The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo

Aphidicolin, an inhibitor of DNA polymerase alpha, blocks DNA synthesis and cell division in sea urchin embryos. The effects of this inhibition appear to be stage dependent. Blastulae treated with aphidicolin before the thickening of the vegetal plate undergo developmental arrest prior to gastrulation. The extent of inhibition of DNA synthesis varies from 60 to 93% in these embryos. However, when aphidicolin is added after the vegetal plate has thickened, development continues normally through pluteus formation, even though DNA synthesis is inhibited by greater than or equal to 90% and cell division has ceased. These observations indicate that, from the vegetal plate stage onward, morphogenesis and overt differentiation are independent of DNA synthesis and cell division.     

Site of the previous meiotic division defines cleavage orientation in the mouse embryo

The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals suggests that even in highly regulative embryos such as the mouse, division patterns might be important for development. Indeed, the first cleavage divides the fertilized mouse egg into two cells: one cell that contributes predominantly to the embryonic part of the blastocyst, and one that contributes to the abembryonic part. Here we show, by removing, transplanting or duplicating the animal or vegetal poles of the mouse egg, that a spatial cue at the animal pole orients the plane of this initial division. Embryos with duplicated animal, but not vegetal, poles show abnormalities in chromosome segregation that compromise their development. Our results show that localized factors in the mammalian egg orient the spindle and so define the initial cleavage plane. In increased dosage, however, these factors are detrimental to the correct execution of division.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

Close Correlation of the Periodicities of the Cleavage Cycle and Cytoplasmic Cycle in Early Newt Embryos

The correlation between autonomous cyclic activity of the cytoplasm (cytoplasmic cycle) and the cleavage cycle was studied by using animal and vegetal half fragments of newt eggs formed by bisecting the uncleaved eggs after those eggs had been rotated through 90° off the vertical axis so as to alter the allocations of the cytoplasmic content in the two halves. When the bisection was made shortly after the rotation (Early Bisection), the resulting vegetal fragment showed 1.5 times longer intervals in the cytoplasmic cycle than its animal partner when cleavage was prevented by injection of colchicine, while when the bisection was made 30 min after the rotation (Late Bisection), the resulting pair of halves displayed equal intervals in the cytoplasmic cycle. The intervals of cell division of vegetal halves formed by the two kinds of bisection in the absence of colchicine were then examined. In these conditions, the vegetal half formed by Early Bisection still showed 1.5 times longer intervals in the cleavage cycle than its counterpart, and the half formed by Late Bisection displayed the same intervals in the cleavage cycle as its animal partner.     

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [3H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.     

Maternal Paracentrotus lividus RNAs are differentially localized during the first cell division

In Paracentrotus lividus eggs, there are RNAs localized at the animal and vegetal poles. During the first cell division, some of these RNAs are associated with the mitotic spindle, whereas others are free in the cytoplasm. Among the RNAs bound to mitotic apparatus (MA), we have found the mitochondrial 16S rRNA. By immunohistochemistry we have also detected hsp60, a mitochondrial membrane protein, localized around the MA, suggesting that the entire mitochondria are associated with it. Western blotting of proteins prepared by cellular fractionation after detergent treatment of P. lividus eggs revealed that both hsp60 and cytochrome c are not associated with cytoskeletal elements. All the above data have been confirmed by immunoblot analyses of preparations of microtubules and MA in which the presence of hsp60 and cytochrome c were detected only in the MA fraction. Moreover, mitochondrial succinate dehydrogenase activity was determined in MA and cytoplasm fractions during the first cell division, and the localization and vitality of the organelles were also confirmed by in vivo staining with Mito red. A possible role for mitochondria in the asymmetric distribution of RNAs and in cell division is discussed.     

