ABSTRACT
Gastrulation in the sea urchin involves an extensive rearrangement of cells of the archenteron giving rise to secondary mesenchyme at the archenteron tip followed by the foregut, midgut and hindgut. To examine the regulative capacity of this structure, pieces of the archenteron were removed or transplanted at different stages of gastrulation. After removal of any or all parts of the archenteron, the remaining veg 1 and/or veg 2 tissue regulated to replace the missing parts. Endoderm transplanted to ectopic positions also regulated to that new position in the archenteron. This ability to replace or regulate endoderm did not decline until after full elongation of the archenteron was completed. When replacement occurred, the new gut was smaller relative to the remaining embryo but the recognizable morphology of the archenteron was re-established. Long after the archenteron reveals territorial specification through expression of specific markers, the endodermal cells remain capable of being respecified to other gut regions. Thus, for much of gastrulation, the gut is conditionally specified. We propose that this regulative ability requires extensive and continuous short-range communication between cells of the archenteron in order to reorganize the tissues and position the boundaries of this structure even after experimental alterations.
INTRODUCTION
Gastrulation in all organisms involves a highly regulated sequence of movements that places the three germ layers in appropriate spatial contexts. In the sea urchin, formation of the archenteron is a multistep process that is accompanied by the eventual differentiation of many distinct cell types. Initially, precursors of the gut lie as a flat epithelial sheet that buckles and then forms a short tube (Ettensohn, 1985b). This is followed by an extensive series of cell rearrangements as the archenteron elongates (Ettensohn, 1985a; Hardin, 1989; Hardin and Cheng, 1986). Protrusive secondary mesenchyme cells (SMCs) form at the archenteron tip during the midgastrula stage and a subset of these migrate away to become pigment and blastocoelar cells. Other SMCs remain associated with the gut and form the circumesophageal muscle and coelomic sacs. After full elongation of the archenteron, constrictions subdivide the endoderm into foregut, midgut and hindgut, and this regionalization becomes evident not only morphologically but also by patterns of region-specific gene expression.
This study began with an interest in learning more about the specification and commitment of cells of the archenteron in the sea urchin embryo. Experiments have suggested that the micromeres that form at the fourth cleavage are not only sufficient but necessary for the initial specification of endodermal and mesodermal fate. Ectopic placement of micromeres can induce a secondary archenteron and removal of micromeres can prevent the normal formation of gut tissues (Horstadius, 1973; Ransick and Davidson, 1993, 1995) This suggests that specification of the archenteron begins with an early signal that emanates from the vegetal pole.
Archenteron cells themselves arise from the macromere lineage which at the sixth cleavage is subdivided into two layers, veg 1 and veg 2. The veg 2 layer lies immediately above the micromeres and was projected by Hörstadius, and in the lineage study of Cameron, to form the entire archenteron (Cameron et al., 1987; Hörstadius, 1935). The veg 1 layer of cells (the upper layer of macromere progeny) was projected to form ectoderm in the anal plate. We show elsewhere in marking studies that veg 1 contributes to the gut as well as to the anal ectoderm in Lytechinus variegatus (McClay and Logan, unpublished data). The veg 2 layer invaginates early during archenteron formation and cells of the veg 1 layer invaginate later, leaving some remaining veg 1 cells to form vegetal plate ectoderm. In vitro studies have shown that veg 1 and veg 2 cells differ in their ability to make endodermal cell types (Khaner and Wilt, 1990; Livingston and Wilt, 1990), indicating that refinement of vegetal cell fates is progressive and is accompanied by veg1 and veg2 becoming different from each other. Later, once progenitors of vegetal cell types are formed, gastrulation movements are followed by subsequent regionalization and region-specific differentiation of the gut.
When cells in an embryo are rearranged with some unpredictability of cell position as occurs during archenteron morphogenesis, a problem exists for the embryo: how can it correctly specify and commit cells and put them in the proper locations. Fate mapping of the vegetal plate prior to invagination indicates that archenteron cells are arranged in a pattern that roughly matches their final position (Ruffins and Ettensohn, 1995). Cell marking studies have shown that these same cells are rearranged to some extent during invagination and archenteron elongation, so there is no absolute predictability of final location. (Hardin, 1989, 1990) The earlier experimental studies reveal an early conditional specification of endoderm, and expression of cell markers reveals that overt specification can be demonstrated by the beginning of gastrulation (Wessel and McClay, 1985). Nevertheless, the experiments to be described here reveal an extensive and persistent series of cell interactions throughout this active morphogenetic period. These lead us to conclude that, while endoderm progenitor cells are conditionally specified by early gastrulation, cells of the different regions of the archenteron retain an ability to become any other part of the archenteron for an extended period of time that coincides with the active period of cell rearrangement. It may well be that morphogenetic processes involving massive cell rearrangements have co-evolved with mechanisms of information transfer between cells and with mechanisms that delay cell commitment events.
MATERIALS AND METHODS
Animals and embryos
Lytechinus variegatus was obtained from Beaufort, NC, or from Key Biscayne, Florida. Gametes were obtained by injection of 0.5 M KCl. Eggs were fertilized in artificial sea water and embryos were grown at temperatures ranging from 17° to 24°C. Growth at several different temperatures assured a staggered supply of embryos at a single stage throughout the day of an operation. Lower temperatures had no adverse effects on development aside from slowing the overall rate. After a surgical operation, embryos were grown in Millipore-filtered sea water in the wells of 96-well plates, a single embryo per well. Older embryos were fed with Duniella tertiolecta.
