A Two-Step Mechanism for Cavitation in the Vertebrate Embryo

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Cell, Vol. 83, 279-287, October 20, 1995, Copyright 0 1995 by Cell Press Signals for Death and Survival: A Two-Step Mechanism for Cavitation in the Vertebrate Embryo

Electra Coucouvanis and Gail Ft. Martin CellS had been documented in the developing embryo. He Department of Anatomy identified several examples of developing organs in which and Program in Developmental Biology Cell deaths are correlated with lumen formation in a solid School of Medicine primordium and placed these in the general category of University of California at San Francisco morphogenetic deaths, defined as those contributing to San Francisco, California 94143-0452 the generation of form or structure. Gliicksmann regarded cell death as a normal aspect of embryogenesis, a somewhat controversial view at the Summary time. Now, however, it is recognized that cell death plays a major role in many developmental processes ranging Conversion of a solid primordium to a hollow tube of from the separation of the digits to the fine tuning of the Cells is a morphogenetic process used frequently dur- immune system (reviewed by Saunders, 1966; Hinchliffe, ing vertebrate embryogenesis. In the early mouse em- 1981; Snow, 1987; Cohen et al., 1992). Cell death that bryo, this process of cavitation transforms the solid occurs naturally as part of the normal functioning of a embryonic ectoderm into a columnar epithelium sur- tissue or organ is termed programmed cell death (PO) rounding a cavity. Using both established cell lines to distinguish it from accidental death of cells due to injury and normal embryos, we provide evidence that cavita- or toxicity. PCD is an active process, sometimes requiring tion in the early mouse embryo is the result of the inter- new mRNA or protein synthesis (reviewed by Wyllie et play of two signals, one from an outer layer of endo- al., 1980). In general, PCD occurs by the mechanism of derm cells that acts over short distances to create a apoptosis, which is characterized by shrinkage of the cyto- cavity by inducing apoptosis of the inner ectodermal plasm and condensation of nuclear chromatin into very cells, and the other a rescue signal mediated by con- dense, sharply defined masses. Biochemical features of tact with the basement membrane that is required for apoptosis include endonuclease digestion of DNA to indi- the survival of the columnar cells that line the cavity. vidual nucleosomes (Arends et al., 1990) and activation of This simple model provides a paradigm for investigat- proteases (Miura et al., 1993). Cells are ultimately broken ing tube morphogenesis in diverse developmental set- down into numerous apoptotic bodies, which are then elim- tings. inated by phagocytosis. Recently, many of Gliicksmann’s examples of PCD have been shown to occur by apoptosis, Introduction including cell death during palate formation (Ferguson, 1988) and in the interdigital zone of developing limbs (Gar- One of the most common structural elements in the devel- cia-Martinez et al., 1993). Growth factor deprivation ap- oping vertebrate embryo is the tube. Simple tubes are pears to be involved in the initiation of some apoptotic progenitors of the alimentary canal and the central ner- deaths during development (reviewed by Raff et al., 1993), vous system, and they are used to construct many of the but in many cases the triggering events are unknown. other organs of the body. For example, the lungs and kid- To investigate the general mechanism of the cavitation neys are composed of vast networks of ducts, tubules, process, we have focused on proamniotic cavity formation and blind-ended tube-like vessels. There are two basic in the rodent embryo, which has previously been hypothe- mechanisms by which such tubes are generated. They sized to involve PCD (Snow, 1987). In both mice and rats, form either by the rolling up of an established epithelial cavity formation occurs shortly after implantation, when sheet or by the creation of a lumen or cavity in a solid the embryonic portion of the conceptus consists of a solid structure. The former mechanism is used to generate mass of pluripotent embryonic ectoderm that is separated much of the neural tube and primitive gut, whereas the by a basement membrane from a surrounding outer layer latter is used in portions of the female reproductive tract of endoderm. The appearance of the proamniotic cavity (Crosby and Hill, 1962) and in exocrine glands, where solid is accompanied by differentiation of the embryonic ecto- epithelial buds are transformed into tubes of cells. The dermal cells lining the nascent cavity into a pseudostrati- latter process has been termed canalization, lumeniza- fied columnar epithelium, resulting in the formation of a tion, or cavitation. The earliest developmental stage at hollow, two-layered egg cylinder (Figure 1A). PCD has also which this cavitation process occurs in the rodent embryo been reported to occur in the preimplantation mouse blas- is just prior to the start of gastrulation, when the initially tocyst, but at that stage, it is not obviously associated with solid embryonic ectoderm is transformed into a hollow egg any morphogenetic process (El-Shershaby and Hinchliffe, cylinder. 1974). In several cases, degenerating or pyknotic cells have The process of proamniotic cavity formation can be stud- been noted in the region where a cavity forms (Koff, 1933; ied in vitro using lines of mouse embryonal carcinoma (EC) Borghese, 1955), raising the possibility that cell death or embryonic stem (ES) cells. EC cell lines are derived plays a role in the process of cavitation. This hypothesis from pluripotent stem cells of teratocarcinomas (reviewed was formally proposed over 40 years ago by Glticksmann by Martin, 1980), whereas ES cells are derived directly (1951) in a review of the times and places where dying from the pluripotent cells of early embryos (Evans and Cell 280

