DEVELOPMENTALBIOLOGY147, 157-173 (1991)

The Bases for and Timing of Regional Specification during Larval Development in Phoronis GARY FREEMAN Friday Harbor Laboratories, University of Washington and Center for Developmental Biology, Department of Zoology, University of Texas at Austin, Texas 78712 Accepted May 13, 1991

A fate map has been constructed for Phoremis vancouverensis. The animal pole of the egg gives rise to the apical plate in the hood of the actinotroch larva. The vegetal pole of the egg marks the site of gastrulation. During the initiation of gastrulation the cells of the animal pole of the embryo are directly opposite those at the vegetal pole of the embryo. The plane of the first cleavage always goes through the animal-vegetal pole of the egg. In about 70% of the cases the plane of the first cleavage is perpendicular to the future anterior-posterior axis of the actinotroch larva; in the remaining cases the plane of the first cleavage is either oblique with reference to, or occurs along, the future anterior-posterior axis of the larva. Following gastrulation catecholamine-containing cells first make their appearance in the apical plate and gut cells first produce esterase. The timing of regional specification in these embryos has been examined by isolating animal or vegetal, anterior or posterior, or lateral regions at different time periods between the initiation of cleavage and gastrulation and examining their ability to differentiate. Animal halves isolated from early cleavage through late blastula stages do not gastrulate and do not form catecholamine-containing cells. When animal halves are isolated with endoderm during gastrulation, they differentiate catecholamine-containing cells. Vegetal halves isolated at the 8- to 16-cell stage gastrulate and form normal actinotroch larvae with esterase-positive gut and catecholamine-containing apical plate cells. When this same region is isolated at blastula stages it does not gastrulate and does not differentiate these cell types. Vegetal halves isolated during gastrulation subsequently form esterase-positive gut cells, but they do not form catecholamine-containing apical plate cells. When presumptive anterior, posterior, or lateral halves are isolated from early cleavage through blastula stages, each half forms a normal actinotroch larva. Lateral halves isolated during gastrulation also form normal larvae. Anterior halves isolated during late gastrulation differentiate only the anterior end of the actinotroch larva. These isolates have a hood with catecholamine-containing apical plate cells and the first part of an esterase-positive gut but lack the anlagen of the intestine and protonephridia. Posterior halves isolated during late gastrulation differentiate only the posterior end of the actinotroch which lacks a hood with catecholamine-containing cells but has an esterase-positive gut, protonephridia, and the anlagen of the intestine. These results suggest that there are regional differences built into the animal and vegetal poles of the cleavage stage phoronid embryo. Interactions between the animal and vegetal zones are necessary for gastrulation and subsequent regional specification. The effect of these interactions is seen at the time gastrulation is initiated. The axis along which anteriorposterior specification occurs appears to be set up during early cleavage. The factors leading to the decision about which end of the axis will differentiate as anterior versus posterior appear to be quite labile. This decision appears to be finalized about the time of gastrulation; the anterior region of the axis appears to be specified before the posterior region is. © 1991 Academic Press, Inc. INTRODUCTION The Phoronida are a small phylum of animals that are f r e q u e n t l y i n c l u d e d in a g r o u p of p h y l a k n o w n a s t h e Lophophorota. The other two phyla that are traditiona l l y i n c l u d e d in t h i s g r o u p a r e t h e B r a c h i o p o d a a n d t h e Bryozoa. Many students of animal phylogeny regard the lophophorates as an independent offshoot from an anc e s t r a l a c o e l o m a t e s t o c k t h a t b e l o n g to n e i t h e r t h e p r o tostome nor the deuterostome super phyla (Willmer, 1990). There are several good descriptions of embryogenesis in p h o r o n i d s f r o m p o l a r b o d y f o r m a t i o n to t h e d e v e l o p m e n t o f a f e e d i n g a c t i n o t r o c h l a r v a ( E m i g , 1977). A t t h i s point an adequate fate map does not exist for the embryos of these animals. The animal-vegetal axis of an egg and the embryo derived from it has its animal pole

d e f i n e d a s t h e s i t e w h e r e t h e p o l a r b o d i e s a r e g i v e n off w h i l e t h e v e g e t a l p o l e is d i r e c t l y o p p o s i t e t h e a n i m a l pole. I n p h o r o n i d s b o t h p o l a r b o d i e s a r e g i v e n off a t t h e s a m e p o i n t on t h e e g g s u r f a c e ( R a t t e n b u r y , 1954; Z i m m e r , 1964). T h e r e a r e s e v e r a l s p e c i e s in w h i c h t h e p l a n e s of t h e i n i t i a l c l e a v a g e s of t h e e g g h a v e b e e n r e l a t e d to the site of polar body formation (Brooks and Cowles, 1905; I k e d a , 1901; R a t t e n b u r y , 1954; Z i m m e r , 1964). I n these species the first cleavage takes place along the a n i m a l - v e g e t a l a x i s o f t h e egg. T h e s e c o n d c l e a v a g e a l s o takes place along the animal-vegetal axis of the egg and is p e r p e n d i c u l a r to t h e f i r s t p l a n e o f c l e a v a g e . T h e t h i r d c l e a v a g e is e q u a t o r i a l w i t h r e f e r e n c e to t h e a n i m a l - v e getal axis. Following cleavage and formation of a blast u l a , g a s t r u l a t i o n is i n i t i a t e d . V i r t u a l l y e v e r y s t u d y o n e m b r y o g e n e s i s in t h e s e a n i m a l s h a s c l a i m e d t h a t g a s 157

0012-1606/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

trulation occurs at the vegetal pole of the embryo and t h a t the apical plate or larval nervous system originates from the animal pole of the embryo (Emig, 1977). There is only one study on P. viridis that provides evidence t h a t bears on this point (Rattenbury, 1954). She states t h a t the forming apical plate in the gastrulating embryo lies directly beneath the polar bodies and indicates that gastrulation is initiated opposite this site. Unfortunately it is not clear how long this association between the developing apical plate and the polar bodies exists. This relationship is not shown in an illustration. Subsequently the apical plate will end up in the preoral hood at the anterior end of the actinotroch larva. The actinotroch larva is bilaterally symmetrical. No attempt has been made to see if there is a relationship between the plane of the first cleavage during embryogenesis and the plane of bilateral symmetry of the actinotroch larva. The only work t h a t has been done to assess the state of regional specification in the phoronid embryo has involved the isolation of blastomeres of early cleavage stages in Phoronis vancouverensis and Phoronopsis hameri and the rearing of these isolates to see whether or not they form normal or partial larvae (Zimmer, 1964). In one set of experiments, blastomeres of two-cell stage embryos were isolated. In some cases both isolates from an embryo developed into actinotroch larvae. In an extension of this experiment blastomeres from an embryo were isolated at the two-cell stage and when each of these isolated blastomeres divided again the two daughter blastomeres were isolated. Some of these quarter embryos formed small actinotroch larvae; however, it is not clear whether or not all four isolates from a single embryo formed actinotroch larvae. In the last experiment in this series another halving was done to give one-eighth isolates. Some of the one-eighth isolates generated in this experiment should have an animal or vegetal origin. These isolates cleaved; however, none of them completed gastrulation. On the basis of these experiments Zimmer concluded that half and quarter embryo could regulate. The lack of development of oneeighth embryos was ascribed to mechanical difficulties associated with the small size of the isolates and not the absence of regulative potential. There are two problems t h a t one is confronted with in interpreting these experiments. (1) Since a fate map has yet to be constructed for phoronid embryos it is not clear whether one would expect any of the blastomere isolates at the two- and fourcell stage to develop into partial embryos. (2) Zimmer does not indicate how many isolates of each type were produced and the percentage of these isolates that developed into actinotroch larvae. This paper will employ the local application of vital dyes to construct a fate map for P. vancouverensis. This fate map will then be used to interpret the results of