Embryo manipulations
Single embryos were transferred by mouth pipet to a Kiehart Chamber (Kiehart, 1982). Glass pipets were used to perform surgery on the embryos. Thin-walled glass capillary tubes were pulled on a Narashige glass pipette puller, then the tips were broken to achieve a hollow blunt tip with an opening of 4–15 μm depending upon the particular operation being performed. The capillary pipet was attached to a micromanipulator with a fine hydraulic control. The back of the pipet was attached via intramedic tubing to a Gilmont glass syringe (0.2 ml). Dow silicon oil entirely filled the tubing and pipet to provide a fine level of suction control. When the pipet tip was introduced into the Kiehart chamber, the tip was frontfilled to about 30 μm with sea water from the chamber, at all times avoiding introduction of air bubbles. This arrangement provided a fine control of suction on the needle. The Kiehart chamber was modified so that double-stick Scotch tape (3M Company) was placed on a poly-l-lysine-coated coverslip. Coverslips were dipped into a solution of poly-l-lysine (Sigma #P 8920), rinsed in dH2O and dried. Then poly-l-lysine-coated coverslip fragments were placed over the Scotch tape so that a shallow chamber provided a narrow space for embryo confinement. The Scotch tape also provided a backstop for the needle to push the embryo against, and it provided a sharp 90° corner that became the cutting edge for surgical separation of different parts of the embryo.
In a typical operation, an embryo was introduced to the area just outside the coverslip-tape-coverslip fragment sandwich. The embryo was swept between the coverslip and the coverslip fragment with the capillary pipet and maneuvered into place for an operation. The embryo was stabbed to penetrate to the appropriate tissue. Tissue was aspirated slowly with the hydraulic syringe and tissues were cut using the corner of the Scotch tape as the edge of the saw. After an operation the embryo was swept out of the chamber and transferred to a microtiter plate well using a mouth pipet. Tissue transfers could also be performed by carefully pulling tissue into the pipette and then transferring it into the appropriate donor position. For ‘intensive care’, some embryos were temporarily held between two small oil droplets in the chamber for a few minutes while a cut surface healed over. Fortunately for these operations, the sea urchin embryo has great resilience to the insults of the operations performed. Most of the embryos dissected recovered fully and could be handled within minutes of an operation.
Markers and labels
For experiments that involved donor and host combinations, donor embryos were labeled by incubation of cultures in rhodamine isothiocyanate (Sigma #R 1755) for 30 minutes to 2 hours (Ettensohn and McClay, 1988). Analysis of embryos with regionally specific monoclonal antibodies were performed by standard immunofluorescent methods on whole-mount preparations (Ettensohn and McClay, 1988). Briefly, embryos were fixed for 15-20 minutes in MeOH (−20°C). Following several washes in sea water at room temperature, the embryos were incubated in monoclonal antibody for 2 hours to overnight. They were washed in sea water containing normal goat serum (1:30) and incubated in Cy3-goat antimouse IgG (Jackson Labs) (1:50), for 30 minutes to 2 hours. Alternatively, the embryos were stained with FITC-labeled goat anti-mouse IgG (Jackson Labs). Embryos were photographed or frames were grabbed electronically for a videorecord of the operations and their consequences.
RESULTS
The vegetal plate is highly regulative
From lineage marking studies, most cells normally fated to become secondary mesenchyme cells are within 15° of the vegetal pole, and progenitors of the foregut, midgut and hindgut are concentrically arranged progressively more distant from the vegetal pole (Ruffins and Ettensohn, 1995) (Fig. 1). However, such a fate map does not reveal the degree to which cells at this time are specified or committed to their respective normal fates. Our first set of experiments, therefore, was designed to ask whether the fates of vegetal plate cells were fixed or whether these cells would regulate under experimental perturbations.
Diagram of vegetal plate dissections. At the top is a view of the vegetal plate showing three concentric rings of cells roughly representing the distribution of the presumptive secondary mesenchyme (in the center), the endodermal component of the veg 2 layer and the veg 1 layer as they are placed on the vegetal plate prior to gastrulation (from Ruffins and Ettensohn, 1995). Three different dissections removed portions of those areas as represented by three partial vegetal plates below. The embryos were then followed individually to determine what was replaced.
Diagram of vegetal plate dissections. At the top is a view of the vegetal plate showing three concentric rings of cells roughly representing the distribution of the presumptive secondary mesenchyme (in the center), the endodermal component of the veg 2 layer and the veg 1 layer as they are placed on the vegetal plate prior to gastrulation (from Ruffins and Ettensohn, 1995). Three different dissections removed portions of those areas as represented by three partial vegetal plates below. The embryos were then followed individually to determine what was replaced.
To investigate these questions, a blunt tipped glass needle was used to dissect portions of the vegetal plate (Fig. 1). The operation was performed in two ways, both with the same outcome. In the first operation, the embryo was approached from the bottom, portions of the vegetal plate were sucked into the blunt pipet and the piece was sheared from the embryo using a corner of the coverslip spacer as a knife. In the second operation, the glass pipet was introduced through the animal pole and the piece of the vegetal plate was removed, using the pipet like a cookie cutter to push a plug of vegetal plate against the coverslip spacer backstop.