Figure 1, Proamniotic Cavity Formation in the E 4.0 E 5.0 E 6.0 Mouse Embryo and the Analogous ProceSS in BLASTOCYST EGG CYLINDER PSAl Embryoid Bodies (A) Schematic diagrams of mouse embryos at jr, stages prior to and just after the completion of proamniotic cavity formation illustrating the different tissue types present. The upper row depicts sagittal sections; the lower row depicts inner transverse sections through the planes indi- cell cated by dotted lines. Prior to implantation mass (E4.0), the blastocyst has an outer layer of \ trophectoderm surrounding a fluid-filled space, blastocoel the blastocoel, and an ICM. By this stage, the trophectoderm primitive endoderm has formed by delamina- tion and differentiation of those cells located m parietal endoderm on the outer surface of the ICM facing the blas- A visceral endoderm tocoel. As the cells of the primitive endoderm proliferate, some are displaced or migrate embryonic ectoderm away from the ICM, along the inner surface of A n basement membrane the trophectoderm. It is thought that subse- quent interactions of primitive endodermal cells with adjacent cell populationsgovern their differentiation (Hogan and Tilly, 1981). Those cells remaining in contact with the ectoderm form the VE, whereas those cells that are in contact with the mural trophectoderm form the PE. Shortly after implantation (E5.0), the ICM- derived ectodermal cells have proliferated to form a solid bud that is surrounded by VE. By E6.0, the proamniotic cavity has formed and is lined by a pseudostratified columnar ectoderm. For simplicity, no distinction is made between the extraembryonic ectoderm and the trophectoderm at the postimplantation stages. Al- though a basement membrane underlies all endoderm cells, including the PE, we have il- lustrated it (thick purple line) only between the VE and the embryonic ectoderm. (B-D) Aggregates of PSAl ceils cultured in suspension were collected on successive days during the cavitation process. Sections were stained with toluidine blue and also with periodic acid-Schiff reagent, which stains basement membrane pink. (B) Embryoid body in which an outer layer of endoderm (en) is visible. There is little evidence of cavitation in the inner ectodermal (ec) cell core. Arrowheads indicate the basement membrane between the two cell populations. (C and D) Embryoid bodies at intermediate stages of cavitation. The ectoderm cells adjacent to the basement membrane form a pseudostratified columnar epithelium (col). Arrows in (C) point to numerous small cavities near the periphery of the embryoid body. (E) A fully cavitated embryoid body. Some cell debris is evident in the cavity. (F and G) A higher magnification view of an embryoid body at an early intermediate stage of cavitation. Section was stained with DAPI. Cells with apoptotic morphology are present in the cavity. (F) bright field; (G) fluorescence. White arrow points to condensed chromatin in an apoptotic cell fragment. (H and I) Electrophoretic analysis of DNA isolated from the following: embryoid bodies at intermediate stages of cavitation (H, lane 1); embryoid bodies prior to the onset of cavitation (H, lane 2); S2 embryoid bodies treated with staurosporine (I).

Kaufman, 1981; Martin, 1981). When cultured as aggre- ROSA-1 1 ES cells cavitate, whereas those formed by S2 gates, both EC and ES cell lines form embryoid bodies, EC cells remain uncavitated. From our data, we conclude structures that recapitulate early steps of periimplantation that cavity formation in embryoid bodies and embryos oc- development, including the formation of endoderm on the curs by apoptosis. This PCD is triggered by a “death signal” surface of the inner cell mass (ICM), differentiation of co- produced by a specific endodermal cell type, visceral en- lumnar epithelium, and formation of a central cavity (Mar- doderm (VE). Moreover, survival of the single layer of co- tin et al., 1977; Martin, 1980, 1981). lumnar epithelium lining the cavity is dependent on a “sur- In this study, we have made use of three cell lines that vival signal” mediated by attachment to the extracellular form embryoid bodies. Those formed by PSAl EC and matrix. Thus, a two-step mechanism of selective cell sur- Mechanism of Cavitation 281

viva1 is responsible for the formation of the proamniotic bryoid bodies, a sizable proportion of the cells die during cavity in the early embryo. In light of Gliicksmann’s hypoth- cavity formation, yet at the end this debris is usually gone. esis that PCD plays a key role in the generation of many This is likely due to phagocytosis of the dead cell debris cavitated structures, we suggest that our data have re- by neighboring cells, a process typical of PCD. Our failure vealed a fundamental mechanism of tube morphogenesis to detect intermediate stages of cavitation in small em- that is widely utilized during vertebrate development. bryoid bodies can be explained by the fact that in a small structure relatively few cells need to die to create a cavity. Because apoptosis is a very rapid process, with as little as 1 hr elapsing between initiation of death and removal Cavity Formation in PSAl Embryoid Bodies Occurs of debris by phagocytosis (Barres et al., 1992), the death by Apoptosis That Starts near the Periphery of a small number of cells can be inconspicuous. and Proceeds Inward One of the hallmarks of apoptotic death is internucleoso- PSAl EC cells are maintained