VOLUME147, 1991

experiments in which halves containing both the animal and the vegetal poles of the embryo or only the animal or the vegetal half of the embryo were isolated at different time intervals during development from early cleavage stages through gastrulation and assayed for their ability to develop. In this way the timing of regional specification can be assessed and one can get an insight into the mechanisms that lead to regional specification. MATERIALS AND METHODS

The biological material P. vancouverensis were collected at low tide on San Juan island and maintained in aquaria with running seawater. Kozloff (1987) was used for species identification. Eggs were obtained by examining clumps of individuals for specimens with a large number of oocytes in their trunk coelom. The region of an individual containing a large number of oocytes was cut off from the rest of the animal and transferred to a small dish of Millipore-filtered pasteurized seawater (PSW). PSW was prepared by filtering seawater through a 0.45-ttm Millipore filter, followed by heating to 80°C for 15 min. Pressure was applied to the top of the body column to squeeze the oocytes out of the opening created by the cut. In most cases these oocytes are already fertilized. After the eggs of an individual were obtained they were transferred through several changes of PSW to dilute out bacteria and then transferred to PSW with streptomycin (1 mg/10 ml) in sterile fourwell plastic dishes (Nunclon) and raised at ca. 13°C in an incubator. The egg batches of some individuals have a tendency to develop abnormally; therefore, a part of the egg batch of a given individual was always set aside as an untreated control to see how it would develop. If fewer than 75% of the eggs in a batch developed normally any experiments done on the remainder of the eggs in the batch were discarded. Histochemical methods for tissue-specific proteins. During the course of this work histochemical markers were used to assay for apical plate and gut differentiation, two tissues found in actinotroch larvae. The apical plate has a tuft of long cilia associated with it; unfortunately it is frequently difficult to distinguish the apical plate cilia from other cilia in P. vancouverensis. There are also a number of nerve cells that originate from the apical plate; these nerve cells have been described at an ultrastructural level (Hay-Schmidt, 1989; Lacalli, 1990). They contain catecholamines, serotonin-like and FMRFamide-like moieties (Hay-Schmidt, 1990a,b). On the basis of Hay-Schmidt's and Lacalli's work it is possible to tentatively assign a given neurotransmitter to nerve cells with a given ultrastructure and position. In this study the catecholamine-containing nerve cells have been assayed for using the sucrose-potassium

GARYFREEMAN

RegionalSpecification in Phoronis

phosphate-glyoxylic acid method of de la Torres and Surgeon (1976); this is the same procedure that HaySchmidt used for this species. Following the staining and mounting procedure this material was examined using fluorescence microscopy. A survey was done of P. vancouverensis actinotroch larvae for gut-specific enzymes t ha t could be assayed for histochemically. One enzyme activity t hat met this condition was esterase. Prior to the esterase assay the material was fixed in 10% formalin in seawater (4°C) for 1 hr. Esterase activity was then assayed using the indoxyl acetate method (Pearse, 1961). This reaction was allowed to run for 10 min (pH 8) at 22-25°C for actinotroch larvae and for 30-60 min for material at early stages of development. Following the reaction the material was dehydrated through a series of alcohols, cleared in xylene, and mounted in Canada balsam. The coverslips were supported so t ha t the material would not flatten. Fate mapping. Two methods were used for marking eggs and embryos. The first method utilized the vital stains nile blue and neutral red; 1% solutions of these dyes were prepared in distilled water. Specific regions on the surface of eggs or embryos were marked using a Singer micromanipulator to bring the open end of a fine capillary filled with 2% agar containing the dye into contact with the surface to be stained. (Directions for preparing the capillary tubes can be found in Novikoff (1938).) The embryos were kept in wells with 2% agar bottoms during staining. The staining process was monitored with a dissecting microscope using reflected light because these eggs and embryos are opaque. In those cases where the site of polar body formation was stained or the region opposite this site was stained it was necessary to switch back and forth at frequent intervals between reflected light and t r a ns m i t t e d light because polar bodies can be most easily visualized using transmitted light. The second method of marking these embryos involved the injection of fluorescein, lysine-conjugated dextran (Molecular Probes) (Gimlich and Braun, 1986) into one blastomere of the two-cell stage embryo to mark its internal contents. The two-cell stage embryos to be injected were placed in a Kiehardt chamber (Kiehardt, 1982) on a compound microscope. The fluorescein-labeled dextran was made up at 10 mg/100 tL1in 0.2 M KC1. The micropipet used for the injection was made of monofilament glass and fabricated on a mechanical puller. It was back-filled with the injection medium. The micropipet was placed in a Leitz instrument holder on a Leitz micromanipulator. The instrument holder was connected to a compressed air tank. A rise in air pressure, activated by a solenoid valve, was used to force fluid from the micropipet after the blastomere was im-

159

paled. After the injection the embryo was examined briefly using fluorescence microscopy to make sure t h a t just one blastomere had been labeled. These cases were reared in the dark and examined at different times during development and as living or fixed actinotroch larvae using fluorescence microscopy so t hat the position of the labeled cells could be ascertained. If there was any indication t h a t the injection procedure slowed the rate of development or arrested development of any of the descendants of the injected cell the case was discarded. Operative procedures. P. vancouverensis eggs and embryos are surrounded by an envelope which persists through gastrulation. Early P. vancouverensis embryos will not survive outside of their envelope. Many of the embryos t hat were operated on had previously been marked with a vital dye so t hat they could be oriented. These embryos were t ransferred to a dish with a 2% agar bottom. A fine glass microneedle was used to cut through the embryo in its envelope by pressing down on the embryo as it was rolled on the agar surface. In those cases where I was interested in isolating only half of the embryo another microneedle was used to make a small hole in the envelope of one of the halves and this half embryo was forced out of the hole leaving the other half of the embryo in the envelope. In those cases where I was interested in the development of both halves of the same embryo a fine nylon monofilament loop was used to separate both halves by positioning it so th a t it fit into the groove created by the cut. The loop was then tightened to separate the halves. The nylon monofilament was obtained by teasing individual filaments out from POH dental floss (Oral Health Products, Inc.). After the isolates had developed to late gastrula stages the ligature was broken, and in those cases where the isolates had different presumptive fates, the identified halves were placed in different wells. Isolates were carefully examined at frequent intervals after they were created to make sure t h a t the operative procedure did not have a deleterious effect and daily or twice daily after t h a t to monitor their developmental progress. If there was a loss of cells the case was discarded. Histological procedures. Experimental embryos and larvae were fixed in 1% osmium in PSW (4°C) for I hr, washed, dehydrated, and embedded in Epon. Sections were cut at 2 #m and stained with methylene blue and azure II (Richardson et al., 1960). RESULTS

Normal Development of P. vancouverensis and the Onset of the Histochemical Differentiation of the Gut and Apical Plate When P. vancouverensis oocytes are removed from the coelom neither polar body has been given off even

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DEVELOPMENTALBIOLOGY

VOLUME147, 1991 t~

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Time (hrs) FIG. 1. Time course of early development from oocyte procurement (0 hr) through gastrulation at 13°C. The top part of the diagram indicates when different developmental events occur. The bars at the bottom indicate the developmental stages and times when different regions of the embryo were isolated in experiments.

