The role of secondary mesenchyme cells during sea urchin gastrulation studied by laser ablation

It has long been thought that traction exerted by filopodia of secondary mesenchyme cells (SMCs) is a sufficient mechanism to account for elongation of the archenteron during sea urchin gastrulation. The filopodial traction hypothesis has been directly tested here by laser ablation of SMCs in gastrulae of the sea urchin, Lytechinus pictus. When SMCs are ablated at the onset of secondary invagination, the archenteron doubles in length at the normal rate of elongation, but advance of the tip of the archenteron stops at the 2/3 gastrula stage. In contrast, when all SMCs are ablated at or following the 2/3 gastrula stage, further elongation does not occur. However, if a few SMCs are allowed to remain in 2/3-3/4 gastrulae, elongation continues, although more slowly than in controls. The final length of archenterons in embryos ablated at the 1/3-1/2 gastrula stage is virtually identical to the final length of everted archenterons in LiCl-induced exogastrulae; since filopodial traction is not exerted in either case, an alternate, common mechanism of elongation probably operates in both cases. These results suggest that archenteron elongation involves two processes: (1) active, filopodia-independent elongation, which depends on active cell rearrangement and (2) filopodia-dependent elongation, which depends on mechanical tension exerted by the filopodia.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

Behavior of pigment cells closely correlates the manner of gastrulation in sea urchin embryos

To know whether behavior of pigment cells correlates the process of gastrulation or not, gastrulating embryos of several species of regular echinoids (Anthocidaris crassispina, Mespilia globulus and Toxopneustes pileolus) and irregular echinoids (Clypeaster japonicus and Astriclypeus manni) were examined. In M. globulus and A. crassispina, the archenteron elongated stepwise like in well-known sea urchins. In the embryos of both species, fluorescent pigment cells left the archenteron tip and migrated into the blastocoel during gastrulation. In T. pileolus, C. japonicus and A. manni, on the other hand, the archenteron elongated at a constant rate throughout gastrulation. In these species, no pigment cell was observed at the archenteron tip during invagination processes; pigment cells began to migrate in the ectoderm from the vegetal pole side toward the apical plate without entering the blastocoel. These results clearly indicate that the behavior of pigment cells closely correlated the manner of gastrulation. Further, it was examined whether the archenteron cells are rearranged during invagination, by comparing the number of cells observed on cross sections of the archenteron at the early and late gastrula stages. The rearrangement was not conspicuous in A. crassispina and M. globulus, in which archenteron elongated stepwise. In contrast, the archenteron cells were remarkably rearranged in C. japonicus, alothough the archenteron elongated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.     

Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells.

Secondary mesenchyme in sea urchin embryos is released into the blastocoel after primary mesenchyme, and although these cells have been recognized for some time, we lack knowledge about many fundamental aspects of their origin and fate. Here we documented the ontogeny of one of the principal, and least well-known, types of cells derived from secondary mesenchyme. The blastocoelar cells arise from mesenchyme released from the tip of the archenteron following the initial phase of gastrulation. The cells migrate with their cell bodies suspended in the blastocoel, rather than being apposed to the basal lamina like primary mesenchyme. The cells extend numerous fine filopodia to form a network of cytoplasmic processes around the gut, along the skeletal rods, and within the larval arms. Once the network is formed, the cells maintain their positions, although they actively translocate vesicles and cytoplasm along their filopodia. Cell counts indicate there is an initial recruitment of cells during gastrulation, followed by a more gradual increase in cell number after the larva begins to feed. Lineage studies in which 16-cell-stage macromeres were injected with horseradish peroxidase indicate that almost all of the macromere-derived mesenchyme forms pigment cells and blastocoelar cells. We propose that blastocoelar cells are a distinct subset of secondary mesenchyme that forms fibroblast-like cells in the blastocoel of sea urchin embryos.     

Mesendoderm cells induce oriented cell division and invagination in the marine shrimp Sicyonia ingentis

The mesendoderm (ME) cells are the two most vegetal blastomeres in the early developing embryo of the marine shrimp Sicyonia ingentis. These two cells enter mitotic arrest for three cycles after the 5th cell cycle (32-cell stage) and ingress into the blastocoel at the 6th cycle (62-cell stage). Circumjacent to the ingressing ME cells are nine presumptive naupliar mesoderm (PNM) cells that exhibit a predictable pattern of spindle orientation into the blastopore, followed by invagination. We examined the role of ME cells and PNM cells in gastrulation using blastomere recombinations and confocal microscopy. Removal of ME progenitors prevented gastrulation. Removal of any other blastomeres, including PNM progenitors, did not interfere with normal invagination. Altered spindle orientations occurred in blastomeres that had direct contact with one of the ME cells; one spindle pole localized to the cytoplasmic region closest to ME cell contact. In recombined embryos, this resulted in an extension of the region of ME-embryo contact. Our results show that ME cells direct the spindle orientations of their adjacent cells and are consistent with a mechanism of oriented cell division being a responsible force for archenteron elongation.     