in the undifferentiated state mal cleavage of DNA, which can be detected by a charac- by culture on a feeder cell layer. When plated at high den- teristic ladder pattern observed when DNA isolated from sity in the absence of feeder cells, they form aggregates, apoptotic cells is analyzed by electrophoresis. To confirm which when detached from the substratum and cultured that the apoptotic cell morphology observed in cavitating in suspension spontaneously develop an outer layer of embryoid bodies correlates with this biochemical feature endoderm. Over the course of several days, these em- of PCD, we isolated DNA from PSAl embryoid bodies bryoid bodies develop a central cavity. Although there is before and during cavitation and analyzed it by gel electro- considerable variation in the size of the embryoid bodies phoresis. In DNA isolated from embryoid bodies harvested (- 100 urn to 1 mm in diameter), at the end of the cavitation at late stages, when a substantial number of cells with process they all consist of two distinct cell layers separated apoptotic morphology were detected, a ladder was clearly by a basement membrane, an outer endoderm and inner evident (Figure 1 H, lane 1). In contrast, there was little if columnar ectoderm, surrounding the central cavity. any ladder detected in DNA isolated from embryoid bodies To gain insight into the mechanism of cavity formation, harvested at early stages (Figure lH, lane 2), when few we looked for embryoid bodies at intermediate stages of cells with apoptotic morphology are observed. the process by fixing, embedding, and serially sectioning As a further test that the mode of cell death is apoptosis, samples harvested at various times during cavitation (Fig- we treated PSAl embryoid bodies with the protein synthe- ures 1 B-1 E). The smallest embryoid bodies were always sis inhibitor cycloheximide, which prevents apoptosis in found to be either uncavitated or fully cavitated. However, many cell types (for example, see Pratt and Greene, 1976). among the larger embryoid bodies, we found some in Embryoid bodies treated with cycloheximide (0.25 pglml which cavity formation was still in progress. Large em- for 4 days) were viable, but failed to cavitate even several bryoid bodies harvested early were found to contain nu- days after control embryoid bodies had completed the pro- merous small cavities located near the periphery (Figure cess (data not shown). 1C). These initial small cavities were never located in the Taken together, these data demonstrate that cavitation center of the embryoid body. A layer of cells with columnar in PSAl embryoid bodies occurs by apoptotic PCD. The morphology was always found between the nascent cavity fact that apoptosis is first detected near the periphery of and the basement membrane. We were unable to deter- the embryoid bodies and then proceeds inward raises the mine which occurs first, the appearance of a cavity or possibility that a signal from the outermost cell layers (ei- ectoderm columnarization, as neither was observed in the ther endoderm or the ectoderm cells that remain viable) absence of the other. At later times, the small cavities is responsible for this PCD. appeared to have merged into a single peripheral space surrounding a solid core of cells (Figure 1 D). At even later Proamniotic Cavity Formation in Mouse and Rat times, the internal solid core was reduced in size, and by Embryos Involves Apoptosis the end of the process, it was usually gone (Figure 1E). To test whether PCD is also involved in cavity formation These results indicate that cavitation begins near the pe- in the early embryo, we examined DAPI-stained serial sec- riphery of the embryoid body and proceeds inward. tions of uteri containing mouse embryos at embryonic day A substantial amount of cellular debris was frequently 5.0-5.5 (E5.0-E5.5) the time when the proamniotic cavity observed in the embryoid bodies at all intermediatestages forms in vivo (Figure 1A). At this stage of development, the of cavitation. A similar observation led Martin et al. (1977) embryonic ectoderm contains less than 120 cells (Snow, to propose that PCD plays a role in embryoid body cavity 1977) and therefore, very few cells would need to die formation. To test this hypothesis, we stained sections of (as few as 3 or 4) to form a central cavity. Based on our embryoid bodies with DAPI, a dye that binds to DNA and observations of cavitation in small PSAl embryoid bodies, enables one to distinguish cells with the condensed chro- we anticipated some difficulty in detecting embryonic cells matin diagnostic of apoptosis from those with normal nu- in the process of dying. Indeed, within a single litter, most clear morphology. DAPI staining clearly revealed cells with embryos either had not yet begun cavitation or had com- apoptotic morphology in the nascent cavities (Figures 1 F pleted the process. However, in 2 of the approximately and 1G). Thus, it appears that the cellular debris detected 60 embryos analyzed, we detected a single apoptotic cell in the cavities is the consequence of PCD. In large em- in the nascent cavity (Figures 2A and 28; data not shown). Cl?ll 282

