Experimental Induction of BMP-4 Expression Leads to Apoptosis In

DEVELOPMENTAL BIOLOGY 202, 253–263 (1998) ARTICLE NO. DB989011 CORE Metadata, citation and similar papers at core.ac.uk Provided by ElsevierExperimental - Publisher Connector Induction of BMP-4 Expression Leads to Apoptosis in the Paraxial and Lateral Plate Mesoderm

Corina Schmidt,* Bodo Christ,*,1 Ketan Patel,† and Beate Brand-Saberi* *Institut of Anatomy, University of Freiburg, D-79001 Freiburg, Germany; and †Zoology Division, School of Animal and Microbial Sciences, University of Reading, P.O. Box 228, Reading RG6 6AJ, United Kingdom

In the avian embryo, epithelialization of the segmental plate and formation of an epithelial dermomyotome depend on signals from the neural tube and the ectoderm overlying the paraxial mesoderm. In this study, we report that ectoderm removal in combination with barrier insertion between the axial organs and the segmental plate leads to an induction of BMP-4 expression in the paraxial mesoderm. In the lateral plate, ectoderm removal alone leads to an increase of BMP-4 expression. Application of BMP-4 protein results in a lack of epithelialization of the paraxial mesoderm. In order to investigate whether the loss of epithelial structures after these manipulations can be attributed to a change in cell fate, a change in cell proliferation, or the induction of apoptosis, the paraxial mesoderm was tested for expression of Msx-2, BMP-2, BMP-4, and BMP-7. Moreover, BrdU and TUNEL staining were carried out. The inhibition of epithelialization after ectoderm removal alone and after segregation of the axial organs is accompanied neither by an increase in apoptosis nor by a reduction of the proliferation rate in the paraxial mesoderm. On the other hand, an ectopic BMP-4 expression in the paraxial mesoderm after ectoderm removal in combination with barrier insertion coincides with the occurrence of apoptotic cells and reduction of proliferation rate in this tissue. Increase of apoptosis and decrease in cell proliferation are observed in the paraxial and lateral plate mesoderm also after application of BMP-4 protein. © 1998 Academic Press Key Words: BMP-4; apoptosis; somite; TUNEL staining; proliferation.

INTRODUCTION et al., 1996), as well as MyoD and myf 5 during myotome formation (Pownall and Emerson, 1992; Weintraub, 1993; In vertebrate embryos, mesoderm is formed during gas- Olson and Klein, 1994). The somites themselves are com- trulation from epiblast cells that ingress through the primi- partmentalized along the mediolateral axis and are charac- tive streak. The newly formed mesoderm is not only terized by the restricted expression of c-Sim 1 (a homologue patterned along the craniocaudal axis, which is impres- of the Drosophila single-minded gene) in the lateral half. sively reflected by the segmentally arranged somites, but The intermediate mesoderm, which lies between the lateral also along the mediolateral axis. The mesoderm forms three plate and the paraxial mesoderm, expresses c-Sim 1 in the different compartments along the mediolateral axis: the Wolffian duct. The lateral plate mesoderm strongly ex- paraxial, intermediate, and lateral plate mesoderm. These presses BMP-4, a member of the TGF-␤ superfamily (Pour- compartments are characterized at the molecular levels by quie´ et al., 1996). the expression of distinct genetic markers. The paraxial Although prospective paraxial, intermediate, and lateral mesoderm from which the somites arise lies adjacent to the plate mesoderm cells ingress through the primitive streak axial organs and is characterized by the expression of Pax-1, at differing anterior–posterior levels, this in itself does not -3, -7, and -9 (Bober et al., 1994; Goulding et al., 1994; determine their fate. Extrinsic signals, however, seem to be Williams and Ordahl, 1994; Ebensperger et al., 1995; Mu¨ller responsible for definitive mediolateral mesoderm patterning. Members of the TGF-␤ family are good candidates for 1 To whom correspondence should be addressed. E-mail: being the extrinsic signals. In Xenopus, it has been shown [email protected]. that one of the members of this family, BMP-4, is able