POLARITIES, CELL DIFFERENTIATION AND PRIMARY INDUCTION IN THE AMPHIBIAN EMBRYO

1. Amphibian eggs are spherical, while the embryos are bilaterally symmetrical. The latter is manifested morphologically when gastrulation begins with the formation of the blastopore at a bilaterally symmetrical (vegetal-dorsal) location on the surface of the embryo. To account for this change in symmetry two polarities (vectors or axes) are required. These need not go through the centre, but if they do, one will go through two poles, called ‘animal’ and ‘vegetal’ in the amphibian embryo, and the other will pass through two points on opposite sides of the egg, one at the ‘dorsal’ and one at the ‘ventral’ side. Together these two polarities define a plane of bilateral symmetry. 2. It may be assumed that one polarity determines that gastrulation begins in the vegetal hemisphere, and the other that it begins at the dorsal side. 3. Judging from the distribution of pigment in the cortex of the egg and that of the yolk-hyaloplasm in the interior, an animal-vegetal polarity is already present in the unfertilized egg. That cytoplasmic components are actually part of the material substrate of this polarity is evident from the fact that the pattern of gastrulation may be upset if the distribution of yolk-hyaloplasm is deranged. 4. At fertilization the pigment border is raised at the side opposite the fertilizing sperm, giving rise to the ‘grey crescent’. The latter confers the first visible bilateral symmetry on the egg, and in fact it determines the presumptive median plane, for blastopore formation begins in the midline of the grey crescent. The dorso-ventral polarity imposed by the sperm is not irreversibly determined. By various experimental means, e.g. restriction of the oxygen supply, it may be inverted. 5. In order to understand the mechanism of the polarities it is necessary to study the processes on which the effects of the polarities are exerted, viz. the process of invagination associated with the formation of the blastopore. It has been known for a long time that at the bottom of the blastoporal groove are located some large flask-shaped cells, called ‘Ruffini's cells’. Various arguments can be mobilized to support the notion that these cells actually are engaged in pulling in the embryonic surface. 6. These cells are the first representatives of a cell type different from the spherical cells which are typical of the early embryo. It may therefore be presumed that Ruffini's cells are the products of the first cell differentiation occurring during amphibian embryogenesis. And it may further be assumed that the polarities somehow control this process. 7. A number of observations suggest that the animal-vegetal polarity is in direct control of the differentiation, ensuring that Ruffini's cells are formed only in the vegetal hemisphere. This point has been corroborated by isolating in cultures small aggregates from various regions of the blastula. When this is done it is found that the only path of differentiation available to animal cells is the formation of small spherical aggregates composed of a mixture of ciliated and non-ciliated cells. In contrast, in cultures of vegetal cells an outgrowth of cells occurs, and these cells share a number of properties with Ruffini's cells, and it is suggested that they are representatives of this cell type. 8. The formation of these cells is suppressed by inhibitors of RNA synthesis and by anaerobiosis induced by KCN. Since oxidative metabolism is apparently required for the differentiation of Ruffini's cells - gastrulation in the intact embryo is suppressed by anaerobiosis - a number of carbohydrate metabolites were scrutinized for their effect on the formation on Ruffini's cells. It was found that at 10 mm lactate completely suppresses their appearance, and indeed all the other cell differentiations that can otherwise be observed in our cell cultures. Since there is a very steep animal-vegetal cytoplasmic gradient in carbohydrate, the content being lowest at the vegetal pole, lactate might potentially be the agent of the animal-vegetal polarity, but there are a number of facts which do not readily support this idea. 9. If animal cells are explanted together with a few vegetal cells, some of the aggregates do not become ciliated, but rather exhibit an outgrowth similar to the one observed with vegetal cells. These animal cells have the same general shape as the vegetal Ruffini's cells, but they are smaller and more pigmented, typical ‘animal’ features. When the cultures are preserved, the cells undergo further differentiation, becoming either ‘mesenchyme’ cells, nerve cells, pigment cells and sometimes even muscle cells may be observed. In the normal embryo these differentiation patterns occur in that part of the animal hemisphere which becomes induced through contact with the vegetal material entering the blastocoel during gastrulation. Thus there is reason to assume that the induction occurring in our cultures is a miniature of the normal induction process. 