The fine structure of Comanthus japonica (Echinodermata: Crinoidea) from zygote through early gastrula

The fine structure of the early embryo of Comanthus has been described by scanning and transmission electron microscopy at approximately 20-min intervals from zygote (20 min) through early gastrula (260 min). In normally developing (and presumably monospermic) embryos, some non-fertilizing sperm were invariably trapped in the perivitelline space; this suggests that there is an effective block to polyspermy at the level of the plasma membrane. No trace of a hyaline layer is encountered in the pervitelline space. At first cleavage, which begins unilaterally at the animal pole, the contractile ring filaments are rather thick (50–150 Å) in comparison to those known for other marine invertebrates. From first cleavage through early gastrula, the lateral surfaces of the blastomeres are broadly adherent, and there is an intercellular material, presumably an adhesive, in the intercellular space. The blastocoel first appears during the four-cell stage. From the eight-cell stage through the start of gastrulation, only one opening, the vegetal pore, connects the blastocoel with the perivitelline space. Gastrulation begins at the 50–100-cell stage, while the vegetal pore is still open, and a clearly defined blastula stage is bypassed. Gastrulation is by a novel process, which I have called holoblastic involution. At gastrulation the eight most vegetal blastomeres, which encircle the vegetal pore, shoot out erect, unbranched filopodia for many microns through the blastocoel. The filopodia adhere to the blastocoelic surfaces of the animal blastomeres and contract, pulling the vegetal blastomeres into the blastocoel. The migrated vegetal blastomeres adhere to one another, forming the entoderm in the vegetal region of the embryo; the remaining blastomeres become the ectoderm. Soon after the completion of cell migration, the entodermal blastomeres appear to cast off their contractile microappendages and adhesive membranes into the blastocoel.     

Behavior of pigment cells in gastrula-stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis

The behavior of pigment cells in sea urchin embryos, especially at the gastrula stage, is not well understood, due to the lack of an appropriate method to detect pigment cells. We found that pigment cells emanated autofluorescence when they were fixed with formalin and irradiated with ultraviolet or green light. In Hemicentrotus pulcherrimus, fluorescent pigment cells became visible at the archenteron tip at the mid-gastrula stage. The cells detached from the archenteron slightly before the initiation of secondary invagination and migrated toward the apical plate. Most pigment cells entered the apical plate. This entry site seemed to be restricted, because pigment cells could not enter the ectoderm and remained in the blastocoele at the vegetal pole side when elongation of archenteron was blocked. Pigment cells that had entered the apical plate soon began to migrate in the aboral ectoderm toward the vegetal pole. In contrast, pigment cells of Scaphechinus mirabilis embryos were first detected in the vegetal plate before the onset of gastrulation. Without entering the blastocoele, these cells began to migrate preferentially in the aboral ectoderm toward the animal pole. When the archenteron tip reached the apical plate, pigment cells had already distributed throughout the aboral ectoderm. Thus, the behavior of pigment cells was quite different between H. pulcherrimus and S. mirabilis.     

Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements of the superficial layer.

The disposition of prospective areas and the course of morphogenetic movements during gastrulation and neurulation were investigated by vital staining. The prospective lining of the archenteron, the prospective neural area, and the prospective epidermal area are represented on the surface of the early gastrula. The prospective lining of the archenteron occupies the area within 65–70° of the vegetal pole and is divided into prospective archenteron roof and prospective archenteron floor by the blastopore pigment line which functions as the locus of invagination. A crescent-shaped neural area lies immediately above the prospective archenteron roof, rising from it at 125° lateral to the dorsal midline to a point 130° above the vegetal pole in the dorsal midline. In the early gastrula, most, if not all, mesoderm is deep to the surface layer and is mapped by the insertion of dyed agar spikes. Results thus far indicate that the prospective notochord lies in the dorsal deep marginal zone, followed laterally by the medial region of the somites, the lateral region of the somites, and the lateral plate.The morphogenetic significance of the comparative disposition of the anlagen in Xenopus is discussed.     

When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?

During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.     