though the egg has been fertilized (Zimmer, 1964). The time of oocyte procurement will be regarded as the initiation of embryogenesis. Figure 1 indicates when developmental events occur during embryogenesis at ca. 13°C in P. vancouverensis. Zimmer (1964) gives a developmental time table for this same species reared at 13-15°C. The timetable presented here is a bit faster than his, especially at later stages. Figure 2 shows some of the developmental stages that we will be concerned with in this study and the actinotroch larva. Following procurement

of the oocytes the first visible events of development are the formation of the polar bodies. The formation of the second polar body takes place over about a 30-min period about 4 hr after the oocytes are obtained (Fig. 2A). During this stage the egg is somewhat flattened along its animal-vegetal axis and a bulge appears where the second polar body will be given off which is larger than the polar body that is actually formed. The first cleavage begins at about 6 hr of development. The plane of the first cleavage passes through the animal-vegetal axis of

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FIG. 2. Diagrammatic views of selected developmental stages from second polar body formation through gastrulation and the 8-day actinotroch larva. (A) Oocyte in the process of producing its second polar body. (B) Two-cell stage. (C) Four-cell stage. (D) Eight-cell stage. Note the polar bodies in A-D. A, B, and D are side views; C is an oblique view. (E, F) Sixteen-cell stage. (E) oblique view of open tier of eight cells. (F) oblique view of closed tier of eight cells. (G) Blastula with opening into its blastocoel (b). (G-L) Lateral views of sections through embryos. G shows the membrane around the embryo. This membrane is present at all stages from A-M. (H) Closed blastula. (I) Blastula with gastral plate (gp). (J) Shallow gastrula. (K) Mid gastrula with a central blastopore (bl). (L, M) Gastrula elongating along future anterior-posterior axis of embryo (a-p, anterior-posterior axis; d-v, dorsal-ventral axis; m, mesenchyme cells; ap, apical plate; r, raphe). (M) External view of gastrula shown in L from its ventral surface. (N) External view of actinotroch larva from side (h, preoral hood; g, gut; i, primordium of intestine; t, tentacle; n, nephric system).

GARY FREEMAN

Regional Specification in Phoronis

the egg as defined by the polar bodies and generates two equal or nearly equal sized daughter blastomeres (Fig. 2B). Immediately after the cytokinesis the two daughter cells are spherical and in minimal contact with each other; however, the area of contact soon increases. During subsequent cleavages which occur about every 2~ hours, the time period when blastomeres are in minimal contact is immediately after cytokinesis. From second cleavage on, cases in which daughter blastomeres do not divide synchronously are relatively common. The plane of the second cleavage is perpendicular to the first and also passes through the animal-vegetal axis of the egg generating four equal or nearly equal blastomeres (Fig. 2C). The plane of the third cleavage is equatorial and equal or slightly unequal so that the daughter blastomeres given off at the animal pole are the same size or slightly smaller than those given off at the vegetal pole (Fig. 2D). After the first cleavage it is difficult to visualize the polar bodies in most living embryos. The fourth cleavage of embryogenesis divides the cells of the embryo into two plates of 8 cells each (Figs. 2E and 2F). There is frequently some intercalation of blastomeres between these two plates. One plate of blastomeres is almost invariably open so that none of the blastomeres contact one another in the center of the plate while the blastomeres of the other plate make contact with each other in the center of the plate. Subsequent cleavages tend to build up layers of cells along the axis perpendicular to the two plates of 8 cells. As these cleavages occur a small blastocoel forms in the embryo. The blastocoel is initially continuous with an opening in the surface of the embryo which was first seen at the 16-cell stage when one plate of 8 cells was open (Fig. 2G). By 40 hr of development the opening has usually closed up (Fig. 2H). The blastula wall is thinner at the site of the former opening. Cilia appear between 24 and 36 hr of development; however there is not enough ciliary movement to cause significant movement of the embryos until after gastrulation. The first indication of gastrulation is the flattening of one region of the surface of the blastula, the gastral plate. This always occurs opposite the thin-walled side of the blastula. This generally forms after about 66 hr of development (Fig. 2I). This plate of cells invaginates, generating a large round blastopore and a shallow archenteron (Fig. 2J). During this process the wall of the archenteron is closely opposed to the cells on the opposite side of the embryo and the blastocoel virtually disappears. During the initial stages of gastrulation the embryo has flattened along the axis defined by the invaginating gastral plate. Now the embryo begins to round up; as this occurs the opening of the btastopore becomes somewhat smaller and the archenteron be-

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comes deeper; at this point the blastopore is still in a central position (Fig. 2K). Shortly after this event begins to occur the embryo also begins to elongate in a plane perpendicular to the initial axis of gastrulation. As the embryo elongates the position of the blastopore shifts along the axis of elongation toward what will become the anterior end of the actinotroch larva. This is accomplished, at least in part, by fusion of the blastopore lips at their posterior extremity. The consequences of the localized fusion of the blastopore can frequently be seen as a raphe which extends posteriorly in living embryos at late stages of gastrulation (Figs. 2L and 2M). During the course of gastrulation the mesodermal precursor cells of the larva form at the junction between the ectoderm and endoderm (Zimmer, 1980). Figure 2N shows an actinotroch larva oriented in the same way as the late gastrula in Fig. 2L. The anterior part of the gastrula has transformed into the preoral hood position at least in part as a consequence of the elongation of the dorsal ectoderm along the future anterior-posterior axis of the larva (Hermann, 1986) (note the position of the apical plate in Figs. 2L and 2N). The blastopore of the gastrula becomes the mouth of the actinotroch larva. The mouth is located ventrally under the preoral hood. The undersurface of the preoral hood and the region of ventral epidermis overhung by the hood form a vestibule outside of and surrounding the mouth. The mouth leads into the gut which is a hollow ciliated sack that extends posteriorly. At the posterior end of the sack there is a plug of cells that is the primordium of the larval intestine, which is connected to the posterior ectoderm. Between 6 and 7 days of development the plug of cells hollows out to form a cavity, the intestine, which is continuous with the gut and the posterior ectoderm, forming the anus of the larva. As the gastrula transforms into a larva a tentacular ridge forms as a thickening of the ectoderm which runs obliquely around the body in such a way t h a t it has a more anterior position on the dorsal surface of the larva than it does on its ventral surface. Because the cells t h a t make up the ridge are more elongated than other epithelial cells, there are more cells per unit surface area and ciliary density is higher along the ridge. Tentacles form as evaginations along the ridge. The first pair of tentacles are formed at either side of the ventral midline; successive pairs are added dorsally. By 9 days of development the larva has two pairs of tentacle buds. There is one other larval structure t h a t will be used as a marker in this study, the protonephridia (Fig. 2N). The protonephridia originate as a ventral ectodermal invagination in the future posterior end of the larva after gastrulation has been completed. Subsequently solenocytes can be seen capping the nephridial ducts. Protonephridia can be identified with assurance only in sectioned mate-

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TABLE 1 THE ONSET OF HISTOSPECIFICMOLECULESIN THE APICALPLATE AND GUT DURINGDEVELOPMENT Time (hr) Histospecific molecule