0012-1606/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 253 254 Schmidt et al. to pattern prospective mesoderm in a concentration- MATERIAL AND METHODS dependent manner; exposure of mesoderm to high concentrations of BMP-4 giving rise to ventral mesoderm (blood), Embryos intermediate levels giving lateral mesoderm (muscle) and low levels specifying dorsal mesoderm (notochord) (Dosch et al., Fertilized eggs (White Leghorn) were obtained from a local 1997). In chick, BMP-4 can not only lateralize medial com- breeder and incubated at 38°C and 80% relative humidity for 2 days up to HH stages 12–14 (Hamburger and Hamilton, 1951). partments in a concentration-dependent manner (Tonegawa et al., 1997), but can also influence the mediolateral identity within a compartment (Pourquie´ et al., 1996). Histology: Hematoxylin/Eosin Staining There are at least two sources of signals necessary for Selected embryos were fixed in Serra’s solution (Serra, 1946) normal development of the paraxial mesoderm. One signal overnight and, after dehydration with ethanol, embedded in paraf- emanates from the ectoderm overlying the paraxial meso- fin. Sections were cut at 8 ␮m and hematoxylin/eosin staining was derm, since removal of this tissue leads to the absence of carried out according to Romeis (1968). epithelial dermomyotomes (Sosic et al., 1997; Dietrich et al., 1997). Likewise, epithelialization of the paraxial mesoderm depends on signals from the neural tube, and after TUNEL insertion of impermeable barriers (Sosic et al., 1997) the Embryos were fixed in 4% paraformaldehyde overnight at 4°C. epithelialization gene paraxis is not expressed. They were rinsed in 0.1 mol KH2PO4 buffer three times prior to The present study was undertaken to investigate mecha- infiltration with 5 and 15% sucrose for 8 and 20 h, respectively, at nisms that lead to a disruption of epithelialization of the 4°C. They were embedded in Tissue Tek (Miles, U.S.A.) for paraxial mesoderm and the role of the BMPs in this process. cryosectioning. Sections were cut at 20 ␮m. For TUNEL staining One of the effects of ectopic application of BMPs to unseg- the In Situ Cell Death Detection Kit, AP (Boehringer, Germany) was used according to the recommendations of the producer. mented paraxial mesoderm was to inhibit the formation of epithelialized structures. We wanted to determine whether the surgical manipulations that resulted in lack of epithe- In Situ Hybridization lial somites (ectoderm removal or isolation of axial organs) Embryos were washed in PBS and then fixed overnight in 4% were mediated by an induction of BMP following these paraformaldehyde at 4°C. The antisense RNA probe was labeled procedures. The action of BMPs resulting in the disruption with digoxigenin and whole-mount in situ hybridization was of epithelialization could be direct, involving an interfer- performed as described by Nieto et al. (1996). The following probes ence with patterning of the mesoderm so that the paraxial were used in this study: an avian probe of Pax-3 (a 660-bp insert mesoderm was induced to adopt a more lateral fate. An cloned into Bluescript KS); BMP-2, PCR cloned fragment (nucleo- alternative direct response could alter cellular characteris- tides 1–797 [Francis-West et al., 1994]), gift from Professor Paul tics such as the rate of cell division or the induction of Brickell; BMP-4, PCR cloned fragment (nucleotides 1–953 [Francis- apoptosis, both of which could influence the formation of West et al., 1994]); and BMP-7, full-length fragment 1.1 kb, and epithelial somites. Alternatively, BMP could act indirectly Msx-2, 1-kb fragment, gift from Dr. Anthony Graham. The embryos were cryosectioned for further examination. by interferring with signaling molecules originating from the ectoderm and dorsal neural tube. A good candidate for a target molecule is Noggin, a secreted glycoprotein able to BrdU Labeling bind BMPs. This molecule has been implicated in the Twenty-five minutes before fixation, 100 ␮l of 40 mM 5-bromo- formation of the dermomyotomal lip through its ability to 2Ј-desoxyuridine (BrdU; Sigma) dissolved in destilled water was induce members of the Wnt gene family (Marcelle et al., applied onto the vitelline membrane and the embryos were rein- 1997). cubated at 37.8°C. Embryos were fixed overnight in Serra (60% In this study we have compared the response of the ethanol abs., 30% formaldehyde, 10% glacial acetic acid) at 4°C, medial and lateral mesodermal compartments to surgical dehydrated, embedded in paraffin, and sectioned (Serra, 1946). manipulations at both molecular and cellular levels. Our Antibody staining was preceded by hydrolysis for 30 min with 2 N study shows that lateral and paraxial mesoderm show HCl. Immunohistochemistry was performed by indirect immuno- dramatically different responses to tissue manipulations at peroxidase method with monoclonal anti-BrdU antibody (Dako) both molecular and cellular levels. Ectoderm ablation alone and peroxidase-conjugated goat anti-mouse IgG (Sigma) as second antibody. DAB was used as chromogen; a weak counterstaining causes an induction of BMP-4 together with an increase in was achieved with True red. the rate of apoptosis and decrease in the rate in cell division in the lateral plate mesoderm but not in the paraxial mesoderm. Even though epithelialization is prevented in BMP-4 Protein the paraxial mesoderm by ectoderm removal, induction of BMP-4 protein was obtained from Genetics Institute (MA). It BMP-4 and apoptosis only occurs after additional isolation was used at concentrations of 1.25–5 ␮g/ml. Heparin-coated acryl- of the neural tube. Neither manipulation in isolation or in amide beads of approximately 80 ␮m in diameter were obtained conjunction was able to induce BMP-2 or BMP-7 or the from Sigma (Deisenhofen, Germany). They were washed in PBS, lateral plate marker Msx-2. dried, and incubated with BMP-4 protein at 4°C for at least 30 min