When any region of the vegetal plate was removed, the embryos regulated to form an archenteron, complete with secondary mesenchyme at the tip. The embryos appeared normal (Fig. 2), other than having archenterons that were smaller than controls. At least 12 replicates of the same manipulation were performed for each of several manipulations. The operations removed different-sized pieces from the central and more lateral regions of the vegetal plate. For example, when the entire presumptive secondary mesenchyme had been removed, the embryo regulated to replace most secondary mesenchyme derivatives, as seen by the presence of blastocoelar cells, coelomic sacs and contracting circumesophageal muscle tissues (an exception to complete replacement of secondary mesenchyme is documented below). In cases where most or all of the veg 2 descendants were removed, the embryo regulated to replace secondary mesenchyme and a complete gut. When a wedge of presumptive endoderm was removed from one side of the basal plate, the embryo regulated to produce a straight archenteron rather than a bent tube, which might have formed had the cells not adjusted for the missing parts.
A pluteus larva from a vegetal plate dissection. The central portion of the vegetal plate had been removed from this embryo. After the operation, the embryo was cultured for an additional 24 hours and then fixed and stained with Endo 1, a monoclonal antibody that is specific for the midgut and hindgut. Shown is a lateral view of the embryo (upper, Nomarski; lower, fluorescence) which has replaced the dissected tissue and made a normal appearing pluteus larva with the marker staining the appropriate regions of the archenteron. Bar = 10 μm.
A pluteus larva from a vegetal plate dissection. The central portion of the vegetal plate had been removed from this embryo. After the operation, the embryo was cultured for an additional 24 hours and then fixed and stained with Endo 1, a monoclonal antibody that is specific for the midgut and hindgut. Shown is a lateral view of the embryo (upper, Nomarski; lower, fluorescence) which has replaced the dissected tissue and made a normal appearing pluteus larva with the marker staining the appropriate regions of the archenteron. Bar = 10 μm.
To ask if replacement was accompanied by appropriate region-specific gene expression, we stained embryos with two different region-specific antibody markers. Endo 1 is a monoclonal antibody that recognizes the midgut and hindgut (Wessel and McClay, 1985). It is first expressed early in gastrulation as the archenteron invaginates and it sharply delineates the foregut-midgut boundary. Ecto V is a monoclonal antibody that specifically stains the foregut and ectoderm surrounding the oral opening (Coffman and McClay, 1990). It has a sharp, non-overlapping boundary with Endo 1 at the foregutmidgut constriction. The embryos from each of the several types of operations expressed these markers in the correct territories (Fig. 2), indicating that respecification had occurred. Further, some of the experimental embryos were cultured to feeding stages. If fed, these embryos consumed algae and grew like unoperated controls. Thus we conclude that the vegetal cells that remain after vegetal plate removals are highly plastic and able to adjust to tissue deletions.
One exception to complete replacement was found. Of the four cell types that arise from the secondary mesenchyme coelomic sacs, pharyngeal muscle, pigment cells and blastocoelar cells, we found that, in operations where the central third of the vegetal plate was removed, the embryos failed to replace pigment cells (Fig. 3). The larvae that resulted from such dissections were otherwise normal but were albino. Therefore, cells of the vegetal plate can replace any missing part of the archenteron except the pigment cell population which, by this time, is excluded from the regulative capacity of the remaining cells.
Dissection of the central region of the vegetal plate. The central region is the location of the presumptive pigment cells. When this area is removed, the embryos recover and replace some secondary mesenchyme cells (coelomic pouches, muscle and blastocoelar cells). However, these embryos do not replace pigment cells as shown by the pluteus in B. A control embryo showing the same abanal view with pigment cells is shown in A. Bar, 10 μm.
Dissection of the central region of the vegetal plate. The central region is the location of the presumptive pigment cells. When this area is removed, the embryos recover and replace some secondary mesenchyme cells (coelomic pouches, muscle and blastocoelar cells). However, these embryos do not replace pigment cells as shown by the pluteus in B. A control embryo showing the same abanal view with pigment cells is shown in A. Bar, 10 μm.
The archenteron replaces missing parts throughout gastrulation
To ask how long the embryo was capable of replacing missing vegetal tissues, dissections were performed at different stages during gastrulation by cutting out invaginated endoderm at the blastopore (Fig. 4). Archenterons were removed at early, middle and late gastrulation, with the latest period being about three hours after the archenteron had made contact with the roof of the blastocoel. Full replacement occurred in most cases as judged by tripartite morphology (Fig. 5). In addition, new coelomic sacs were seen on either side of the archenteron tip and circumesophageal muscle was replaced, as judged by muscular contractions of the esophagus as well as staining for myosin heavy chain which specifically stains these muscles (Wessel et al., 1990) (data not shown). In cases where replacement was incomplete, foregut was missing along with coelomic sacs and muscle. The failure to replace foregut in these cases may be due simply to reduction of tissue mass below a certain level, or it is possible that we removed all cells competent to make a foregut and secondary mesenchyme in these cases. There was no specific manipulation that we could find, however, that would guarantee failure to replace foregut. It should be noted that pigment cells were present in most of the later gut replacements because the first wave of pigment cells is known to leave the secondary mesenchyme early in gastrulation (Gibson and Burke, 1985), and therefore was not removed by our later dissections.