Cohort number

72

84

96

108

Catecholamines

1 2 3 4

0/6 0/6 0/5 0/6

0/4 0/6 0/4 1/8

2/5 1/5 4/4 8/8

5/5 3/3 5/5 7/7

Esterases

rial (see Hay-Schmidt, 1987, for a description of these structures). The time of first appearance during development of catecholamine-containing apical plate cells and esterase-containing gut cells was examined by taking large cohorts of embryos from single females and assaying them for these markers of cell differentiation at 12-hr intervals from 72 to 108 hours of development. The results of these experiments are presented in Table 1. There were some embryos where catecholamine-containing cells were present at 96 hr. In every case examined there were catecholamine-containing cells present at 108 hr of development. There are some embryos that contained esterase in some endodermal cells at 84 hr of development. All of the embryos assayed at 96 hr had endoderm-specific esterase activity and the activity was seen in more endodermal cells. Previous work of HaySchmidt (1990b) on the development of catecholaminecontaining cells in P. vancouverensis has shown that in early 0-tentacle larvae there are five or six catecholamine-containing cell bodies near each other at the site of the developing apical plate. While the developmental age of the early 0-tentacle larval stage is not given, I estimate that it is somewhere between 108 and 136 hr. This is the earliest developmental stage that he examined. The catecholamine-containing cells seen here look just like those that Hay-Schmidt (1990b) shows in his Fig. 10A. By 5 days of development these cell bodies have started to produce catecholamine-containing cell processes, and by 8 days a network of brightly staining cell processes is present, primarily in the hood (see HaySchmidt 1990b, Figs. 11-16). Figure 3A shows the distribution of esterase in an 8-day actinotroch larva; note that the entire gut is stained but that the intestine is unstained. Figure 3B shows esterase activity in only the endoderm of a 96-hr gastrula.

The Fate Map of the P. vancouverensis Embryo The animal-vegetal axis and the site of gastrulation. To define the relationship between the animal-vegetal axis of the egg and the site of blastopore formation during

VOLUME147, 1991

gastrulation a set of eggs were marked at the time of second polar body formation, at the site of second polar body formation (the animal pole), or directly opposite the site of polar body formation (the vegetal pole) with nile blue; the embryos were reared through gastrulation, and the position of the stain was noted. Sixteen eggs stained at the site of polar body formation and seven eggs stained on the side of the egg opposite the polar bodies had enough stain at 3 days of development so that they could be analyzed. When eggs are stained at the site of polar body formation, the polar bodies do not stain; this means t h a t the polar bodies can frequently be picked out at later stages of development above the blue-stained cells. In each of these 23 eggs the plane of the first cleavage passed along the animal-vegetal axis, the plane of the second cleavage also passed along the animal-vegetal axis perpendicular to the plane of the first cleavage, and the plane of the third cleavage was equatorial with reference to the animal-vegetal axis. At the fourth cleavage the eight cell tiers always formed along the animal-vegetal axis. The open tier of eight cells was always at the animal pole and the closed tier of eight cells was always at the vegetal pole. At the end of the first day of development the opening into the blastocoel was always at the animal pole. In nine of the cases stained at the animal pole, the polar bodies could be seen adjacent to the opening. During the initial stages of gastrulation (Figs. 2J and 2K), in every case the vegetal pole corresponded to the site of blastopore formation and the animal pole was opposite this site. The relationship between the symmetry properties of the late gastrula and the symmetry properties of the actinotroch larva was established by marking selected regions of the late gastrula with the vital dye nile blue

FIG. 3. Histochemical localization of esterase in the embryo and actinotroch larva. (A) Eight-day larva; reaction run for 10 min. The arrow points to the unstained intestine. (B) Ninety-six-hour-old gastrula; note the esterase activity in the anterior region of the developing gut; reaction run for 60 rain. Both photographs are at the same magnification; the bar indicates 50 #m.

GARY FREEMAN

Regional Specification in Phoronis

A

33

9

4

5

10

32

2

1

C

D 4

7

FIG. 4. The design and results of the nile blue vital staining experiments designed to elucidate the relationship between the first cleavage plane and the axis of bilateral symmetry. (A) Diagram showing the operation of marking one blastomere of a two-cell embryo furthest from the plane of the first cleavage. (B) The distribution of the nile blue marks in late gastrulae; the different categories from left to right are anterior, anterior-lateral, lateral, posterior-lateral, and posterior. The mark can either be on the left or the right side of the embryo for the categories anterior-lateral, lateral, or posterior-lateral. The marks were equally distributed among both sides. The number of cases in each category is given immediately below B. (C) Diagram showing the operation of marking the plane of the first cleavage at the equator of the embryo. (D) The distribution of these marks in late gastrulae. The categories t h a t these marks were placed in are the same as in B. The distribution of marks on the right and left sides of these embryos were equal.

and noting where the dye mark ended up in the actinotroch larva. When the anterior end of the late gastrula (Figs. 2L and 2M) was marked (eight cases), the mark always ended up on the underside of the oral hood. When the posterior end of the late gastrula was marked (six cases), this region invariably ended up in the ventral posterior region of the actinotroch where the anlagen of the nephric system and intestine form. When a lateral region of the late gastrula is marked, the stain is always located ventrally just under the tentacle ridge on one side of the actinotroch.

The plane of the first cleavage and the anterior-posterior axis of the actinotroch larva. In the initial set of experiments that was done, embryos were allowed to cleave to form two cells. A nile blue mark was now placed on the surface of one blastomere furthest from the plane of cleavage or in an equatorial region of the embryo along the plane of the first cleavage (Fig. 4). The embryos were raised to the late gastrula stage when

163

they had elongated in their future anterior-posterior axis and the blastopore had an eccentric position (Fig. 2M). The position of the stained region in these embryos was then recorded (Fig. 4). When the mark was placed furthest from the plane of the first cleavage, the mark ended up at one end of the anterior-posterior axis of the gastrula in 70% of the cases, in the lateral region of the gastrula in 7% of the cases, and at an oblique angle with reference to the anterior-posterior axis in 23% of the cases. If the first cleavage always separates the presumptive anterior from the presumptive posterior half of the gastrula, one would expect the anterior region of the gastrula to be marked in one-half of the cases and the posterior region of the gastrula to be labeled in the other half of the cases. The fact that the anterior region of the gastrula is marked 2.8 times more frequently than the posterior region suggests that the process of staining the blastomere may play a role in biasing the anterior-posterior axis of the future larva. When the equatorial region of the first cleavage plane was marked, the mark ended up at one end of the anterior-posterior axis in 11% of the cases, in the lateral region of the gastrula in 70% of the cases, and at an oblique angle with reference to the anterior-posterior axis in 20% of the cases. The fact that staining is lateral in the majority of cases supports the conclusion that the first cleavage normally separates the presumptive anterior from the presumptive posterior half of the gastrula in about 70% of the cases. In other cases the angle of the plane of the first cleavage appears to be random with reference to the presumptive anterior-posterior axis. The fact t h a t marks at the animal or the vegetal pole of the embryo had no effect on development indicates t h a t this polarity is stable. In the second set of experiments, one blastomere of a two-cell embryo was injected with a fluorescein-conjugated dextran. Figure 5A shows a two-cell embryo shortly after injection; this fluorescent marker is inherited by all of the cells generated by the injected blastomere. When regions of the embryo are marked at the two-cell stage with a vital dye such as nile blue, because of the relatively slow development of these embryos and the expansion of the epithelial covering as the gastrula turns into a larva, it is not possible to follow the stain mark after gastrulation because it fades. One advantage of the fluorescein-conjugated dextran is t h a t it can be visualized in whole mount preparations of larvae. When one blastomere of a two-cell embryo is injected with a fluorescent marker, the cellular region t h a t inherits the marker can occupy a number of different positions in the actinotroch larva. I have characterized these regions as anterior, anterior-lateral, lateral, and posterior; they correspond roughly to the stained regions of the later gastrulae shown in Fig. 4. The most

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FIG. 5. The distribution of fluorescein-conjugated dextran in actinotroch larvae after filling one blastomere at the two-cell stage. The photographs in this plate are arranged in pairs; the image on the left shows the embryo in transmitted light and the image on the right shows