before use. Control bead implantations were carried out in the same way except for the use of PBS as control substance.

Polycarbonate Filter The polycarbonate ﬁlter was obtained from Millipore (Eschborn, Germany). The ﬁlter used in this study had a pore size of 0.2 ␮m and a thickness of 10 ␮m.

Operations The following types of operations were carried out in ovo (Fig. 1): (A) The surface ectoderm overlying the paraxial mesoderm, the lateral plate, and the dorsal neural tube at the level of the segmental plate on one side of the embryo was removed (n ϭ 13). (B) In a number of specimens the removed ectoderm was re- placed by a 1-␮m-thick gold foil (n ϭ 12). (C) A polycarbonate ﬁlter (pore size 0.2 ␮m) was inserted between the neural tube/notochord and the caudal segmental plate extending over a length of four to eight prospective somites (n ϭ 11). (D) After removal of the surface ectoderm overlying the segmental plate and the lateral plate on one side of the embryo, a polycarbonate ﬁlter (pore size 0.2 ␮m) was inserted between the neural tube/notochord and the caudal part of the segmental plate (n ϭ 13). (E) Acrylamide beads soaked in BMP-4 protein were placed laterally to the segmental plate at caudal level (n ϭ 30). For controls, acrylamide beads with PBS were grafted in the same way (n ϭ 15). The operated specimens were incubated for 18–24 h.

Quantification Data obtained by TUNEL staining and BrdU incorporation were quantified using the Wilcoxon signed rank test at a 5% level. A defined area of 0.04909 mm2 at the operated and at the control side was evaluated in which the total cell number, BrdU-positive cell number, and TUNEL-positive cell number were counted. Sections from four to six different specimens were evaluated.