Local shifts in position and polarized motility drive cell rearrangement during sea urchin gastrulation

This study examines the mechanisms of epithelial cell rearrangement during archenteron elongation in the sea urchin embryo using scanning electron microscopy, differential interference contrast videomicroscopy, cell marking, and fluorescently labeled chimaeric clones. Archenteron elongation involves two major processes: local shifts in position of cells in the archenteron wall and polarized motility of the cells as they rearrange. Fluorescently labeled chimaeric clones introduced into the archenteron of Lytechinus pictus are initially 4-5 cells wide; by the end of gastrulation the clones elongate and narrow, so that they are one cell wide in the narrowest region of the archenteron. The extent of clonal mixing indicates that cells in the archenteron change their relative positions by only 1-2 cell diameters during cell rearrangement. Cells at the blastopore rearrange concomitantly with cells in the archenteron, resulting in a 35% decrease in blastopore diameter. Endoderm cells undergo polarized, stage-specific changes in shape and motility as they rearrange; (1) they flatten markedly along their apical-basal axis throughout archenteron elongation; (2) just prior to the onset of cell rearrangement, basal surfaces of all cells in the archenteron extend long, polarized lamellipodial protrusions along the axis of extension of the archenteron; (3) as cell rearrangement begins, basal surfaces round up and the cells become isodiametric; (4) by the 3/4 gastrula stage the cells become stretched along the animal-vegetal axis, apparently due to filopodial traction, and finally (5) they continue to rearrange, returning to a less elongated shape by the end of gastrulation. Direct observation of gastrulation in the cidaroid Eucidaris tribuloides indicates that in this species cell rearrangement is accomplished by progressive circumferential intercalation of cells without upwardly directed filopodia. This intercalation is accompanied by explosive, apparently stochastic, cortical blebbing activity at the boundaries between cells, suggesting that in addition to whatever cell rearrangement may be generated by filopodial tension, such activity is an important component of the active rearrangement process.     

Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.

A main achievement of gastrulation is the movement of the endoderm and mesoderm from the surface of the embryo to the interior. Despite its fundamental importance, this internalization process is not well understood in amphibians. We show that in Xenopus, an active distortion of the vegetal cell mass, vegetal rotation, leads to a dramatic expansion of the blastocoel floor and a concomitant turning around of the marginal zone which constitutes the first and major step of mesoderm involution. This vigorous inward surging of the vegetal region into the blastocoel can be analyzed in explanted slices of the gastrula, and is apparently driven by cell rearrangement. Thus, the prospective endoderm, previously thought to be moved passively, provides the main driving force for the internalization of the mesendoderm during the first half of gastrulation. For further involution, and for normal positioning of the involuted mesoderm and its rapid advance toward the animal pole, fibronectin-independent interaction with the blastocoel roof is required.     

Cleavage and gastrulation in the shrimp Sicyonia ingentis: invagination is accompanied by oriented cell division.

Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.     

The Spemann organizer meets the anterior‐most neuroectoderm at the equator of early gastrulae in amphibian species

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid‐ to late‐gastrula. However, we found that this physical contact was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the “subduction and zippering” movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.     

Ambystoma maculatum gastrulae have an oriented, fibronectin-containing extracellular matrix.

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin-containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastula (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip-animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN-containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN-coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge.     

Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze-Dried Newt Embryos Before and During Gastrulation

A scanning electron-microscopic study was carried out on the extracellular matrices (ECMs) in freeze-dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.     

The very rapid induction of filopodia in insect cells

Many insect cells, including epidermis, fat body, ocnocytcs and pericardial cells, can very easily be induced to form long fine processes or filopodia. Filopodia contain microfilaments hut differ from epidermal feet in lacking microtubules and in having a much smaller and uniform diameter. Although they may be 10-30 mum long they are less than 0.1 mum wide. They often form straight connections like guy-ropes between their origins and their tips, and when freed from their surface attachments they may contract into helices, as though capable of generating tension. The basal lamina helps to keep the basal surfaces of epidermal cells together. In Rhodnius epidermis, filopodia form only seconds after its removal. They arise at the cell margins and extend to distant part of neighbouring cells where they adhere particularly at their tips. Such filopodia retract and disappear in 20-60 min with the reformation of the basal lamina as though they have functioned to pull neighbouring cells back together. In Calpodes epidermis, filopodia form from the lateral faces as well as the cell margins after trypsin digestion of desmosomes and hemidesmosomes. The observations suggest that filopodia are induced in response to cell separation and function to restore cell to cell continuity. Filopodia also form in the normal course of development where cells separate prior to their rearrangement to make new tissues as in epidermal and fat body metamorphosis. Filopodia are probably ubiquitous agents for the sensing and movement of cells relative to one another in tissue morphogenesis.     

Cell adhesion during gastrulation : A new approach

In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.