A sequence to show an archenteron dissection. (A) A pipet is filled with ASW to within 30 μm of the tip, and placed near the embryo. (B) The archenteron has been partly sucked into the pipet. (C) The entire archenteron has been pulled into the pipet without disturbing the primary mesenchyme cells. (D) Finally, the pipet is manipulated to shear the archenteron from the embryo and the embryo quickly recovers.
A sequence to show an archenteron dissection. (A) A pipet is filled with ASW to within 30 μm of the tip, and placed near the embryo. (B) The archenteron has been partly sucked into the pipet. (C) The entire archenteron has been pulled into the pipet without disturbing the primary mesenchyme cells. (D) Finally, the pipet is manipulated to shear the archenteron from the embryo and the embryo quickly recovers.
Two embryos from which archenterons were removed were given 24 hours to replace their archenterons. (A) A lateral view of an embryo that was stained with Endo 1 marking the midgut (M) and hindgut (H). The foregut is present also (F) and is not stained with the Endo1 marker. (B) A different embryo from an anal view. In this case, the embryo was stained with Ecto V, an antibody that stains the foregut (F), in addition to the oral ectoderm surrounding the mouth. Also shown but not stained is the midgut and hindgut (M and H).
Two embryos from which archenterons were removed were given 24 hours to replace their archenterons. (A) A lateral view of an embryo that was stained with Endo 1 marking the midgut (M) and hindgut (H). The foregut is present also (F) and is not stained with the Endo1 marker. (B) A different embryo from an anal view. In this case, the embryo was stained with Ecto V, an antibody that stains the foregut (F), in addition to the oral ectoderm surrounding the mouth. Also shown but not stained is the midgut and hindgut (M and H).
In addition to inspection of the morphology, each embryo was treated with one of two monoclonal antibodies. Again, Endo 1 and Ecto V were used to determine whether the replaced archenterons also regulated marker expression. In each case, the pattern of Endo 1 staining was confined to the replaced midgut and hindgut after a day of culture, and foreguts regulated to express Ecto V (Fig. 5).
To ask which cells had the capacity to replace archenterons after they had been removed, we eliminated varying amounts of vegetal plate tissue surrounding the base of the archenteron. In 37 embryos of 40 scored, small archenterons were replaced. In three cases where we removed the entire archenteron plus the surrounding vegetal plate without removing primary mesenchyme cells, a gut was not replaced (Fig. 6). These experiments are difficult to interpret because we did not know the limits of the veg 1 lineage at the time of dissection. This is important because part of the veg 1 lineage normally becomes ectoderm, yet when archenterons are removed it is likely that those veg 1 ectodermal cells retain the capacity to become endoderm (Logan and McClay, unpublished data). In the three cases without a gut, the skeleton was normal and the remaining embryo had a fairly normal morphology, although the ectoderm appeared quite taut over the skeleton, as would be expected if the veg 1 ectodermal contribution were gone. We may have removed all the cells that are capable of converting to endoderm in these three cases but alternative explanations are also possible. For example, the surgical insult or the removal of too much mass from the embryo might have prevented replacement. Nevertheless, since veg1 contributes to both archenteron and to vegetal plate ectoderm, it is possible that we eliminated all veg 1 cells only in these three cases. Thus, we can conclude at this point only that competence to replace archenterons may be restricted to cells of the veg 1 and veg 2 lineages during gastrulation.
An embryo with no archenteron. The embryo shown in brightfield (upper) and by fluorescence (lower) was dissected at about the stage shown in Fig. 4. In addition to the archenteron, additional vegetal plate around the circumblastoporal area was removed without disturbing the primary mesenchyme cells. The embryo made a normal skeleton indicating that both the ectoderm, which provides positional information, and the PMCs, which make the skeleton, were unperturbed. However, there is no archenteron. The embryo was stained with Endo 1 and the photograph highly overexposed to bring up a pale background that indicates the shape of the embryo.
An embryo with no archenteron. The embryo shown in brightfield (upper) and by fluorescence (lower) was dissected at about the stage shown in Fig. 4. In addition to the archenteron, additional vegetal plate around the circumblastoporal area was removed without disturbing the primary mesenchyme cells. The embryo made a normal skeleton indicating that both the ectoderm, which provides positional information, and the PMCs, which make the skeleton, were unperturbed. However, there is no archenteron. The embryo was stained with Endo 1 and the photograph highly overexposed to bring up a pale background that indicates the shape of the embryo.
Sequence of archenteron replacement
Having established that the embryo retains a capacity to replace the archenteron, we asked how the new gut formed. Embryos were dissected (n=7) (Fig. 7) and examined at intervals during the replacement process. The sequence in Fig. 7 shows one embryo as it replaced its archenteron. Within 1 hour of dissecting a midgastrula embryo, a well-defined invagination was present at the vegetal plate. However, the new archenteron appeared different from normal gut tissues because there were very few extensions of filopodia from presumptive new secondary mesenchyme cells in the experimental embryos. Normally, secondary mesenchyme cells are highly dynamic and extend long filopodia that make contact with the wall of the blastocoel. As can be seen in Fig. 7C-E, the archenteron tip appears smooth and devoid of protrusive activity. The archenterons themselves elongated more slowly and, in addition, exhibited a broad-based dome shape for a longer period than is seen in normal archenteron elongation (see Fig. 7C).