GARY FREEMAN

Regional Specification in Phoronis

common labeling pattern observed in the actinotroch larva is shown in Fig. 5C (19/32 cases). The epithelium that makes up the hood of the actinotroch is labeled; the labeled epithelium extends to the tentacle ridges; frequently the anterior surface of the tentacles are labeled while the posterior surface of the tentacles are unlabeled. Along the ventral side of the larva there is a region of unlabeled cells that frequently extends to the mouth. In most of these cases all or part of the gut is labeled from its anterior to its posterior end; however, there were three cases where the gut was unlabeled. Six of 32 cases showed an anterior-lateral staining pattern (Fig. 5D). In these cases the hood epithelium on one side and the tentacles on the same side were labeled, while the hood epithelium was only partially labeled on the other side and the tentacles on that side were unlabeled. The gut was unlabeled in one case; in the other five cases the gut was partially labeled, the label was invariably on the side of the larva with the stained tentacles. Frequently a few labeled mesenchymal cells could be seen in parts of the larva where the gut or external epithelium were not stained. A lateral staining pattern was observed in three cases (Fig. 5E). In these cases a lateral half of the epithelial covering from the hood to below the tentacle ridge or to the posterior end of the larva was labeled. In each of these cases the gut was labeled on the side of the larva with the labeled epithelium. Frequently labeled mesenchyme cells were observed in parts of the larvae where the gut and external epithelium were not labeled. The posterior staining pattern was observed in four cases (Fig. 5F). In every case a narrow band of epithelial cells ran along the dorsal midline from the base of the hood to the tentacle ridge; the epithelium on the dorsal surface posterior to the tentacle ridge to the posterior end of the larva including the anlagen of the intestine, the epithelium t h a t makes up the ventral posterior covering of the larva, and a strip of ventral epithelium going all or part way to the mouth was labeled. There was only one case where the gut was labeled. In every case scattered labeled mesenchymal cells could be seen in the hood and tentacular regions of the larva. Twenty of these larvae were examined for the distribution of fluorescent label when they were late gastru-

165

lae. One of these cases is shown in Fig. 5B, which corresponds to an anterior distribution of label as indicated in Fig. 4; this case developed into the larva shown in Fig. 5C. In every case there was a one to one correspondence between the position of the label in the late gastrula and the position of the label in the larva. Table 2 compares the percentage of larvae that show a given labeling pattern following staining of part of one blastomere furthest from the cleavage furrow at the two-cell stage with nile blue versus the injection of fluorescein-conjugated dextran. The percentage of larvae showing each labeling pattern is comparable. Together these two different kinds of marking experiments suggest t h a t in about 70% of the cases the plane of the first cleavage is perpendicular to the anterior-posterior axis of the larva, while in about 8% of the cases it corresponds to the anterior-posterior axis and in the remaining cases it has an oblique angle with reference to this axis. The experiments involving the injection of fluorescein-conjugated dextran show t h a t this treatment, like the vital staining, biases the treated blastomere to become the anterior end of the larva when the first cleavage plane is perpendicular to the anterior axis. Given the histological descriptions of gastrulation, the fate mapping studies which indicate the vegetal origin of the gut, and the pattern of early cleavage, it is interesting that descendants of only two of the four vegetal blastomeres at the eight-cell stage can form the entire gut and t h a t these blastomeres can be in either the presumptive anterior or posterior half of the embryo. Figure 6 presents a fate map of the external surface of the larva, based on the marking of its animal half and future anterior half at the eight-cell stage under conditions where the plane of the first cleavage is perpendicular to the larva's anterior-posterior axis. Because of the use of fluorescein-labeled dextran the demarcation between the larval contributions of the anterior and posterior blastomeres at the eight-cell stage is well defined. Since nile blue was used to mark the animal and vegetal regions of the embryo the demarcation between the two halves shown here represents a guess. An analysis of sectioned material where smaller regions such as individual identified eight-cell stage blastomeres are labeled with fixable fluorescein-labeled dextran is needed to further refine this fate map.

the embryo in the same orientation viewed with the appropriate fluorescence excitation wave length. (A) Two-cell embryo after the injection of one blastomere with fluorescein-labeled dextran. (B) Late gastrula stage embryo (similar to Fig. 2L); the blastopore is on the left in this figure but cannot be made out with transmitted light. The fluorescein label is in the anterior end of the embryo and the labeled gut can be seen in outline in the posterior end of the embryo. (C) The same embryo shown in B at 8 days of development. In the left pair of panels the larva is oriented with its ventral side facing the viewer. In the right pair of panels the same larva is viewed from its side. The label shows an anterior distribution. (D) Larva showing an anterior-lateral distribution of label. In the left pair of panels the larva is oriented with its ventral side facing the viewer. In the right pair of panels the same larva is viewed from the side. (E) Larva showing a lateral distribution of label. The larva is oriented with its dorsal side facing the viewer. (F) Larva showing a posterior distribution of label. The larva is oriented with its dorsal side facing the viewer. All photographs are at the same magnification. The bar indicates 50 ~m.

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TABLE 2 A COMPARISONOF LABELINGPATTERNS SEEN IN EMBRYOSAND LARVAE FOLLOWING MARKING WITH NILE BLUE OR FLUORESCEIN-CONJUGATED DEXTRAN % Distribution Treatment Nile Blue Fluorescein Dextran

Number cases Anterior

Anteriorlateral Lateral

Posteriorlateral Posterior

61

54

15

7

8

16

32

59

19

9

0

13

Regional Specification in the Embryo at Different Times between First Cleavage and Gastrulation The timing of regional specification in developing embryos of P. vancouverensis has been examined by isolating animal or vegetal, anterior or posterior, or lateral regions of comparable size at different time intervals between the initiation of cleavage and gastrulation and examining their ability to differentiate. The times when these isolations were done are indicated in Fig. 1; these stages include the 2- to 16-cell stage, early and late blastula stages, and gastrula stages. The isolations were done either by destroying one half of the embryo or by separating both halves of an embryo (see Materials and Methods). The way in which these isolates differentiated was compared to what one would expect for t hat region given the fate map of these embryos.

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Animal halves isolated at the cleavage stage and the two blastula stages lived for a maximum of about 6 days and then dissociated into single cells. These cases showed no indication of gastrulation or larval morphogenesis; the only indication of cell differentiation was cilia formation by the cleavage and early blastula isolates; however, longer cilia characteristic of the apical plate were not seen. At 5 days of development these isolates consisted of solid epithelial masses with no indication of internal mesoderm or endodermal cells (Figs. 8A and 8B). All of the cases assayed were catecholamine negative; only 2 of 21 cases were esterase positive, these cases had only a small number of weakly staining cells. The esterase assays were run for 30-60 min which overstains the gut in intact embryos at 5 days of development. Animal halves isolated during gastrulation usually survived until the eighth day. Only one of these cases formed a normal actinotroch larva; most of the cases completed gastrulation and formed a ciliated vesicle with an internal gut and mesodermal cells (Fig. 8D). Some of the cases had long cilia of the type associated with the apical plate. Every case assayed was catecholamine or esterase positive. Vegetal halves isolated at the 8- or 16-cell stage frequently lived for at least 8 days. Some of these cases did not gastrulate; they invariably dissociated by 6 days. The majority of the cases gastrulated; 60% of these ap

Regional specification along the animal-vegetal axis. To produce animal and vegetal isolates, oocytes t hat were giving off their second polar body were marked at either the site of polar body formation or directly opposite this site with a vital dye. This allows one to identify the animal or vegetal half unambiguously at the time the isolate is created. Figure 7A indicates the regions t h a t were isolated at the different developmental stages. Most of the animal and vegetal isolates made at cleavage stages were created during the fourth cleavage. Operations were also done during the third cleavage; during this division there is a greater chance of killing one or more blastomeres, for this reason there are fewer cases for this stage. Since the results of both operations are similar they have been lumped together. The animal and vegetal isolates obtained at the different developmental stages were treated in one of four ways. Some of the isolates for each developmental stage were reared for as long as 8 days to see how they would differentiate. The rest of the isolates were assayed at different time intervals for catecholamines or esterase or fixed, sectioned, and examined for histological indications of differentiation. The results of these experiments are summarized in Table 3.