RESULTS Expression Pattern of BMP-4 in Normal Embryos In the trunk of 2-day chick embryos BMP-4 is expressed throughout the somatic and splanchnic mesoderm (Fig. 2A) FIG. 1. Operation scheme. (A) Ectoderm removal. (B) Ectoderm removal followed by covering the defect with gold foil. (C) Medial and in the mesenchymal portion of the mesonephric tissue. barrier insertion. (D) Medial barrier insertion and ectoderm re- At 2.5 days (HH stage 19), BMP-4 is strongly expressed in moval. (E) Implantation of BMP-4 or control beads. the limb bud mesenchyme and in the ectoderm overlying the neural tube (Figs. 2B and 2C). The dorsal neural tube also expresses BMP-4 (Fig. 2E), which is in line with reports in the literature (Liem et al., 1995). It is also still detectable was inhibited and the dermomyotome was absent (Figs. in the lateral plate mesoderm and mesonephric tissue (Fig. 2F–2H) or reduced in size. Removal of ectoderm over the 2D). BMP-4 is not expressed in the paraxial mesoderm or lateral plate resulted in a decrease in thickness of the lateral the Wolfﬁan duct. plate mesoderm (Figs. 2F, 2I, 2J). In situ hybridization revealed that BMP-4 expression in the Effect of Ectoderm Removal (Figs. 1A and 1B) lateral plate was upregulated after ectoderm removal, whereas After ectoderm removal in the caudal portion of the the BMP-4 expression pattern in the paraxial mesoderm was segmental plate, epithelialization of the paraxial mesoderm not altered after ectoderm removal in 21 of 25 embryos (Figs.

2M, 2N, 2O, 2Q). BMP-2 or -7 expression was never altered expression of BMP-4 in the paraxial tissue. However, after after surgery (data not shown). Furthermore, induction of ectoderm removal in combination with barrier implanta- BMP-4 expression was not accompanied by the induction of tion between the segmental plate and the axial organs, the lateral plate marker Msx-2 (data not shown). ectopic BMP-4 expression was induced in the paraxial TUNEL staining of specimens that had been reincubated mesoderm in 10 of 13 embryos (Figs. 3F and 3G). Expression for 24 h after ectoderm removal showed an increase in of BMP-2 or -7 on the other hand was not induced (data not number of apoptotic cells in the lateral plate mesoderm shown). Msx-2 expression was not altered by these manipu- (Figs. 2I, 2J, 2P), but no increase of apoptotic cells in the lations (data not shown). paraxial mesoderm (Figs. 2I, 2L, 2P). TUNEL staining showed that apoptosis now occurred Likewise, BrdU application showed a reduction of cells under these experimental conditions throughout the par- entering the S phase of the cell cycle in the lateral plate in axial mesoderm (Fig. 3J). This was paralleled by a complete comparison to the contralateral side and no alterations in absence of BrdU-labeled nuclei after an exposure of 25 min, the paraxial mesoderm (Fig. 2R). The described effects indicating a decrease in mitotic activity (Fig. 3H). These (TUNEL/BrdU) were seen in all embryos. effects (TUNEL/BrdU) were seen in all embryos. There was no significant difference in the total number of Effect of Barrier Insertion (Pore Size: 0.2 ␮m) cells on both sides at a 5% level in TUNEL and anti-BrdU between Axial Organs and Segmental Plate staining. When comparing numbers of apoptotic cells/ (Fig. 1C) BrdU-positive cells on the operation and the control side we find no significant difference in the operations after isolated After insertion of a polycarbonate membrane (pore size: ectoderm removal or barrier insertion. Only the combina- 0.2 ␮m) between the caudal segmental plate and the axial tion of ectoderm removal and barrier insertion leads to organs and subsequent reincubation, epithelialization of more apoptotic cells and to a decrease in BrdU-positive cells the paraxial mesoderm was inhibited and epithelial dermo- on the operation side compared to the control side (Fig. 4; myotomes were absent. Similar to the results after ecto- Tables 1 and 2). derm removal, in situ hybridization for BMP-4 did not reveal any changes in BMP-4 expression in 10 of 11 embryos; i.e., there was still no expression in the paraxial BMP-4 Bead Implantation (Fig. 1E) mesoderm (Figs. 3A and 3B). TUNEL staining showed that apoptosis did not occur In order to test whether the reported effects on the cell after this manipulation (Fig. 3E). BrdU application in ovo cycle were caused by BMP-4 or just independently accom- and subsequent analysis with anti-BrdU antibodies showed panied by an increase in BMP-4 expression, we applied no differences in the number of cells that entered the S heparin-coated beads soaked in BMP-4 at concentrations of phase of the cell cycle (Fig. 3C). The described effects 1.25–5 ␮g/ml lateral to the segmental plate. Using Pax-3 as (TUNEL/BrdU) were seen in all embryos. a marker for the epithelial somite, we found that somite size was decreased at the site of operation (Fig. 3K). The dermomyotomes were partially absent (Fig. 3L). The de- Combination of Barrier Insertion and Ectoderm scribed effect was seen in all embryos. Removal (Fig. 1D) TUNEL staining revealed in all cases the presence of Ectoderm ablation resulted in paraxial mesoderm unable apoptotic cells in the neighborhood of the bead and to a to form epithelial structures, but there was no ectopic lesser extent throughout the operated side in the paraxial as