Sequence of archenteron replacement. (A) The embryo had its archenteron removed as shown immediately after the operation in B. That embryo was photographed at 1 hour (C), 4 hours (D), 8 hours (E), 16 hours (F), and 20 hours (G) after the operation. The three embryos (H-J) are of the same embryonic age as the experimental embryos shown immediately above each panel.
Sequence of archenteron replacement. (A) The embryo had its archenteron removed as shown immediately after the operation in B. That embryo was photographed at 1 hour (C), 4 hours (D), 8 hours (E), 16 hours (F), and 20 hours (G) after the operation. The three embryos (H-J) are of the same embryonic age as the experimental embryos shown immediately above each panel.
In normal embryos, there is a highly dynamic set of events that occur after contact with the animal pole (Gustafson and Wolpert, 1963; Hardin and McClay, 1990) In the replacement animals, correct association with the stomodaem occurred to form a mouth and foregut. Despite an alteration in the timing of contact between the new archenteron and the target at the animal pole, the archenteron morphology regulated to appear normal. Thus, the process of replacement occurred without fully reiterating the normal developmental sequence.
Specified tissues regulate to replace missing archenteron parts
Endo 1 expression delineates the foregut-midgut border by the mid to late gastrula stage indicating that the endoderm is specified prior to that time. To investigate if the specified tissue would replace portions of the archenteron, we removed the prospective foregut/secondary mesenchyme (Endo 1 negative) to determine if the prospective midgut or hindgut (Endo 1 positive) would convert and replace the foregut. The foregut region was removed from 12 cases at the midgastrula stage. We stained six of the embryos 18 hours after the foregut removal. Those embryos had used presumptive midgut (and perhaps some hindgut) to replace the foregut, and still expressed Endo1 in the newly reorganized foregut (Fig. 8). After an additional 12–24 hours of culture, staining of the remaining cases with monoclonal markers revealed that the foregut now no longer had any remnants of the Endo 1 marker and expressed the Ecto V marker. Thus, the midgut had already begun to express Endo 1 but remained competent to replace the foregut, turn off Endo 1 expression and turn on Ecto V expression. The Endo 1 staining that we saw 18 hours after the operation in the foregut was antigen that presumably had yet to completely turn over.
A lateral view of an embryo from which the prospective foregut and secondary mesenchyme had been removed, cultured 18 hours, fixed and stained. As shown, the foregut (F) was replaced and is positive with Endo 1. In this case, the presumptive midgut has replaced the foregut and the Endo 1 marker has yet to be lost from the new foregut cells.
A lateral view of an embryo from which the prospective foregut and secondary mesenchyme had been removed, cultured 18 hours, fixed and stained. As shown, the foregut (F) was replaced and is positive with Endo 1. In this case, the presumptive midgut has replaced the foregut and the Endo 1 marker has yet to be lost from the new foregut cells.
A second tissue that appears to be specified during gastrulation is the secondary mesenchyme cells. These cells differ in their behavior from the rest of the archenteron by exhibiting a highly protrusive phenotype beginning at the early to midgastrula stage. Though specified, SMCs are not committed to their fates. Several years ago, we established that if skeletogenic primary mesenchyme cells (PMCs) were removed from the embryo, SMCs could convert to replace them (Ettensohn and McClay, 1988). This led us to ask whether experimentally replaced SMCs could then replace PMCs. To perform this experiment, we first eliminated PMCs from embryos (Fig. 9). Then, at the midgastrula stage, we removed the archenteron, which eliminates presumptive SMCs. In all 15 cases, the SMCs were replaced along with the missing PMCs. A completely normal skeleton was constructed in 13 of the 15 cases scored (Fig. 9). These results show that the newly formed gut replaces missing SMCs, and we assume these are the cells that then convert to become PMCs.
PMC and archenteron replacement. (A) A mesenchyme blastula embryo prior to a two step operation. (B) First, the PMCs were eliminated from the embryo. (C) Then the embryo was cultured until the midgastrula stage. (D) At that point, the archenteron was removed. This embryo was then cultured in ASW for an additional 36 hours, fixed and stained for Endo 1. (E) The embryo replaced its archenteron and the PMCs. The PMCs made a normal skeleton.
PMC and archenteron replacement. (A) A mesenchyme blastula embryo prior to a two step operation. (B) First, the PMCs were eliminated from the embryo. (C) Then the embryo was cultured until the midgastrula stage. (D) At that point, the archenteron was removed. This embryo was then cultured in ASW for an additional 36 hours, fixed and stained for Endo 1. (E) The embryo replaced its archenteron and the PMCs. The PMCs made a normal skeleton.
Competence for replacement persists throughout gastrulation
To ask how long after specification portions of the archenteron retain the capacity to assume new fates, we set out to incubate donor pieces of archenteron in the blastocoel of hosts to see if the isolated archenteron pieces would differentiate autonomously. Unexpectedly, the donor pieces attached to the host gut, sorted out and became part of the host gut. This phenomenon was exploited to probe the duration of competence by labeling donor embryos with rhodamine isothiocyanate (RITC) at early gastrulation, then transplanting a donor piece into the blastocoel of unlabeled hosts. The donor piece was directed to a specific site of insertion by pushing the donor tissue against the side of the host archenteron at the desired site. After one or two minutes, the tissue stuck in that position, the pipet was withdrawn and the embryo removed from the Kiehart chamber. After allowing the host embryo to develop to the pluteus stage, we fixed the embryos and stained with the midgut-hindgut marker. The RITC allowed us to identify the donor tissue after fusion with the host gut and the green fluorescence of the marker protein allowed us to assess for regulation of correct gene expression.