Eap~ FIG. 6. Reconstruction based on vital staining and fluorescein tagging experiments indicating the presumptive fate of the animal half and the anterior half of an embryo when the first cleavage plane is perpendicular to the future anterior-posterior axis of the embryo. (A) Eight-cell embryo. The animal half is indicated by stippling and the presumptive anterior half is indicated by cross-hatching. Half of the animal region of the embryo is simultaneously half of the anterior region. (B) Cross-sectioned view of early gastrula. (C-E) Eight-day larva in three views. (C) Larva with its hood extended and its dorsal side facing the viewer. (D) The same larva with its hood extended and its ventral side facing the viewer. (E) The same larva with its hood in the normal position, viewed laterally (ap, apical plate; m, mouth).

GARYFREEMAN

Regional Specification in Phoronis

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FIG. 7. Operations done to isolate animal, vegetal, anterior, posterior, and lateral halves at different times during development. In each case the region along which the embryo was bisected is indicated by the dashed line. (A) Animal-vegetal isolations. All of these embryos were marked at the site of polar body formation. Column a shows an external side view of an eight-cell embryo. Column b shows a lateral cross section view of an early blastula. Column c shows a lateral cross section view of a late blastula. Column d shows a lateral cross sectional view of a mid gastrula. (B) Anterior-posterior isolations along the plane of the first cleavage. All of these embryos had a mark placed on one blastomere of a two-cell embryo at a point furthest from the first plane of cleavage. All of the diagrams under columns a-d show external views of embryos. Column a shows a four-cell embryo; 1 marks the first cleavage plane. Column b shows an early blastula. The view looks down into the animal opening of the blastocoel. Column c shows a late blastula. The view looks down on the animal pole of the embryo. In these cases this site was marked with another vital dye at the time of polar body formation. The left panel of column d shows a mid gastrula viewed from the site of invagination. The right panel of column d shows a late gastrula viewed from the ventral surface of the embryo. (C) Lateral isolations along the plane of the second cleavage. All of these embryos had a mark placed on one blastomere of a two-cell embryo at a point furthest from the first plane of cleavage. All of the diagrams in columns a-d show external views of the embryos. Column a shows a four-cell embryo; 2 marks the plane of the second cleavage. In columns b-d the embryos are oriented in the same way that they are in row B.

w e n t on to d e v e l o p i n t o h a l f - s i z e d a c t i n o t r o c h l a r v a e ( F i g . 8C). T h e d e v e l o p m e n t o f t h e s e l a r v a e a f t e r t h e i n i t i a t i o n of g a s t r u l a t i o n w a s d e l a y e d s l i g h t l y w i t h r e f e r ence to i n t a c t c o n t r o l s . A l l of t h e s e l a r v a e t h a t w e r e e x a m i n e d h i s t o l o g i c a l l y w e r e n o r m a l in e v e r y r e s p e c t . Every case that developed into an actinotroch larva that was assayed was catecholamine and esterase positive. Those cases that gastrulated but did not form a hood a p p e a r e d to s t o p d e v e l o p i n g f o l l o w i n g g a s t r u l a t i o n . When they were examined histologically a gut, mesenc h y m a l cells, a n d a n e x t e r n a l c i l i a t e d e p i t h e l i u m w e r e present. Every case that was assayed was esterase positive and catecholamine negative. A sample of cases that d i d n o t g a s t r u l a t e w a s fixed w h e n i t w a s 5 d a y s old. These cases resembled animal halves; histological exami n a t i o n of t h e s e i s o l a t e s i n d i c a t e d t h a t t h e y c o n s i s t e d o f a s o l i d c i l i a t e d c e l l u l a r m a s s w i t h no i n d i c a t i o n of m e s o derm or endoderm. Both cases assayed for catecholamines were negative; however, one of four cases s h o w e d a low level o f e s t e r a s e a c t i v i t y in a f e w cells. When vegetal halves were isolated from early or late blastulae most cases lived through 5 days of developm e n t a n d t h e n d i s s o c i a t e d i n t o s i n g l e cells. O n l y f o u r of these cases gastrulated. Those cases that did not gastrul a t e w h i c h w e r e e x a m i n e d h i s t o l o g i c a l l y c o n s i s t e d of a s o l i d m a s s of c i l i a t e d c e l l s w i t h no i n d i c a t i o n of m e s o -

derm or endoderm. All cases assayed for catecholamines or esterase were negative. While vegetal halves isolated during gastrulation never formed actinotroch larvae, those isolates that were examined histologically had a g u t , m e s o d e r m a l cells, a n d a n e x t e r n a l c i l i a t e d e p i t h e l i u m ( F i g . 8E). E v e r y c a s e t h a t w a s t e s t e d w a s e s t e r a s e positive while those cases that were assayed for catecholamines were negative.

R e g i o n a l specification along the a n t e r i o r - p o s t e r i o r axis. P r e s u m p t i v e a n t e r i o r a n d p o s t e r i o r h a l v e s of e m b r y o s w e r e o b t a i n e d b y c u t t i n g e m b r y o s in h a l f a l o n g t h e p l a n e o f t h e f i r s t c l e a v a g e in a s m u c h a s t h i s p l a n e is p e r p e n d i c u l a r to t h e a n t e r i o r - p o s t e r i o r a x i s o f t h e e m b r y o in r o u g h l y 70% of t h e c a s e s . A s a c o n t r o l f o r t h i s e x p e r i m e n t e m b r y o s w e r e a l s o c u t in h a l f a l o n g t h e i r second plane of cleavage since this cleavage plane corres p o n d s to t h e a n t e r i o r - p o s t e r i o r a x i s in a b o u t 70% of t h e c a s e s . P r i o r to m o s t of t h e s e o p e r a t i o n s t h e e m b r y o s were marked with neutral red at the site of polar body formation and equatorially along part of the first cleava g e p l a n e o r a t t h e e n d of a t w o - c e l l s t a g e b l a s t o m e r e furthest from the cleavage plane, with nile blue. These m a r k s a l l o w e d o n e to o r i e n t t h e e m b r y o a t t h e t i m e i t w a s c u t i n t o t w o h a l v e s . F i g u r e s 7B a n d 7C i n d i c a t e t h e regions isolated at the different developmental stages. The isolates created at the cleavage stages were made