10. Just as in the sea-urchin embryo, the animal cells in amphibia may become ‘vegetalized’ by addition of Li⁺ to the culture medium. 11. For various reasons it is likely that Ruffini's cells contain heparan sulphate, and in the belief that this substance might be the inductor proper, its effect was tested on animal cells. It turned out that in a concentration of 0·1 ppm it can alter the differentiation pattern of these cells, and we suggest that heparan sulphate, for the time being, is the most likely candidate for the role of primary inductor in the amphibian embryo. 12. The edges of the blastoporal groove, and hence the formation of Ruffini's cells, proceeds gradually around the circumference of the embryo. The effect of the dorso-ventral polarity therefore appears to be concerned with the time at which the cells undergo differentiation, imposing a spatial and a temporal gradient on this phenomenon. The second overt manifestation of the dorso-ventral polarity, next to the formation of the grey crescent, concerns the size of the embryonic cells, the dorsal ones being always smaller than the ventral. This fact suggests the possibility that the polarity may exert its effect by interfering with the process of cell division. 13. The cell divisions in the early embryo are distinguished by being synchronous; all cells are either undergoing mitosis or they are in interphase. The duration of the latter is typically very short. After a certain number of cell divisions, around 10, when the embryos are in the mid-blastula stage, the synchrony is gradually lost, while the interphase becomes considerably prolonged. This peculiar behaviour suggests that the cytoplasm of the early embryonic cells contain some factor which ensures the synchrony. The well-known presence in the early embryo of deoxyriboside-containing material, in an amount corresponding roughly to the total amount of DNA residing in the cell nuclei after 10 cell divisions hinted that deoxyribosides might indeed be the ‘synchrony factor’. 14. This idea was tested first on intact embryos. An excess of deoxyribonucleotides was injected into very early embryos. The result was developmental arrest at a pregastrula stage (no Ruffini's cells formed) in a large percentage of embryos. However, the number of cells was greater than in the controls, and the rate of cell division higher, indicating a delay in the transition to synchrony, thus supporting the proposed mechanism. Furthermore, the deoxynucleotides inhibited cell differentiation and an explanation of this was found in the fact that they also strongly inhibited RNA synthesis. 15. The studies were extended to cell cultures. It was found that deoxyribosides inhibit the differentiation of animal as well as vegetal cells; instead, the cells go on dividing at least for another two rounds. The utilization of added deoxyribosides does not demonstrate that the endogenous substances are similarly utilized. That they are, was indicated by the following experiment: In the presence of cytosine arabinoside, an inhibitor of DNA synthesis de novo, the explanted cells go on dividing an unknown number of times, and then they, animal as well as vegetal cells, undergo differentiation. But in either case these cells are larger (about four times) than the controls. This result suggests that in the experimental cultures the cells go on dividing as long as the cytoplasmic deoxyribosides last and then stop, while the controls synthesize their own DNA for two rounds of division before they undergo differentiation. 16. It is now possible to suggest a mechanism for the dorso-ventral polarity. First it affects the cell size such that the dorsal cells are the smallest. If the cytoplasmic deoxyribosides are evenly distributed at the outset, then small cells must be nearer exhaustion than large ones. A dorso-ventral gradient in cell sue will therefore automatically imply a dorso-ventral gradient in the time at which the cells reach the state in which they can undergo differentiation.     