In the test shown in Fig. 10, donor tissues were taken from the presumptive foregut/midgut and added to hosts in the presumptive midgut region. At the time of the operation, the host embryos were selected to be at a constant age (the host archenteron received donor tissue about an hour before contact with the roof of the blastocoel). The age of the donor foregut was the variable. In most cases (32 out of 40 scored), donor pieces merged with the host tissue, but we found an age-related response. Younger presumptive foregut/midgut donors (from archenterons that had not yet reached the blastocoel roof) fully regulated to their host position (Fig. 10). Older donor pieces (beginning an hour or two after archenteron contact with the animal pole) were increasingly less able to regulate (Fig. 10). In some cases the older donor tissue expressed the midguthindgut marker but morphologically were abnormal in that they formed a pouch along side the host tissue (Fig. 10). Even later, donor tissues began to express markers autonomously regardless of the host site of insertion. This experiment shows that the prospective foregut/midgut tissues eventually lose full competence to regulate to form other parts of the archenteron, and this loss occurs beginning around the time the archenteron reaches the roof of the blastocoel.
Archenteron transplant experiment. Donor embryos were labeled with rhodamine isothiocyanate. At midgastrula (A) and late gastrula (B), pieces of archenteron from the distal half were transplanted into the presumptive foregut-midgut region of unlabeled host gastrulae at a stage just prior to contact of the archenteron with the roof of the blastocoel. A day later, the embryos were fixed and stained with Endo 1. (A) A lateral view of an embryo in which the donor archenteron contributed to SMCs, foregut and midgut in the host embryo, in each case regulating correctly to the position occupied by the cells. (B) A later transplant (in an abanal view) of the same kind, incompletely regulates. The donor tissue regulates to express the Endo 1 marker correctly (yellow, which indicates the red donor cells are expressing the stained lineage marker) but it forms an abnormal pouch off to the side of the midgut.
Archenteron transplant experiment. Donor embryos were labeled with rhodamine isothiocyanate. At midgastrula (A) and late gastrula (B), pieces of archenteron from the distal half were transplanted into the presumptive foregut-midgut region of unlabeled host gastrulae at a stage just prior to contact of the archenteron with the roof of the blastocoel. A day later, the embryos were fixed and stained with Endo 1. (A) A lateral view of an embryo in which the donor archenteron contributed to SMCs, foregut and midgut in the host embryo, in each case regulating correctly to the position occupied by the cells. (B) A later transplant (in an abanal view) of the same kind, incompletely regulates. The donor tissue regulates to express the Endo 1 marker correctly (yellow, which indicates the red donor cells are expressing the stained lineage marker) but it forms an abnormal pouch off to the side of the midgut.
Archenteron replacement does not show directionality
In the experiments described so far, all of the manipulations removed the archenteron back to a certain level by removing from the tip. Replacement then occurred with remaining vegetal tissues replacing more distal archenteron parts. To learn if replacement were only unidirectional, we challenged the embryo by removing just the presumptive midgut region (Fig. 11). This operation was performed by insertion of a needle through the side of the embryo and carefully sucking up the desired tissue. The prospective midgut then was removed by pushing the pipet through the embryo to the tape backstop to cut it from the remaining archenteron. The pipet was partially withdrawn and, on the way out of the embryo, used to push the presumptive foregut into contact with the remaining stump of tissue at the bottom of the archenteron. After about a minute, the two tissues stuck together, the pipet was withdrawn and the embryo cultured. The missing piece was replaced and a normal-appearing three part gut was produced (Fig. 11). A similar series of operations removed just the presumptive hindgut. It too was replaced. Thus the embryo has an ability to recognize the absence of single parts of the archenteron and can replace that missing part. From its appearance, we propose that the midgut was replaced by contributions both from the distal end and from the basal end of the archenteron because the correct relative proportions of the three parts of the gut were re-established in the plutei that developed. Had it been otherwise, we would have seen a foregut or hindgut that was out of proportion with the other two parts.
Presumptive midgut removal. (A) A pipet was introduced into the side of an embryo at the late gastrula stage (the anus is toward the top in this view). (B) The embryo after the presumptive midgut has been removed and before the injury has been repaired. (C) Even after this insult, the embryo replaces its midgut as shown by staining of the embryo 24 hours later with Endo 1. The hindgut also stained. A phase-contrast image of the embryo in C is shown in D. The only abnormality in this embryo was a slight mispatterning of the skeleton, probably as a result of the insult of the operation to the ectoderm, which supplies positional information for skeletal pattern.
Presumptive midgut removal. (A) A pipet was introduced into the side of an embryo at the late gastrula stage (the anus is toward the top in this view). (B) The embryo after the presumptive midgut has been removed and before the injury has been repaired. (C) Even after this insult, the embryo replaces its midgut as shown by staining of the embryo 24 hours later with Endo 1. The hindgut also stained. A phase-contrast image of the embryo in C is shown in D. The only abnormality in this embryo was a slight mispatterning of the skeleton, probably as a result of the insult of the operation to the ectoderm, which supplies positional information for skeletal pattern.