FIG. 2. Whole-mount in situ hybridization for BMP-4 in normal chick embryos. (A) HH stage 15. (B) HH stage 19. (C) HH stage 19, detail at forelimb level. (D) Cross section through the caudal portion of a HH-19 chick embryo. There is no BMP-4 expression seen in the neural tube at this level. (E) Cross section through the forelimb level of a HH-19 chick embryo. At this level BMP-4 expression is seen in the neural tube. Cross sections of chick embryos after ectoderm removal from the right segmental plate and the lateral plate in combination with gold foil covering (F,G) or without gold foil (H); reincubation of 18 h and HE staining. (F and G) Gold foil covers the mesodermal compartments instead of surface ectoderm (arrow), the dermomyotome is missing. Asterisk: Wolffian duct. (H) No epithelium is found (arrowheads). (I–L) TUNEL staining shows an increase of apoptotic cells (darkly stained) in the lateral plate (arrow) and lateral portion of the paraxial mesoderm (arrowhead) after ectoderm removal (I,J). The dermomyotome is smaller (L) than on the control side (K). The extent of the dermomyotome is indicated by arrowheads. J, K, and L are details at higher magnification of I. (M) Whole-mount in situ hybridization for BMP-4 of a chick embryo after ectoderm removal over the right segmental plate and lateral plate. BMP-4 expression is stronger at the operation site (arrowheads). (N) Detail of the caudal part of the embryo in M. (O) Cross section of the embryo in M and N. Stronger BMP-4 expression is found in the lateral plate (covered with regenerated ectoderm) but not in the paraxial mesoderm on the operated side; an epithelium is missing (asterisk). (P–R) Cross section of a chick embryo after removal of surface ectoderm and covering with gold foil. TUNEL staining reveals more apoptotic cells in the lateral plate (arrow) at the operation site (P); in this area we see a stronger BMP-4 expression (Q). An enlarged Wolffian duct is found and the BMP-4 expression in its vicinity is lost (Q). Anti-BrdU staining shows no difference in proliferation between manipulated and control side (R, arrows: corresponding areas; asterisk: lateral plate).