EC cells do not cavitate. We reasoned that such cells could be useful for gaining insight into the mechanism of cavitation if they are competent to undergo cavitation but fail to produce the required initiating signals. Alternatively, they might be incapable of undergoing PCD, or some other aspect of the cavitation process. We first tested whether S2 cells are able to undergo PCD by treating 52 embryoid bodies with staurosporine (1 FM for 4 days), a potent in- ducer of apoptosis (Bertrand et al., 1994). In DAPI-stained sections, we found that virtually all cells in the treated embryoid bodies were apoptotic (data not shown). In addition, DNA isolated from staurosporine-treated S2 cells dis- plays the ladder pattern characteristic of apoptotic cells (Figure 1 I). We next asked whether S2 cells are capable of partici- pating in the cavitation process by performing cell-mixing experiments with cells that form cavitated embryoid bodies. In the first experiment, we used ROSA-1 1 ES cells as the cavitation-competent partner. These cells are marked by constitutive expression of the /acZ gene (Friedrich and Soriano, 1991) and, therefore, appear blue when stained with X-Gal, a chromogenic substrate for f%galactosidase. When cultured separately, ROSA-1 1 cells form cavitated embryoid bodies in which all cells are stained blue (Figure 3A), whereas S2 cells form embryoid bodies that do not cavitate and are not stained with X-Gal (Figure 38). When undifferentiated stem cells of the two lines were mixed in equal proportions and plated to form aggregates, we obtained chimeric embryoid bodies containing both unmarked S2 cells (which appear pink after staining with neutral red) and X-Gal-positive (blue) ROSA-11 cells. Among these chimeric embryoid bodies, some were cavitating and contained 52 cells (pink) in the columnar epithelium bordering the cavity (Figure 3C). Although these data provide clear evidence that S2 cells can differentiate into the columnar epithelium characteristic of cavitated embryoid bodies, it was unclear whether they had undergone Figure 2. Apoptotic Cells Are Present during Proamniotic Cavity For- mation in the Mouse and Rat Embryo apoptosis in response to signals operating during cavita- (A-D) DAPI-stained sections of an E5.0-E5.5 mouse embryo (A, bright tion because it was difficult to tell whether any of the debris field; B, fluorescence) and an E7.0-E7.5 rat embryo (C, bright field; in the cavity was derived from the unmarked S2 cells. D, fluorescence). Arrows point to condensed chromatin in apoptotic We therefore performed a mixing experiment in which cell fragments in the proamniotic cavity. Abbreviations: ec, ectoderm; the S2 cells were marked by f3-galactosidase expression ve, visceral endoderm. Mouse and rat embryos are shown at the same (S2//acZcells, see Experimental Procedures) and the cavi- magnification. tation-competent partner was unmarked ICM cells (iso- The rat embryo is much larger than the mouse embryo lated from normal embryos). In these experiments, small at the stage when the proamniotic cavity forms (compare clumps of undifferentiated S2llacZ cells were aggregated Figures 2A and 2C). By analogy with the large versus small with several ICMs. We also separately cultured S2//acZ embryoid bodies, we expected it would be easier to detect aggregates and aggregates of three to four ICMs. As ex- apoptotic cells during proamniotic cavity formation in the pected, the aggregates of ICMs merged into a single ICM rat. In DAPI-stained serial sections of a single litter, we that cavitated and did not stain with X-Gal (Figure 3D), observed several embryos in which numerous cells with whereas SPllacZ cells formed embryoid bodies that failed the condensed chromatin characteristic of apoptosis were to cavitate and in which all cells were stained (blue) with present in the nascent proamniotic cavity (Figures 2C and X-Gal (Figure 3E). The mixed aggregates formed individ- 2D; data not shown). These data show that the formation ual chimeric embryoid bodies in which we could detect of the proamniotic cavity in rodent embryos involves PCD. not only X-Gal-positive S2//acZ cells in the columnar epithelium (Figure 3F; data not shown), but also S2//acZcells S2 Cell Aggregates Can Respond to but Do Not with apoptotic morphology in the cavity (Figure 3F). Produce the Signals Required for Cavitation These results demonstrate that S2 cells are competent One of the most intriguing findings reported by Martin et both to undergo apoptosis and to differentiate into colum- al. (1977) was the fact that embryoid bodies formed by S2 nar epithelium in response to signals for cavitation. Fur- Mechanism of Cavitation 283

Figure 3. Chimeric Embryoid Bodies Pro- duced by Mixing S2 Cells with Cavitation- Competent Partners Intact embryoid bodies were stained with X-Gal to identify /a&expressing cells. Sections of these embryoid bodies were stained with neutral red. (A) A fully cavitated embryoid body formed by ROSA-1 1 cells, which constitutively express LecZ. In this embryoid body, X-Gal staining is less intense in flattened endodermal cells than in columnar ectoderm. (B) A noncavitated embryoid body formed by 52 cells, which do not express the /acZ gene. (C)A chimeric embryoid body containing a mixture of ROSA-I 1 and S2 cells. Note that the 52 cells (pink) have participated in the formation of the columnar epithelium. (D) A cavitating aggregate of three to four mouse embryo ICMs. (E) An X-Gal-stained, noncavitated embryoid body formed by a subclone of S2 cells that constitutively express /acZ. (F) A chimeric embryoid body containing a mixture of ICM cells and /aci’-expressing S2 cells. An arrowhead points to an S2 cell in the columnar epithelium. X-Gal-positive S2 cell debris is evident in the cavity. The white box marks the region I rhown at higher magnification in the inset in the lower right corner. thermore, the signals that induce these two responses by Significantly, little if any differentiation of columnar epi- S2 cells are produced not only by ES cell-derived em- thelium was detected in S2/END-2 combination cultures. bryoid bodies but also by normal embryo ICMs. Indeed, many of the outermost S2 ectodermal cells (which in a PSAl embryoid body would form columnar epithelium) A