In a second experiment, the presumptive midgut was taken from one embryo and donated to a second, and then the two embryos were cultured together. This meant that one embryo lacked most of the midgut region and the other embryo was enriched with twice as much presumptive midgut. In 20 cases of tissue transfer, both the depleted donor and the host plus its donor gut regulated to attain a normal morphology (Fig. 12) with restoration of the relative proportionality of the three archenteron subdivisions. In becoming smaller or larger, the gut appears to reallocate tissue appropriately to the three parts.
Regulation of cellular allocation to the archenteron compartments. At the late gastrula stage, a presumptive midgut was dissected from the embryo on the left and inserted into the embryo on the right. The left embryo therefore was heavily depleted in distal archenteron cells while the right embryo received extra archenteron cells. Both embryos were cultured an additional 24 hours, then fixed and stained with Endo 1 (upper, fluorescence; lower, bright field). In both the small and large archenterons that were replaced, the relative proportions of the foregut, midgut, and hindgut were retained.
Regulation of cellular allocation to the archenteron compartments. At the late gastrula stage, a presumptive midgut was dissected from the embryo on the left and inserted into the embryo on the right. The left embryo therefore was heavily depleted in distal archenteron cells while the right embryo received extra archenteron cells. Both embryos were cultured an additional 24 hours, then fixed and stained with Endo 1 (upper, fluorescence; lower, bright field). In both the small and large archenterons that were replaced, the relative proportions of the foregut, midgut, and hindgut were retained.
We then asked whether deleted tissue could be replaced by tissue from a very different region of the archenteron. The tip of the archenteron was removed from a host embryo. Then, a rhodamine-labeled donor piece was dissected from the presumptive hindgut region. That donor piece was inserted ectopically where the archenteron tip had been removed. Therefore the prospective midgut initially was surrounded on both sides with presumptive hindgut (Fig. 13). As shown, the donated hindgut tissue not only regulated correctly fill in the morphology of foregut, but it also prevented the prospective midgut from participating in the replacement of more distal parts (which it would have done if no tissue had been added there). The transferred prospective hindgut received the correct information to cause it to change fate and become foregut, proportionality of the three parts of the archenteron was restored and the original hindgut retained its hindgut status. Taken together, these experiments suggest a remarkable level of cell-cell communication in order to correctly regulate the organization of the archenteron in these embryos.
Presumptive hindgut transplanted into the foregut region. Portions of the tip of the archenteron were removed from the host embryo. Then prospective hindgut was isolated from a rhodamine labeled donor embryo from a position at the edge of the archenteron on the vegetal plate prior to its entry into the archenteron tube. The presumptive hindgut was inserted into the archenteron tip position in the host embryo. The embryo was then grown an additional 24 hours, fixed and stained with Endo 1. By fluorescence (lower) and brightfield (upper), the archenteron is normal. The foregut is partially red (arrow) indicating that the hindgut transplant has regulated structurally and it no longer expresses the Endo 1 hindgut marker for which it was originally specified to produce. Thus it has regulated to its distal position.
Presumptive hindgut transplanted into the foregut region. Portions of the tip of the archenteron were removed from the host embryo. Then prospective hindgut was isolated from a rhodamine labeled donor embryo from a position at the edge of the archenteron on the vegetal plate prior to its entry into the archenteron tube. The presumptive hindgut was inserted into the archenteron tip position in the host embryo. The embryo was then grown an additional 24 hours, fixed and stained with Endo 1. By fluorescence (lower) and brightfield (upper), the archenteron is normal. The foregut is partially red (arrow) indicating that the hindgut transplant has regulated structurally and it no longer expresses the Endo 1 hindgut marker for which it was originally specified to produce. Thus it has regulated to its distal position.
DISCUSSION
The sea urchin embryo is well known as a highly regulative organism. A variety of experiments show that, at least until the 64-cell stage, there is great plasticity in the ability of cells to assume identities other than their normal fate (Driesch, 1900; Henry et al., 1989; Hörstadius, 1939; Khaner and Wilt, 1990; Livingston and Wilt, 1990; Ransick and Davidson, 1993). Only the micromeres, precursors of the primary mesenchyme cells, appear to be fixed during these early cleavage stages. More recent evidence has extended the period of known plasticity. Secondary mesenchyme cells were found to have the capacity to replace primary mesenchyme cells if the PMCs were removed (Ettensohn, 1992; Ettensohn and Malinda, 1993; Ettensohn and McClay, 1988; McClay et al., 1992), showing that the SMC lineage is conditional until late in gastrulation. In the present experiments, we have observed a remarkable ability of circumblastoporal and archenteron tissues to regulate to microsurgical removals and transplantations. This means that much, if not all, of the veg 1 and veg 2 lineage retains developmental plasticity throughout gastrulation, even in cases where the cells have begun to express markers that are specific to one of the sub-regions of the archenteron. The secondary mesenchyme lineage is included in that plasticity since it is replaced as well. These broad capabilities extend even further with the demonstration that the endoderm, which converts to become SMCs, can also replace PMCs, just as original SMCs can.