Copyright © 1998 by Academic Press. All rights of reproduction in any form reserved. BMP-4 Expression Leads to Apoptosis 259 well as in the lateral plate mesoderm (Fig. 3M). These case of the paraxial mesoderm, survival factors capable of observations never occurred in controls, in which PBS- inhibiting BMP-4 expression are produced by both the soaked beads had been implanted under the same condi- ectoderm and the neural tube, either of which is sufﬁcient tions (Fig. 3N). to inhibit BMP-4 expression. Since the paraxial mesoderm responds to BMP-4 expression by decreasing mitotic levels and increasing apoptotic events, it seems that it is more DISCUSSION sensitive to BMP-4 than the lateral plate, which can express BMP-4 without displaying these cellular properties. We In the present study, we examined the effect of ectoderm suggest that there could be a gradient in BMP-4 responsive- removal on the paraxial and lateral plate mesoderm, as well ness with the most sensitive tissue located medially and as the effect of medial barrier insertion on the caudal the least sensitive situated more laterally. In addition, there segmental plate. In the paraxial mesoderm both manipula- might be a countergradient of survival factors. At present tions led to inhibition of epithelialization of the segmental we do not know the mechanism by which BMP-4 expres- plate and to an absence of epithelial dermomyotomes. sion is suppressed by ectoderm/neural tube; however, re- Reduction of epithelialization in the paraxial mesoderm cent work in both Drosophila and chicks may offer candi- also occurred after application of BMP-4. We therefore date pathways. In Drosophila signaling mediated by dorsal examined BMP-4 expression after ectoderm removal and leads to the inhibition of Dpp transcription. Dorsal is only barrier insertion by in situ hybridization. active when it translocates to the nucleus from the cyto- We demonstrate that ectoderm removal over the lateral plasm. Dorsal is prevented from entering the nucleus when plate mesoderm leads to an upregulation of BMP-4 expres- bound by Cactus. However, in response to external signals, sion. In the paraxial mesoderm, which normally does not Cactus is phosphorylated and ultimately degraded, releas- express BMP-4, expression of this gene is induced only after ing Dorsal to enter the nucleus and inhibit Dpp transcrip- a combined operation in which ectoderm was removed and tion (Huang et al., 1993; Schwyter et al., 1995; reviewed by a barrier inserted between axial organs and caudal segmen- Tickle, 1998). Recently Bushdid et al. (1998) and Kanegae et tal plate. In the lateral plate and in the paraxial mesoderm, al. (1998) have shown that the protein coded by the chick the number of apoptotic cells is increased after manipula- homolog of cactus (I-␬B) binds NF-␬B (chick homolog of tions that lead to upregulation of BMP-4. Since the appli- Dorsal) and this binding leads to lifting the inhibition of cation of exogenous BMP-4 protein also leads to local BMP-4 transcription. We suggest that factors produced by induction of apoptosis, we conclude that BMP-4 upregula- the dorsal neural tube and ectoderm could make somitic tion and apoptosis did not occur as independent effects of I-␬B labile to degradation, leading to repression of BMP-4 ectoderm removal and barrier insertion, but that apoptosis transcription. When both these tissues are removed I-␬B was caused by an increase in BMP-4 concentration. These forms a stable complex with NF-␬B and this lifts the results suggest that in the case of the lateral plate meso- repression on BMP-4 transcription. derm the ectoderm is the source of a survival signal and one In contrast to the induction of BMP-4, epithelialization of of the functions of this signal is to maintain BMP-4 expres- the segmental plate is inhibited by either ectoderm removal sion levels below those capable of inducing apoptosis. In the or barrier insertion alone. Since application of BMP-4 also

FIG. 3. (A) BMP-4 whole-mount in situ hybridization of a chick embryo after insertion of a polycarbonate membrane between neural tube/notochord and segmental plate; reincubation of 20 h. The polycarbonate membrane has been removed (arrow). (B) In cross section, there is no change in BMP-4 expression in the isolated paraxial mesoderm, although epithelial structures are missing. (C) Anti-BrdU staining of an embryo after the same operation as described in A. No difference in proliferation between manipulated (arrow) and control side. (D and E) Cross section of a chick embryo after insertion of a polycarbonate membrane, TUNEL staining, showing operated side and contralateral control side. No apoptotic cells are found in the paraxial mesoderm, although epithelial structures are missing (E). Control side showing a normal dermomyotome and sclerotome (D). (F) BMP-4 whole-mount in situ hybridization of a chick embryo after ectoderm removal and barrier insertion between neural tube/notochord and segmental plate. In the operation area no somites are found; a stronger BMP-4 expression is observed (arrowheads). (G) Cross section of the embryo shown in F reveals an ectopic BMP-4 expression (arrows) in the paraxial mesoderm. (H) Anti-BrdU staining shows a reduced proliferation rate in the paraxial mesoderm after ectoderm removal and isolation from the axial organs (arrows: corresponding areas). (I and J) Cross section of a chick embryo after removal of ectoderm overlying the segmental plate and barrier insertion between neural tube/notochord and segmental plate, showing operated and contralateral control side. TUNEL staining shows an increase of numerous apoptotic cells on the operated side (J); control side (I). The area of ectopic BMP-4 expression shown in G is comparable to the area with increased apoptosis shown in J. (K) Pax-3 in situ hybridization of a chick embryo after implantation of BMP-4 soaked beads lateral to the segmental plate. The somites next to the beads are reduced in size. (L) Transverse section of the embryo in (K) shows only a rudiment of the dermomyotome on the operated side (arrow). (M) TUNEL staining of a different embryo after BMP-4 bead implantation shows a strong increase of apoptotic cells (arrows) in the tissue surrounding the bead and even across the midline of the embryo. (N) Pax-3 in situ hybridization of a chick embryo after implantation of PBS-soaked beads lateral to the segmental plate. The somites next to the beads show no alteration in size.