Visceral Endoderm Cell Line Can Provide undergo apoptosis (Figure 4A). In contrast, S2 cells in the Signal Required for Apoptosis in S2 chimeric embryoid bodies with ES- or ICM-derived cells Embryoid Bodies do differentiate into columnar epithelium (see Figure 3C). We next explored the question of whether it is the endo- The simplest interpretation of these data is that cells of derm of the embryoid bodies that produces the signals the END-2 line differ from normal embryonic VE in that required for cavitation. Embryoid body endoderm is classi- they fail to produce some factor that permits or instructs fied by comparison with the two types of endoderm found the differentiation of S2 cells to columnar epithelium. How- in the pregastrulation embryo (see Figure lA), visceral ever, we cannot rule out the possibility that, in the mixed endoderm (VE) and parietal endoderm (PE), which are aggregates of S2 and cavitation-competent cells, differen- distinguishable by morphological and biochemical criteria tiation of S2 cells into columnar ectoderm is a conse- (reviewed by Hogan et al., 1986). The outer layer of PSAl quence of interactions with neighboring ES- or ICM-derived embryoid bodies and of ICMs cultured under certain condi- ectoderm. tions in vitro contains a mixture of VE and PE, whereas Differentiation of cells near the basement membrane that of S2 embryoid bodies contains only PE, leading to to columnar ectoderm and apoptosis of other ectodermal the suggestion that VE produces the signals responsible cells in the embryoid bodies usually occur concomitantly. for cavitation (Martin et al., 1977), an idea consistent with The observation that END-2 cells can trigger apoptotic the fact that in vivo the cavitating embryonic ectoderm is death of S2 cells in the absence of ectoderm columnariza- covered with a layer of VE. tion is important, because it provides evidence that there To test this hypothesis, we cultured aggregates of S2 cells with END-2 cells. The END-2 cell line was isolated from a culture of differentiating EC cells and has been found to express many markers of VE (Mummery et al., 1985, 1991). We devised a protocol for obtaining S2 aggregates that contained END-2 cells in their outer layer only (see Experimental Procedures). Sectioning of S2/END-2 embryoid bodies cultured for 3-4 days in collagen gels revealed that a substantial fraction of the S2 ectodermal cells were undergoing apoptotic cell death (Figure 4A). This effect was observed in a majority of S2/END-2 embryoid bodies in 9 of 11 experiments. In contrast, no signifi- Figure 4. Addition of END-2 Cells to the Outer Surface of S2 Embryoid cant amount of apoptosis was observed either when a PE Bodies Causes S2 Cell Apoptosis cell line, PYS (Lehman et al., 1974), was used in place of (A) S2 embryoid body with END-2 cells on its outer surface. Arrows END-2 cells (Figure 48) or in control cultures of S2 em- point to regions with apoptotic cells. bryoid bodies in collagen gels (data not shown). (B) S2 embryoid body with PYS cells on its outer surface. Cell 284

are two independent signals involved in cavitation: one ment membrane were unaffected by treatment with the for cell death and the other for cell differentiation/survival. polyclonal antiserum (Figure 5C). Furthermore, these data support the hypothesis that it is Taken together, these data provide evidence that it is the VE cells in embryoid bodies and embryos that produce interaction with the ECM that enables the cells that form the signal for apoptosis during cavitation. the internal layer of columnar epithelium in cavitated embryoid bodies to survive. In contrast, in S2 embryoid bodies The Columnar Epithelium Is Rescued from Death such interaction is not required for survival, presumably by Interaction with the Extracellular Matrix because VE is absent and thus there is no death signal An important feature of the cavitation process is the sur- from which the cells must be saved. vival and differentiation of the ectodermal cells that form the walls of the cavity. The finding that a death signal Discussion produced by endoderm is operating during cavitation raises the question of how the inner layer of columnar The studies described here elucidate the mechanism of epithelium is able to survive. Although a wide variety of cavitation in the early mouse embryo, a process that sets factors and circumstances prevent cell death in other sys- the stage for gastrulation by converting the solid embry- tems, we were intrigued by a recent report showing that onic ectoderm into a hollow closed tube of columnar epi- interfering with the interaction between mammary epithe- thelial cells. Analysis of PSAl embryoid bodies showed lial cells and the extracellular matrix (ECM) by treatment that cavity formation occurs by apoptosis. Cavitation starts with blocking antibodies results in apoptotic death of the near the periphery of the embryoid bodies and proceeds cells (Boudreau et al., 1995). We used a similar approach inward, suggesting that it is induced by signals produced to explore the possibility that in embryoid bodies the inter- by the cells peripheral to the cavity. A key result was the action of the columnar ectoderm cells with the ECM res- finding that, although S2 embryoid bodies fail to cavitate, cues them from apoptosis. 