. Before this stage, the micromeres and micromere-descendants showed this potential slightly or not at all. The inductive signal emanated from the micromere-descendants almost on time even when the cells were cultured in isolation. The micromere-descendants completed transmission of the signal for inducing the archenteron in the animal cap within 2 h of recombination. The animal cap at between the 28-cell stage and 2 h after the 32-cell stage could react with the inductive signal from the micromere-descendants. Embryos composed of only animal cap mesomeres that had received the inductive signal from micromere-descendants for a limited period had the potential to develop into 8-armed plutei. Each pluteus formed an adult rudiment essentially on the left side of the larval body, and metamorphosed into a juvenile with pentaradiate symmetry.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with a quartet of micromeres

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with micromeres was examined. An animal cap consisting of mesomere-descendants was isolated from a 32-cell stage embryo, and recombined with a quartet of micromeres isolated from a 16-cell stage embryo. The recombined embryos were completely depleted of the progenitors of an archenteron, pigment cells, blastocoelar cells and muscle cells. Secondary mesenchyme-like cells (induced SMC) were released from the archenteron derived from the animal cap cells in the recombined embryos. Some induced SMC differentiated into pigment cells, confirming previous data for another echinoid species. Moreover, three different kinds of mesodermal cells-blastocoelar, muscle and coelomic pouch cells-were formed in the recombined larvae. Experiments using a fluorescent probe confirmed that the pigment, blastocoelar, muscle cells and cells in part of the coelomic pouches in the recombined larvae were derived from the animal cap mesomeres. These results indicated that the animal cap mesomere had the potential to differentiate through cell fate regulation into four mesodermal cell types-pigment, blastocoelar, muscle and coelomic pouch cells-.     

Developmental potential of small micromeres in sea urchin embryos

The large micromeres (lMics) of echinoid embryos are reported to have distinct potentials with regard to inducing endo-mesoderm and autonomous differentiation into skeletogenic cells. However, the developmental potential of small micromeres (sMics), the sibling of lMics, has not been clearly demonstrated. In this study we produced chimeric embryos from an animal cap recombined with various numbers of sMics, in order to investigate the developmental potential of sMics in the sea urchin Hemicentrotus pulcherrimus and the sand dollar Scaphechinus mirabilis. We found that sMics of H. pulcherrimus had weak potential for inducing presumptive ectoderm cells to form endo-mesoderm structures. The inducing potential of ten sMics was almost equivalent to that of one lMic. The sMics also had the potential to differentiate autonomously into skeletogenic cells. Conversely, the sMics of S. mirabilis did not show either inductive or skeletogenic differentiation potential. The sMics of both species had the potential to induce oral-aboral axis establishment. These results suggest that the potential for sMics to differentiate into skeletogenic cells and for inducing the presumptive ectoderm to differentiate into endomesoderm differs across species, while the potential of sMics to induce the oral-aboral axis is conserved among species.     

Micromere descendants at the blastula stage are involved in normal archenteron formation in sea urchin embryos

Several lines of evidence suggest that micromere signaling plays a key role in endo-mesoderm differentiation along the animal-vegetal (A-V) axis in sea urchin embryos. A recent study has suggested that the activity of micromeres of inducing endoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula stage in the echinoids Scaphechinus mirabiris and Hemicentrotus pulcherrimus. In the present study, to confirm the inductive capacity of the micromere descendants in normal development, the timing of initiation of gastrulation and the elongation rate of the archenteron were examined in both micromereless embryos and in micromereless embryos cultured until the hatching blastula stage and then recombined with micromere descendants of the same age. The micromereless embryos consistently exhibited a delay in the initiation of gastrulation and a decrease in elongation rate of the archenteron, as compared with those in controls. In contrast, when the micromereless embryos cultured until the hatching blastula stage were recombined with micromere descendants of the same age, the recombinant embryos exhibited rescue of both the delay in initiation of gastrulation and a decrease in elongation rate of the archenteron. The delayed expression of alkaline phosphatase activity, an endoderm-specific marker, in the micromereless embryos was also rescued in the recombinant embryos. The recombined micromere descendants formed the larval spicules in the same schedule as that observed in the controls. These results indicate that at the hatching blastula stage, micromere descendants emanate a signal(s) required for normal gastrulation of the presumptive endo-mesodermal region.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Potential of veg2 blastomeres to induce endoderm differentiation in sea urchin embryos