FIG. 4. (A) Block diagrams showing the number of BrdU-positive cells and total cell number after ectoderm removal, barrier insertion, and combination of both operations. Operation side in comparison to control side. BrdU-positive cells are decreased in number only after the combined operation at the operated side. (B) Block diagrams showing the number of TUNEL-positive (apoptotic) cells and total cell number after ectoderm removal, barrier insertion, and combination of both operations. There is an increase in apoptotic cells at the operated side after the combined operation.

has the same result, it is possible that both these scenarios Wnts have recently been shown to induce dermomyotome are acting through a common pathway. Two BMP-4 binding markers in vitro (Fan et al., 1997). It has recently been shown proteins have been detected in the neural tube and somites that forced expression of Noggin has an inhibitory effect on (Hogan, 1996; Holley et al., 1996; Zimmerman et al., 1996). BMP-4 expression (Capdevila and Johnson, 1998). This prob- Follistatin is expressed in the neural tube and in the dorsal ably also accounts for our ﬁndings, since barrier insertion somitic compartment, whereas Noggin is expressed in the between axial organs and paraxial mesoderm shuts down dorsal neural tube and notochord (Connolly et al., 1995; Noggin. This results in ectopical BMP-4 expression obviously Amthor et al., 1996; Hogan, 1996). Interestingly, the only when additional signals from neural tube and/or ecto- somites also express a Follistatin-related gene called Flik, derm are also lacking. Hirsinger et al. (1997) showed that Wnt which is reliant on the neural tube for maintaining its expres- 1 induces Noggin in the dorsal somite, the protein of which sion (Amthor et al., 1996). It is not known whether the can bind and antagonize BMP-4. It has also been reported by expression of Flik is sensitive to ectoderm ablation. However, Takahashi (reviewed by Tajbakhsh and Spo¨rle, 1998) that since either neural tube isolation or ectoderm removal leads to Noggin can induce somites in lateral plate tissue, implying lack of epithelialization, it could be possible that a family of that Wnt 1 from the neural tube acts on epithelialization and genes exists whose members are present in both tissues. This mesoderm speciﬁcation indirectly through Noggin. It is not may implicate members of the Wnt family, since Wnt 1 and yet known if ectodermal Wnts have the same effect or Wnt 3a are both expressed in the neural tube and Wnt 4 and whether they act directly on the epithelialization of the Wnt 6 in the ectoderm of mice (Parr et al., 1993). All of these paraxial mesoderm.

TABLE 1 Anti-BrdU Staining

No. of Total cell number, BrdU-positive cells, Total cell number, BrdU-positive cells, Operation specimen operation side operation side control side control side

Ectoderm removal 1 240 64 230 60 2 230 62 242 62 3 234 60 250 62 4 250 64 236 62 5 232 60 240 60 6 254 64 242 62 Mean value Ϯ 240 Ϯ 9.9 62.3 Ϯ 2.0 240 Ϯ 6.7 61.3 Ϯ 1.1 standard deviation Barrier insertion 1 236 56 250 60 2 250 60 238 58 3 240 56 230 56 4 230 52 240 54 Mean value Ϯ 239 Ϯ 8.4 56 Ϯ 3.3 239.5 Ϯ 8.2 57 Ϯ 2.6 standard deviation Ectoderm removal 1 192 10 206 54 ϩ barrier insertion 2 200 10 196 52 3 214 12 210 60 4 206 14 200 54 Mean value Ϯ 203 Ϯ 9.3 11.5 Ϯ 1.9 203 Ϯ 6.2 55 Ϯ 3.4 standard deviation