52 cells are competent to respond to the signals that in- PSAl embryoid bodies that had a well-developed endo- duce cavitation. This enabled us to perform experiments dermal cell layer were incubated for 3-4 days in a poly- that identified VE as the source of a death signal. Using clonal antiserum similar to the one employed by Boudreau an antiserum raised against cell-substratum adhesion et al. (1995). This antiserum was raised against a purified molecules, we have also shown that the selective survival glycoprotein fraction consisting predominantly of cell- of the ectoderm cells lining the cavity is dependent on substratum adhesion molecules. It is known to recognize cell-ECM interactions. Thus, cavitation is the result of the integrins of both the 81 and av families and to interfere interplay of two signals, one that induces apoptosis and with cell-substratum interactions (Albelda et al., 1989). the other that prevents it. Analysis of sections of the antiserum-treated embryoid In addition, our results suggest that this same combina- bodies revealed that nearly all of the ectodermal cells lo- tion of signals causes cavitation in the early embryo. We cated adjacent to the ECM were apoptotic (Figure 5A; data have shown that apoptotic cells are present in the nascent not shown; results of 7 experiments). Although the endo- proamniotic cavity in utero, implicating PCD in proamniotic derm layer of treated embryoid bodies was often detached cavity formation in vivo. We have also found that S2 cells from the ectodermal core, the endoderm cells usually re- are induced to undergo apoptosis and to participate in the mained viable. In control cultures, in which the embryoid formation of a columnar epithelium by signals from either bodies were incubated in the presence of a l/50 dilution ES or ICM cells. This suggests that ICMs produce the of nonimmune rabbit serum (Figure 58; data not shown; same signals for cavitation as do embryoid bodies. results of 12 experiments), increased death was never Based on the data described here, we propose a model observed. From these data, we conclude that interactions in which cavitation occurs by a simple two-step mecha- with the ECM mediate the survival of the ectodermal cells nism involving two interacting cell populations separated lining the cavity in PSAl embryoid bodies. by a basement membrane. In this model (Figure 6) the Evidence that the apoptosis observed in PSAl embryoid outer cells produce a death signal that can act at a dis- bodies is not due to nonspecific toxicity of the polyclonal tance, either by short range diffusion or by cell-to-cell relay antiserum was obtained from experiments with S2 em- (Figure 6A, long black arrows). Interaction with the base- bryoid bodies. In 7 of 7 experiments, 52 embryoid bodies ment membrane (Figure 6A, short red arrows) rescues that had a well-developed layer of endoderm and base- cells from death. The combination of these two signals

Figure 5. Treatment of Embryoid Bodies with an Antiserum that Blocks Cell-ECM Interac- tions (A) Antiserum-treated PSAI embryoid body. Arrows point to apoptotic ectodermal cells near the basement membrane. (B) Control culture. PSAI embryoid body incubated in nonimmune serum. (C) Antiserum-treated S2 embryoid body. No effect on the ectodermal cells is observed. Mechanism of Cavitation 285

(END-2, ROSA-1 1, or ICM cells) induces apoptosis of ~2 Cells is consistent with (but not necessarily indicative of) an active mechanism of the Fas ligand type. Although our data seem inconsistent with a mechanism that involves ref?IOVal of a survival factor, it is possible that the death signal we describe acts to “program” the cells to die by outer cells send rendering them dependent on specific factors that are pre- cells not in contact with cavitation death signal basement membrane complete sumably available only to cells that have appropriate inter- inner cells rescued by undergo apoptosis interaction with actions with the ECM. The mechanism of death in our basement membrane model could also be the consequence of conflicting signals. For example, the signal from the outer cells could Figure 6. A Two-Signal Model for the Morphogenesis of Cavitated Structures instruct all inner cells to differentiate. If contact with the (A) Schematic representation of the first phase of the cavitation pro- basement membrane is absolutely required for differentia- cess. The long black arrows represent a death signal produced by the tion, then inner cells not adjacent to it would be receiving outer cells. The short red arrows represent the survival signal mediated conflicting signals and would therefore be driven into by interaction between inner cells and the basement membrane. apoptosis. (B) An intermediate stage in cavitation. The black dots represent apop- The second key feature of our model is a signal that totic cells. (C) When cavitation is complete, the result is a hollow tube or vessel. appears to be mediated by attachment to ECM and rescues cells from death. There is ample precedent in the literature for cell dependence on ECM for survival. Human results in the death of inner cells not in contact with the endothelial cells, gut epithelial cells, and mammaryepithe- ECM and survival of a single layer of inner cells in contact lial cells all have been shown to undergo apoptosis when with the basement membrane(Figure 6B), therebyproduc- cell-ECM interaction is perturbed (Meredith et al., 1993; ing a hollow tube of cells (Figure 6C). Boudreau et al., 1995). Moreover, in cultures of human A central feature of this model is a signal that causes melanoma cells and in tumor angiogenesis, it has been cell death. Previous studies have suggested three basic shown that specific members of the integrin family of cell mechanisms that account for the control of cell death dur- adhesion molecules mediate such ECM-dependent sur- ing development: first, direct initiation of apoptosis by an vival (Brooks et al., 1994; Montgomery et al., 1994). external signal; second, death due to the absence of tro- Our results provide some insight into the nature of the phic