Two different modes of gastrulation in sea urchin embryos have been reported. The first mode, reported in Hemicentrotus pulcherrimus and some other species, consists of two phases: a primary and a secondary invagination. The second mode involves gastrulation with a continuous convolution of cells near the blastopore; this mode has been reported to occur in the embryos of the sand dollar, Scaphechinus mirabilis. The rudimentary gut is comprised of fewer cells in the embryos of the former species than in the latter. We assumed that the differences in gastrulation modes could be related to the different potentials of the veg2 layer to induce endoderm differentiation in the upper layer. In the present study, we produced chimeric embryos consisting of an animal cap recombined with veg2 layer blastomere(s) to compare the inductive effect of the veg2 layer and/or the blastomere(s) in H. pulcherrimus and S. mirabilis embryos. Our results showed that the inductive effect of the veg2 layer is stronger in S. mirabilis embryos than in H. pulcherrimus embryos. Moreover, it was suggested that the difference in the strength of inductive effects of veg2 layers is related to the difference in gastrulation modes.     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

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

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

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Investigations into the degree of cell mixing that occurs between the 8-cell stage and the blastocyst stage of mouse development.

This study was designed to assess the degree of cell mixing that occurs during the early development of the mouse embryo, and thus provide information which is important in relation to the current theories of differentiation. Previous studies of this nature have involved either chimeric composites, or have only followed a very limited number of cells in the embryo. Here the products of one of the 4-cell stage blastomeres have been labeled with tritiated thymidine, at a level which allows their descendants to be identified three or four cell divisions later, and recombined with the remaining blastomeres of the same embryo. After fixing and sectioning of the embryos at the blastocyst stage the locations of the labelled cells have been analyzed to assess the degree of clumping that they display. A significant tendency for the products of this one 4-cell stage blastomere to be confined to a single area in the blastocyst is demonstrated. This indicates that there is little marked cell movement during the observation period. The relevance of these results to current knowledge of blastocyst development is discussed.     

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.     

The origin of skeleton forming cells in the sea urchin embryo

Summary In embryos of the modern sea urchin species, subclass Euechinoidea, primary mesenchyme cells are derived from the progeny of micromeres formed at the sixteen cell stage of embryogenesis. The micromeres reside within the vegetal plate epithelium and later ingress into the blastocoel as primary mesenchyme cells which form the larval skeleton. Embryos of Eucidaris tribuloides, a member of the primitive subclass Perischoechinoidea, exhibit several noteworthy differences from euechinoid primary mesenchyme cell lineage including variable numbers and sizes of micromeres, the absence of mesenchyme ingression, and the lack of any detectable primary mesenchyme although a larval skeleton forms. In the present study, the cell lineage of the spiculogenic mesenchyme has been studied in Eucidaris tribuloides and in the euechinoid Lytechinus pictus by microinjecting the fluorescent tracer, Lucifer Yellow, into individual blastomeres of the embryo. In addition, wheat germ agglutinin, a lectin which binds only to primary mesenchyme cells of the early euechinoid embryo, was injected into the blastocoel of embryos of both species in order to examine the distribution of cells which possess primary mesenchyme-specific cell surface markers. The results of these experiments demonstrate that the spiculogenic mesenchyme of both Lytechinus and Eucidaris arise from descendants of micromeres formed at the sixteen cell stage, although the temporal and spatial distribution of these mesenchyme cells varies considerably between species. Furthermore, the evidence obtained suggests that the information necessary for spicule formation is already segregated to the vegetal pole by the eight cell stage. The results also suggest that there are no gap junctions present between the blastomeres of the early sea urchin embryo.     

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.     

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p < 0.05), but significantly lower than in TR<>N chimeric blastocysts (p < 0.001). Similarly, a higher proportion (p < 0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.