Clearly, the veg 1 and veg 2 lineages are conditionally specified throughout gastrulation. By marker expression, the archenteron appears to be already specified by early invagination (Nocente-McGrath et al., 1989; Ransick and Davidson, 1993; Wessel and McClay, 1985), but respecification of archenteron cells can occur until late in gastrulation when donor cells in our experiments appear to lose that capacity. Prior to the beginning of invagination, however, the status of the vegetal plate is less clear. It is possible that the vegetal plate is prespecified in concentric rings of SMCs, foregut, midgut and hindgut surrounding the vegetal pole as indicated by the fate map (Ruffins and Ettensohn, 1995) but, without markers at these early stages, one cannot distinguish regional prespecification from a state in which the entire vegetal plate is equivalent. Either way, with the exception of the presumptive pigment cells, the cells remain highly conditional and subject to alteration by cell-cell interactions.
The extraordinary conditionality of archenteron specification was quite surprising to us at first, but its presence might have been anticipated as an adaptation to accommodate the extensive cell rearrangements that characterize gastrulation. The normal sequence of cell rearrangements during invagination is well documented (Ettensohn, 1985a; Gustafson and Wolpert, 1967; Hardin and McClay, 1990; Hardin and Cheng, 1986). Blastomere labeling studies have shown that a small contiguous patch of labeled macromere descendants in the vegetal plate becomes rearranged by convergence-extension movements to form a thin linear strand of cells along the length of the archenteron (Hardin, 1989). If individual cells were rigidly committed prior to invagination, a highly stereotypic sorting out process would be required in order to place each committed cell at its correct location. If such a predestination hypothesis were correct, then it is unlikely that our experiments would have demonstrated such versatility in an ability to replace the gut after a variety of insults. Instead, what appears to occur is an ongoing series of cell communications that help cells refine and localize their specified condition to a precise position in the embryo.
Inferences on the cell-cell signaling process also come from aggregation studies which reveal that gut formation can occur outside of the context of the embryo. For example, when labeled macromere progeny were included in cell aggregates, the labeled cells sorted out from ectoderm, cavitated to form a central lumen, often formed a three part endodermal gut and expressed endodermal markers (Bernacki and McClay, 1989). In addition, our experiments here reveal that, even in the embryo, there is a broad capacity for reorganization that does not require full reiteration of the normal sequence of morphogenetic movements. After removal of the archenteron at almost any time during gastrulation, the cells in the center of what remains of the vegetal plate begin to invaginate again. This means that any group of veg 1 or veg 2 cells can launch the invagination. The elongation and narrowing of the replaced archenteron appears to occur more slowly than normal but, again, apparently any veg 1 or remaining veg 2 cells are capable of participating in this process. Thus, cells of the presumptive archenteron appear to have a remarkable ability to reorganize themselves after a number of manipulations, and the reorganization may not be dependent upon a highly stereotypic sequence of cell behaviors. With such flexibility in morphogenetic rearrangements, it is likely that a continuing cell signaling mechanism plays a major role to assure that the ‘correct’ cell arrangement is attained.
The variety of tissue replacements described in this paper require cells of the veg 1 and veg 2 lineage to respond to some rather complex experimental challenges. When presumptive hindgut was placed in a site originally occupied by foregut and secondary mesenchyme, it left the prospective midgut surrounded on both sides by hindgut. Even so, the transplanted tissue was respecified as foregut. For this kind of regulation to occur, the transplanted cells had to receive information about their new position and then differentiate into foregut. In addition, the archenteron as a whole had to reallocate cell fates to preserve the tripartite morphology of the gut. When presumptive midgut was eliminated, cells on either side had to recognize that the center of the archenteron was missing and had to become respecified in order to replace the missing piece. In turn, when the presumptive midgut was replaced, it must have suppressed the prospective foregut and hindgut from further conversion to a midgut fate. As these and other experiments accumulate, it becomes clear that a number of signals are involved in the organization of the archenteron subdivisions. Further, the duration of the replacement capacity infers that these signals continue throughout gastrulation.
If central vegetal plates are removed, veg 1 and veg 2 cells remain competent to replace other SMC subtypes, but they no longer are able to replace the pigment cell SMC sublineage. Veg 1 and veg 2 also have the capacity to convert and replace PMCs if the PMCs and early archenteron are removed. Oddly, although neither veg 1 nor veg 2 cells can replace pigment cells, the pigments cells themselves can convert to replace PMCs (Ettensohn and Ruffins, 1993). PMCs normally repress SMCs from becoming skeletogenic (Ettensohn and McClay, 1988) and, since SMCs normally are the only cells that convert, they may be the only cells with the appropriate signaling apparatus to respond to missing PMCs. Thus, as the veg 1 and veg 2 lineages become subdivided into the divisions of the archenteron, competence is restricted, but in rather different ways for the several sublineages.
Conceptually, these gut replacement studies support the notion that cell interactions continue to provide spatial information throughout gastrulation. A model for how such information might be distributed is seen in the allocation of information through the notch or wingless pathways. These are cell-cell signalling pathways that are known to relay information in the plane of an epithelium to nearby cells. How such information might be relayed to fix position in space more globally is less clear. Nevertheless, it is apparent that cells can detect their position in the embryo and adjust their direction of differentiation in response to signaling cues. One of the great challenges in studies on regulative processes is to learn, not only at the cellular level but also at the molecular level, how cells read and measure their position in space.
ACKNOWLEDGEMENTS
The authors would like to thank Gary Wessel for use of his myosin heavy chain antibody and to the students of the Woods Hole Embryology Course whose questions led to these experiments. Support was provided by NIH HD 14483