The localization of an inductive response

Combinations of tissues from Xenopus blastulae have been used to identify several mechanisms that limit the number of animal cells forming muscle after induction by vegetal cells. The results disagree with a model in which direct physical contact or very close proximity between animal and vegetal cells restricts the number of cells that receive the inductive signal. Rather it seems that a diffusible inducer is released by vegetal cells, and spreads through 4-8 animal cell diameters, equivalent to a distance of 80 microns, from the nearest vegetal cells. Several factors seem to cooperate to prevent the further spread of the mesoderm-forming induction. These include the slow diffusion and/or instability of the inducer, the time of loss of competence of animal cells to respond to induction, and the amount of vegetal tissue that releases inducer for a limited time. The combination of these, and perhaps other, processes seems to ensure that a consistent minority of animal cells are induced to form muscle, thereby leaving other animal cells available to form the nervous system and epidermis.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids

The mouse uterine epithelium under various hormonal regimes is a good system to identify biochemical events associated with cell growth, DNA synthesis and cell division. This is because estradiol-17β stimulates the cells to undergo a synchronized wave of DNA synthesis and cell division. Estriol, on the other hand, also stimulates DNA synthesis but because of the rapid loss of this hormone from the tissue some of the cells abort, giving a constant epithelial cell number. Three days of progesterone pretreatment, however, completely suppresses the estradiol-17β-induced wave of DNA synthesis and cell proliferation.Using these hormonal treatments we have shown that both estradiol-17β and estriol stimulate protein and rRNA synthesis with the concomitant increase of protein and rRNA per mg of DNA. These macromolecules accumulated in direct proportion to the fraction of cell committed to DNA synthesis. Estriol, however, did not sustain the growth responses and at the peak of DNA synthesis both rRNA and protein synthesis had returned to control levels. Progesterone pretreatment, despite inhibiting the proliferative response, failed to inhibit any of the estradiol-17β-induced increases in protein and rRNA synthesis. Indeed 12 h after estradiol-17β injection the cells had identical protein and rRNA contents, regardless of whether they had been exposed to progesterone or not.The present data therefore suggests that in the uterine epithelium cell growth as defined by protein and rRNA accumulation and DNA synthesis represents two independently regulated pathways.     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the alga<Emphasis Type="Italic">Scenedesmus quadricauda</Emphasis>

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.     

Delayed transition to new cell fates during cellular reprogramming

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.     

Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.     

Regionalized Cell Division during Sea Urchin Gastrulation Contributes to Archenteron Formation and Is Correlated with the Establishment of Larval Symmetry

During gastrulation of the sea urchin, Lytechinus variegutus there is localized proliferation of cells in the vegetal plate region prior to its invagination. Cell counts show that during gastrulation the number of cells per embryo increases 60% from 1025 to 1640. Measurements of cell volumes suggest that some growth may follow these divisions. Feulgen staining shows that the greatest mitotic activity throughout gastrulation occurs in the vegetal plate region. Labelling embryos with 3H-thymidine reveals that incorporation in the vegetal plate is confined to cells that encircle the base of the archenteron. Pulse-chase experiments indicate that these labelled cells contribute descendants to the vegetal half of the archenteron. Additionally, 3-dimensional reconstructions of vegetal regions at different stages reveal that by the end of gastrulation two bilateral clusters of labelled cells lie at the future sites of the post-oral arms of the pluteus larva, thus marking the axes of bilateral and dorso-ventral symmetry. Our findings suggest that two of the principal events of sea urchin gastrulation — the formation of the archenteron and the establishment of symmetry in the larva — are accompanied by distinct patterns of cell division.     

Novel population of embryonic secondary mesenchyme cells in the keyhole sand dollar Astriclypeus manni

We have found a novel embryonic cell population in the keyhole sand dollar Astriclypeus manni, which we refer to as lucent fluorescent cells (LFCs). Live LFCs are transparent, but emit autofluorescence after formaldehyde fixation. LFCs become noticeable in the vegetal plate of early gastrulae immediately after the appearance of pigment cells. As development progresses, LFCs increase in number and migrate from the vegetal plate toward the animal pole in a manner similar to pigment cells. Notably, LFCs also migrate into the oral ectoderm, while pigment cells do not. In addition, we determined that there were nearly 300 LFCs per embryo, which greatly exceeds the number of pigment cells. Treatment with the Notch signaling inhibitor N‐[(3,5‐Difluorophenyl)acetyl]‐l ‐alanyl‐2‐phenyl]glycine‐1,1‐dimethylethyl ester (DAPT) resulted in a marked decrease in pigment cell number, but only a modest decrease in LFCs. In DAPT‐treated embryos, LFCs had a distribution pattern similar to pigment cells and were excluded from the oral ectoderm. Unlike other sea urchins, Nodal signaling was not involved in the specification of pigment cells and LFCs in these embryos. Pulse treatment and measurement of cell diameters revealed that LFCs underwent 13–15 cycles of cell division and were specified during the 11th cleavage, one cell cycle later than observed for pigment cells. At the pluteus stage, a cluster of LFCs was observed in the animal plate in addition to two rows of LFCs running along the ciliary band. In addition, dozens of LFCs aligned at the uppermost level of the stomodaeum. Therefore, though the two cell populations share some features, LFCs are considerably different from pigment cells.