168

DEVELOPMENTAL

BIOLOGY

VOLUME147, 1 9 9 1

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GARYFREEMAN

Regional Specification in Pharonis

169

FIG. 8. Development of animal or vegetal and anterior or posterior halves isolated from embryos at different stages of development. (A) Five-day animal half isolated from a 16-cell embryo incubated for 30 min for esterase. It is esterase negative. (B) Histological section through a 5-day animal half isolated from a 16-cell embryo. (C) Five day vegetal half isolated from a 16-cellembryo incubated for 10 min for esterase. Note the esterase-positive gut, the forming hood and tentacle ridges. (D) Animal half at 5 days of development isolated from a gastrulating embryo. The isolate consists of an epithelial vesicle that surrounds a piece of gut (arrow). After this isolate was photographed it was assayed for catecholamines. It was positive. (E) Histological section through a vegetal half at 5 days of development. Note the gut and mesodermal cells (arrow). (F) Anterior half at 8 days of development isolated from a late gastrula incubated for 10 min for esterase. The isolate has a mouth that opens on a vestibule; it has an esterase-positive gut. There is no indication of a tentacle ridge or intestine. (G) Posterior half at 8 days of development from the same embryo that gave the isolate shown in F. Arrow a indicates the tentacle ridge and a gut is visible which ends in an intestine (arrow b). Photographs A, C, D, F, and G are at the same magnification. The bar indicates 50 t~m.Photographs B and E are at the same magnification. The bar indicates 50 ~m.

f r o m the first t h r o u g h the t h i r d cleavages. The results obtained were independent of the cleavage stage used. The operations done d u r i n g g a s t r u l a t i o n were perf o r m e d either at the stage when the blastopore h a d a central position in the r o u n d g a s t r u l a (when viewed f r o m the vegetal pole) or when the e m b r y o h a d begun to elongate along its f u t u r e a n t e r i o r - p o s t e r i o r axis and the blastopore was closest to the p r e s u m p t i v e a n t e r i o r end. W i t h i n this stage the t i m i n g of the operation h a d an effect on the results. P r e s u m p t i v e anterior, posterior, and lateral halves isolated at early cleavage and early and late blastula stages of embryogenesis developed in essentially the same way. The m a j o r i t y of these cases lived t h r o u g h 8 days of development. A few of these cases d i s i n t e g r a t e d prior to gastrulation; however, a m a j o r i t y of the cases gastrulated. A few of the cases t h a t g a s t r u l a t e d stopped developing a f t e r g a s t r u l a t i o n ; however, the m a j o r i t y developed into n o r m a l half-sized actinotroch larvae (Table 3). The development of these larvae a f t e r the initiation of g a s t r u l a t i o n was delayed slightly with reference to intact controls. E v e r y case t h a t developed into an actinotroch larva t h a t was assayed was c a t e c h o l a m i n e or es-

terase positive. A t each of these developmental stages there were a n u m b e r of e m b r y o s t h a t were cut in half along either their first plane of cleavage or perpendicular to this plane of cleavage w h e r e both halves developed into a c t i n o t r o c h larvae. W h e n a n t e r i o r halves were isolated f r o m late g a s t r u lae, in most cases these isolates differentiated into only the a n t e r i o r end of the a c t i n o t r o c h l a r v a (Fig. 8F). These isolates consisted of the hood region of the actinotroch; in every one of these cases w h e r e catecholamine-cont a i n i n g cells were assayed for t h e y were present. The m o u t h and g u t were also present. The intestine and nephric s y s t e m were i n v a r i a b l y absent. The absence of the intestine was established by an e x a m i n a t i o n of both sectioned m a t e r i a l and isolates stained for esterase (the intestine does not stain for esterase). P o s t e r i o r halves isolated at this stage differentiated into either a n o r m a l a c t i n o t r o c h larva, an a c t i n o t r o c h larva with a small hood, or the posterior end of the a c t i n o t r o c h larva. Those cases t h a t differentiated as posterior halves (Fig. 8G) lacked a hood and c a t e c h o l a m i n e - c o n t a i n i n g cells; these cases had a gut, an intestine, and a nephric system. The posterior isolates t h a t have a small hood are

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DEVELOPMENTAL BIOLOGY

interpreted as halves t h a t have more of the anterior region than the other posterior halves either because of the level of the cut t h a t created them or because they have a greater regulatory potential for differentiating anterior regions due to the developmental stage when they were isolated. There were five operations done on late gastrulae where both halves from the same embryo were successfully reared. In all five cases the presumptive anterior half formed only the anterior region of the actinotroch larva; however, the differentiation of the posterior half was much more variable. In one case the posterior half formed a normal actinotroch larva, in another it formed a posterior half with a small hood, and in three cases it formed only a posterior half. When lateral halves were isolated from late gastrulae these halves invariably developed as normal actinotroch larvae. These cases included eight halves isolated from four embryos. This indicates t h a t isolates made at this stage which include both presumptive anterior and posterior regions as well as animal and vegetal domains can regulate to form normal larvae. When presumptive anterior and posterior halves are isolated from mid gastrulae one also gets halves that differentiate as anterior or posterior regions of the actinotroch larva. Because of the stage when the operation was done one cannot be sure if those cases that differentiated as anterior or posterior regions of the actinotroch larva were derived from the corresponding presumptive anterior and posterior regions of the embryo. Given the fact that this is the case when this operation is done 6 hr later when the embryo has elongated and the blastopore has an eccentric position, it is safe to assume that this is also the case at the mid gastrula stage. This indicates t h a t the anterior and posterior regions of the embryo have already been specified in some cases by this stage. A larger proportion of the presumptive anterior and posterior halves isolated at the mid gastrula stage differentiated into normal but half-sized actinotroch larvae than at the late gastrula stage. There are two possible explanations for this behavior. (1) Since the plane of the first cleavage only separates the presumptive anterior and posterior regions of the embryo in about 70% of the cases, those cases that develop as normal actinotroch larvae may have originated from embryos where the plane of the first cleavage corresponded to the plane of bilateral symmetry. In those cases where mid gastrulae were cut in half perpendicular to the plane of the first cleavage, the majority of the cases developed as normal but half-sized actinotroch larvae as one would expect; however there were 2 of 12 cases that developed as partial actinotroch larvae, they probably originated from embryos cut along their presumptive anterior-posterior axis. (2) The process of specifying the anterior and posterior regions of the embryo may still be taking place at

VOLUME147, 1991

this stage. In those isolates that develop as normal actinotroch larvae this process may not have proceeded far enough at the time of isolation and therefore these halves regulated. There were six operations done on mid gastrulae where both halves from the same embryo were successfully reared. In two of these embryos both halves differentiated as normal actinotroch larvae. In two embryos one half differentiated as the anterior region of an actinotroch larva and the other half differentiated as a normal actinotroch larva. In one embryo one half differentiated as the anterior region of the actinotroch larva and the other half differentiated as a posterior half with a small hood. In the last embryo one half differentiated as the anterior region of the actinotroch larva and the other half differentiated as the posterior half. If this data set and the data set where both halves of late gastrulae differentiated are considered together they suggest that the process of regional specification along the anterior-posterior axis occurs first in the anterior region of the embryo and subsequently in the posterior region. The evidence for this temporal progression is based on the observation t h a t anterior halves of the actinotroch larva can be paired with normal actinotroch larva, but posterior halves are never paired with normal actinotroch larva. In addition to these experiments single blastomeres were isolated from four-cell stage embryos and reared to see how they would differentiate. Thirty-one of these isolates were created; 26 of them lived for 5 or more days of development, all of these cases formed normal quarter-sized actinotroch larvae. The formation of structures like tentacles in these larvae was delayed with reference to normal sized control larvae. This finding confirms Zimmer's (1964) observation that blastomeres isolated at the four-cell stage can form normal actinotrochs. DISCUSSION

Determination along the Animal- Vegetal Axis The fate mapping studies reported here confirm Rattenbury's (1954) observations that gastrulation occurs at the vegetal pole of the embryo and apical plate formation takes place at the animal pole of the embryo. This relationship had been assumed in several papers on the embryology of these animals; however, even after the appearance of Rattenbury's paper the appropriate evidence was never cited. The results of experiments involving the isolation of animal halves at different stages from early cleavage through gastrulation indicate that they only exhibit the capacity to produce catecholamine containing neurons when they are isolated at the mid gastrula stage about 18 hr before catecholamine accumulation is detectable.