factors; third, apoptosis as the response of a cell to signals involved. For example, it is clear that the endo- conflicting signals. There are several examples of mecha- derm-derived death signal must act at some distance, nisms of the first type. In the immune system, binding of since in PSAl embryoid bodies and in embryos it reaches the Fas ligand to its receptor causes apoptotic death of cells beyond those that will form the columnar epithelial the receptor-bearing cell (Itoh et al., 1991). Tumor necrosis layer. However, the range of its effect appears to be lim- factor (TNF) produced by natural killer cells can act in a ited, since in particularly large embryoid bodies we have similar way (Cohen et al., 1992). In the developing eye, sometimes observed a small core of living cells that per- Lang and Bishop (1993) found that ablation of an ocular sists in the middle of the cavity. Furthermore, it is unlikely macrophage subset resulted in the persistence of normally that the death signal is freely diffusible, since preliminary transient eye structures, suggesting that ocular macro- studies suggest that there is little cell death in S2 aggre- phages are required for developmental tissue remodeling gates when they are cultured in the presence of a mono- in the eye, perhaps because they provide an extracellular layer of END-2 cells (unpublished data), whereas signifi- signal that initiates cell death. cant S2 cell death is observed when close contact between An example of the second mechanism comes from stud- END-2 cells and S2 embryoid bodies is maintained by ies of cell survival in the nervous system. The classic ex- culture in collagen gels. periments of Hamburger and Levi-Montalcini (1949) showed In contrast, the rescue signal acts locally, is contact me- that increased survival of neurons resulted when the size diated, and can be blocked by a polyclonal antiserum of their target tissue was increased, whereas ablating the raised against purified cell surface glycoproteins con- target tissue caused neuronal death. Subsequently, it was sisting mainly of integrins. Although this antiserum blocks shown that target-derived trophic factors mediate this ef- integrin-dependent adhesion to the ECM, it is likely that fect (reviewed by Oppenheim, 1991). On the basis of these other cell-ECM interactions are perturbed in the absence and other studies, Raff (1992) has proposed that virtually of normal integrin-mediated adhesion. Thus, the precise all cells in the embryo are programmed to die unless they role of the integrins in cavitation remains to be determined. constantly receivesignals instructing them to remain alive. Even if a specific integrin family member were shown to Finally, cells that receive conflicting signals can also be involved, integrin-ligand binding per se need not be become apoptotic. This was elegantly demonstrated by the survival signal. For example, integrins can modulate Evan et al. (1992) using an in vitrosystem in which constitu- cell responsiveness to growth factors (Elliott et al., 1992). tive expression of c-myc, combined with a block to cell There is increasing evidence that specific develop- proliferation, resulted in apoptosis. mental strategies are reiterated many times during em- Our finding that the addition of specific cell types bryogenesis. We have provided evidence for a two-step Cell 286

mechanism responsible for the conversion of the solid ICM method produced S2 embryoid bodies with the added cells localized to the hollow tube of cells that constitutes the pregastrula- in the outer layer, we used the END-2/lacZ cells described above. X-Gal staining demonstrated that the added END-PIlacZ cells were tion mouse embryo. Since this type of morphogenetic con- located only on the surface of the aggregates and were present on version occurs very frequently in the vertebrate embryo, most but not all aggregates (data not shown). it is tempting to speculate that the model we propose for Mouse ICMs were isolated by immunosurgery (Solter and Knowles, the early embryo is applicable in a wide variety of develop- 1975) from E4.5 blastocysts flushed at E3.5 from the uteri of normal or superovulated CD1 female mice (Charles River, Wilmington, MA) mental settings. The basic requirement of the model is and cultured overnight. ICMs were aggregated together (three to four the interaction of two cell populations separated by a base- ICMs) or with S2llacZ cell aggregates of similar size (two of each) ment membrane. In the case of the early embryo, these using a protocol similar to that described by Nagy and Rossant (1993). two cell populations are VE and embryonic ectoderm, but in other situations, it could be other cell types that interact, Histological Analysis Embryoid bodies were fixed in 4% paraformaldehyde at 4°C or at For example, during exocrine gland morphogenesis, the room temperature for at least 3 hr. They were then dehydrated through overlying ectoderm grows down into the mesoderm and 80%, 95%, and 100% ethanol/distilled HP0 and embedded in glycol is converted from a solid bud to a hollow tube of epithelial methacrylate (Polysciences, Warrington, PA) according to the instruc- cells. In this case, the mesoderm would be the “outer layer” tions of the manufacturer. Sections (2.5-5 Km thick) were cut on a that produces the death signal. Similarly, the specific mo- JB4 microtome with a glass knife. All staining procedures were performed on sectioned material except the assays for f3-galactosidase lecular identity of the signals involved may differ on a case activity. Stains used on sections included neutral red, toluidine blue, by case basis. Future investigationsof thegeneral applica- and periodic acid-Schiff reagent. Stained