Early steps of paraxial mesoderm epithelialization are that several independent pathways for apoptosis converge accompanied by the expression of paraxis and Pax-3, which at BMP-4. was used here as a marker of the epithelial somite and Previous work has shown that although paraxial, inter- dermomyotome. Both Pax-3 and paraxis expression is in- mediate, and lateral plate mesoderm ingress through hibited after ectoderm removal and isolation from the axial differing levels along the primitive streak this in itself is organs (Sosic et al., 1997). When a residual dermomyotome not sufficient to specify the fate of the tissues but was found in our operations, it was Pax-3 positive. There- additional extrinsic factors are required for this to be fore Pax-3 can be used as a marker of epithelialization. achieved (Vakaet, 1995). Ectopic expression of the BMP-4 Neither operation (ectoderm removal or segregation of antagonist Noggin at a similar craniocaudal level leads to medial tissue) in isolation or in combination led to the medialization and inhibits BMP-4 expression (Capdevila upregulation of BMP-2 or BMP-7 expression. Previous and Johnson, 1998). However, after surgical manipulation work in Xenopus has demonstrated that the signaling at the level of the segmental plate resulting in ectopic potential of the BMPs is increased by the utilization of BMP-4 expression, we were unable to detect signs of heterodimers as opposed to monomeric proteins (Suzuki lateralization (monitored by Msx-2 expression). These et al., 1997). These experiments suggest that in some results suggest that the fate of the mesoderm has been cases the responsiveness of the tissues may limit the fixed at the segmental plate level. This might be very choice of active proteins. The inability to induce numer- important since the embryo then seems to redeploy ous BMPs capable of forming highly active heterodimers members of the BMP family of proteins and their antago- could be a means of effecting a limited response. This nists (Noggin and Follistatin) for further patterning pro- could be an important feature, especially when inducing cesses within these compartments (Hogan, 1996). cellular responses such as apoptosis. In this context, it is Epithelialization of the paraxial mesoderm is con- also interesting that apoptosis was observed in sympa- trolled by signals from the axial organs and the ectoderm. thoadrenal progenitor cells after BMP-2 or BMP-4 appli- Both signals are necessary for epithelialization to occur. cation alone, at even lower doses than the ones used by us We conclude not only that BMP-4 is counteracted at (Song et al., 1998). Moreover, BMP-4 expression also protein level but also that its expression is influenced by leads to inhibition of cell proliferation and induction signals from the ectoderm and the axial organs. An of apoptosis in the limb bud (Ferrari et al., 1998). In upregulation of BMP-4 leads to apoptosis. It will be this study, BMP-4 was induced after ectopic expression extremely interesting to see how these findings correlate of Msx-2. In our investigation, we did not find any with the presence of BMP-4 receptors in different meso- evidence for Msx-2 involvement which could indicate dermal compartments.

TABLE 2 TUNEL Staining

No. of Total cell number, Apoptotic cells, Total cell number, Apoptotic cells, Operation specimen operation side operation side control side control side

Ectoderm removal 1 220 0 210 0 2 236 0 218 0 3 215 0 220 0 4 205 0 212 0 5 215 0 223 0 6 225 0 215 0 Mean value Ϯ 219 Ϯ 10.5 0 Ϯ 0 216.3 Ϯ 4.9 0 Ϯ 0 standard deviation Barrier insertion 1 173 0 166 0 2 167 3 172 3 3 180 1 193 1 4 179 0 200 0 5 185 1 195 1 6 200 0 210 0 Mean value Ϯ 180.7 Ϯ 11.3 0.83 Ϯ 1.17 189.3 Ϯ 16.9 0.83 Ϯ 1.17 standard deviation Ectoderm removal 1 205 59 195 1 ϩ barrier insertion 2 187 56 196 2 3 200 60 205 2 4 178 53 192 1 5 193 54 188 1 6 205 61 192 2 Mean value Ϯ 194.7 Ϯ 10.8 57.1 Ϯ 3.3 194.6 Ϯ 5.8 1.5 Ϯ 0.55 standard deviation

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