GARYFREEMAN RegionalSpecification in Phoronis Catecholamine synthesis in these isolates could reflect either the fact t h a t these animal half cells have endoderm cells from the vegetal half of the embryo associated with them or the fact that these cells had been associated with the vegetal half of the embryo long enough prior to isolation so that they acquired the ability to form catecholamine-containing neurons as a consequence of an inductive interaction. The fact that animal halves isolated from cleavage through late blastula always dissociated after a few days but lived much longer when associated with cells from the vegetal half suggests that this region of the embryo may be dependent on the vegetal half for its survival. The catecholamine-containing nerve cells are presumably derived from the apical plate. Descriptive studies of phoronid embryogenesis indicate t h a t the apical plate begins to develop during blastula stages in at least some species (Brooks and Cowles, 1905; Rattenbury, 1954). It can be identified because of the length of its cilia and the columnar shape of the cells t h a t make up the plate. In P. vanconverensis the apical plate is present by at least the late gastrula stage (84 hr) (Zimmer, 1980) and may be present prior to this stage. If it is present at the late blastula stage (54-60 hr), any inductive influence emanating from the vegetal half of the embryo must be doing more than specifying the plate; it also must be influencing its further differentiation. When vegetal halves are isolated at early cleavage stages they gastrulate and go on to differentiate normal actinotroch larvae. The behavior of these isolates shows that at this stage they have the capacity to regulate the loss of the animal cap cells by forming structures normally formed by these cells. When comparable vegetal halves are isolated from blastula stages, in most cases they do not gastrulate and show no sign of forming esterase. One interpretation of this result is t h a t the vegetal half of the embryo needs to interact with the animal region of the embryo to gastrulate and t h a t at the blastula stage the vegetal cells no longer have the ability to regulate the loss of the animal cap by differentiating cells with the properties of animal cap cells. After vegetal halves are isolated during gastrulation they invariably go on to differentiate gut esterase; however, no further differentiation and morphogenesis typical of this part of the embryo occurs and there is no differentiation of catecholamine-containing cells even though the gastrula is covered by a ciliated epithelium. This behavior suggests t h a t at this stage the vegetal region still lacks the ability to regulate for the loss of animal cap cells and t h a t the animal cap is necessary for a number of subsequent developmental events that occur after the initiation of gastrulation; these events might include elongation of the gut and the formation of the intestine and nephric system. HSrstadius (1936) has documented a

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similar decline in the ability of vegetal halves of echinoid embryos to regulate when isolated at different time periods between cleavage and late blastula. The echinold embryos retain their capacity to regulate through later developmental stages. These results suggest t h a t there are factors specifying the animal and vegetal regions of these embryos t h a t are localized in the appropriate halves of the eightcell embryo t h a t are inherited by the cells generated in each of these regions. These factors may give the vegetal half the ability to gastrulate and the animal half the ability to differentiate neurons; these factors may also give the animal half the ability to send unique developmental signals to the vegetal half and to receive unique developmental signals from the vegetal half, and they may give the vegetal half an analogous set of cell interaction properties. The observation t h a t embryo halves which contain both animal and vegetal domains develop into normal larvae when isolated from early cleavage stages through late blastula fits the argument t h a t the localization of cytoplasmic factors and inductive interactions appear to be responsible for regional specification along the animal-vegetal axis of these embryos. D e t e r m i n a t i o n along the A n t e r i o r - P o s t e r i o r A x i s

The fate mapping studies performed here show t h a t the anterior-posterior axis of the actinotroch larva is essentially perpendicular to the animal-vegetal axis of pregastrula embryos. Since the first cleavage plane passes through the animal-vegetal axis of the embryo one can ask whether there is a correlation between the plane of the first cleavage and the anterior-posterior axis of the embryo. These studies show t h a t the first cleavage plane can occur along any angle with respect to the future anterior-posterior axis of the actinotroch; however, in about 70% of the cases this plane of cleavage is perpendicular to the anterior-posterior axis. This percentage is much higher than one would expect on the basis of chance. An attempt has been made to find out if there is a correlation between the plane of the first cleavage and the axis of bilateral symmetry in a number of different kinds of animals. In the embryos of some animals there is a 1:1 correlation between the plane of first cleavage and the axis of bilateral symmetry (e.g., ascidians, Conklin, 1905), in other embryos there is no correlation between the plane of the first cleavage and the axis of bilateral symmetry (e.g., asteroida, Kominami, 1983), and in yet other embryos while there is not a perfect correlation between the plane of first cleavage and the axis of bilateral symmetry, there is a much higher proportion of cases t h a t exhibit this relationship than can be attributed to chance (e.g., cephalochordates, Tung, et

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al., 1958). In many cases where there is a correlation between the plane of the first cleavage and the axis of bilateral symmetry, this reflects an anisotropy that has been built into the egg or embryo which the plane of cleavage reflects or an anisotropy that is generated as a consequence of the cleavage t h a t biases subsequent steps in the process of regional specification (Freeman, 1979). If the anterior-posterior axis of these embryos was determined by the inheritance of a localized cytoplasmic region which ended up in one blastomere in those cases where the first cleavage was perpendicular to the presumptive anterior-posterior axis one would expect that if one marked one blastomere, that blastomere would form the anterior region of the larva in half the cases and the posterior region of the larva in the other half of the cases. This is the case in the direct developing echinold Heliocidaris where the plane of the first cleavage separates the dorsal and ventral sides of the future adult (Henry and Raft, 1990). One of the peculiarities of the P. vancouverensis embryo is that when this experiment is done the mark ends up in the anterior region of the embryo from 2.8 to 6.3 times as often as it ends up in the posterior region, depending on the marking procedure used. This indicates t h a t a cytoplasmic localization mechanism probably does not play a role in specifying either the anterior or the posterior end of the anteriorposterior axis of the embryo. The fact t h a t there is a correlation between the plane of the first cleavage and the anterior-posterior axis suggests that events which occur during early cleavage stages of embryogenesis may play a role in positioning the meridian the future anterior-posterior axis will go through with reference to the animal-vegetal axis of the embryo. Since the act of marking these embryos can cause anterior regional specification, the initial steps leading to anterior and posterior specification must be very labile. The data on the behavior of presumptive anterior, posterior, and lateral halves after isolation at various developmental stages suggest that anterior versus posterior, along this axis, is not determined until gastrulation. These data also suggest that this process starts in the presumptive anterior region of the embryo. Presumably this process requires cell interactions. Oral-aboral axial establishment and regional specification along the axis appear to occur much earlier during echinoid development (HSrstadius, 1973). There are many ways in which the descriptive embryologies of the Phoronida and the Brachiopoda are similar (Zimmer, 1973). Many students of animal phylogeny feel that the Brachiopoda are derived from a phoronid like ancestor (Willmer, 1990). I am currently investigating the bases for and the timing of regional specification during larval development in the Brachi-

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poda. It is hoped that this kind of comparative experimental study will provide part of the basis for understanding the changes that took place in the developmental program of the phoronid lines t h a t evolved into brachipods. I want to t h a n k Dr. A. O. D. Willows, the Director of the Friday Harbor Laboratories, for facilitating my work there, and Robert Goldstein for critically reading this paper. This work was supported by NSF G r a n t DCB-8904333.

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