sections were air dried, bility of our model may be hampered by the inconspicuous dipped in xylene, and coverslipped using xylene-based mounting me- nature of apoptosis. However, as we have shown, the dium. Sections stained with DAPI (4’,6-diamidino-2-phenylindole dihy- drochloride) were rinsed in distilled Hz0 and mounted in glycerol. availability of an in vitro model system can greatly facilitate To detect 8-galactosidase activity, cells or embryoid bodies were the analysis of the cavitation process. Moreover, such fixed in 2% paraformaldehyde, 0.2% glutaraldehyde for 5-10 min at model systems should also provide the means for isolating 0% washed in PBS, and stained with X-Gal as previously described and identifying the specific molecules that are responsible (Saneset al., 1986). Cellswere then postfixed in4% paraformaldehyde for death and survival during tube morphogenesis. and processed for sectioning as described above. DNA Analysis Total cellular DNA was isolated from cells as previously described Experimental Procedures (Sherwood et al., 1994) and end labeled using P*P]dCTP and T4 DNA polymerase prior to electrophoresis on a 2% agarose gel. The gel was Cell Cultures dried under vacuum and exposed to X-ray film overnight at -80%. All cultures were incubated in 5% CO, (in air) at 37%. The END-2 cell line (provided by Dr. C. Mummery) and subclones of it (see below) Antibody Treatment were cultured on gelatin-coated tissue culture dishes in a I:1 mix of The rabbit antiserum used in this study (provided by Dr. C. Damsky) Ham’s F12 and Dulbecco’s modified Eagle’s medium (4.5 g/l glucose; was raised against a purified glycoprotein fraction consisting predomi- DMEM) supplemented with 100 U/ml penicillin and 100 Kg/ml strepto- nantly of cell-substratum adhesion proteins isolated from the rat ceil mycin and with 7.5% fetal calf serum (Hyclone, Logan, UT). All other line L6A. lmmunoprecipitation studies demonstrated that this antise- cell lines, isolated ICMs, and mixed cell cultures were grown in DMEM rum binds to integrin subunits a,, 02, fi3, c5, 6, and 03; it was also shown supplemented with 10% fetal calf serum, penicillin, and streptomycin. to block cell adhesion to laminin, collagen, and fibronectin (Albelda et The method for producing and culturing embryoid bodies has been al., 1989). Before use, the antiserum was heat inactivated at 55°C for described by Martin et al. (1977). In brief, 1 x 1 O’-3 x lo7 undifferenti- 20 min and diluted I:50 with culture medium, ated cells were seeded in 9 cm diameter tissue culture dishes in the absence of feeder cells. On the third day after this passage, clumps of Acknowledgments cells had formed. These were then detached from the dish by pipetting fresh medium over them and transferred to bacteriological petri dishes. Thereafter, the medium was changed daily. We thank Linda Prentice for excellent technical assistance and Drs. Ann Sutherland and Patrick Tam for helpful discussion and advice. To obtain subclones of END-Z and S2 cells that constitutively express @galactosidase, a construct (M. Lewandoski and G. R. M., un- We thank Dr. Caroline Damskyforgenerouslyproviding the antiserum, published data) containing the /acZ gene under the control of the We are also grateful to Drs. Marc Tessier-Lavigne, Cori Bargmann, and Ann Sutherland, as well as our laboratory colleagues for critical human 8-actin promoter (K. Sturm and R. Pedersen, personal com- readings of the manuscript. E. C. is the recipient of an individual Na- munication) and the neomycin resistance gene under control of the tional Research Service Award from the National Institutes of Health. phosphoglycerate kinase promoter (Tybulewicz et al., 1991) was intro- This work was supported by National Institutes of Health grant ROl duced into the cells by electroporation as described by Hebert et al. HD22731 to G. R. M., as well as by a grant from The Lucille P. Markey (1994). After 10-l 2daysof culture inselective medium, G418resistant Charitable Trust to the University of California at San Francisco Pro- clones were isolated. Clones were screened by X-Gal staining (see gram in Biological Sciences, below) for constitutive /acZ expression. In the case of the SPllacZ clones, staining was tested both before and after differentiation to Received August 14, 1995; revised September 7, 1995. embryoid bodies. 52 embryoid bodies with END-2 or PYS cells in their outer layer References were produced by the following method: 52 cells were seeded as described above. When aggregates had formed (after 2 or 3 days of Albelda, S.M., Daise, M., Levine, E.M., and Buck, C.A. (1989). Identifi- culture), a single cell suspension of END-2 or PYS cells was added cation and characterization of celf-substratum adhesion receptors on to the culture dish. After 6-18 hr, when the added cells had settled cultured human endothelial cells. J. Clin. Invest. 83, 1992-2002. to the bottom of the dish or adhered to the 52 cell aggregates, the aggregates were detached from the dish by pipetting. After l-2 days Arends, M.J., Morris, R.G., and Wyllie, A.H. (1990). Apoptosis: the culture in bacteriological petri dishes, the aggregates were transferred role of the endonuclease. Am. J. Pathol. 736, 593-608 to collagen gels, prepared as previously described (Tessier-Lavigne Barres, B.A., Hart, I.K., Coles, H.S., Burne, J.F., Voyvodic, J.T., Rich- et al., 1988). Culture in collagen gels served to keep the END-2 or ardson, W.D., and Raff, M.C. (1992). Cell death and control of cell PYS cells in close proximity to the S2 aggregates. To confirm that this survival in the oligodendrocyte lineage. Cell 70, 31-48. Mechanism of Cavitation 287

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