In Xenopus embryos, the extracellular matrix (ECM) protein tenascin (TN) is expressed dorsally in a very restricted pattern. We have studied the spatial and temporal expression of TN mRNA in tailbud-stage embryos by RNAase protection and in situ hybridization using a cDNA probe for Xenopus TN obtained by PCR amplification. We report that TN transcripts are principally expressed in cells dispersed around the neural tube and notochord as well as in myotome and sclerotome cells. No TN mRNA could be detected in lateral plate mesoderm, but expression was detectable beneath tail fin epidermis. In a second series of experiments, we studied the expression of TN mRNA and protein in combinations between animal and vegetal stage-6 blastomeres and in stage-8 blastula animal caps treated with activin A or basic fibroblastic growth factor (b-FGF). Isolated animal cap tissue cultured alone differentiates into epidermis, which expresses neither TN protein nor TN mRNA. TN expression is, however, elicited in response to isolated dorsal vegetal blastomeres and in response to high concentrations of activin, both of which treatments lead to formation of muscle and/or notochord. Low concentrations of activin, and ventral vegetal blastomeres, treatments that induce mesoderm of ventral character, are poor inducers of TN. However, b-FGF, which also induces ventral mesoderm, elicits strong expression. These results indicate that TN regionalization is a complex process, dependent both on the pattern of differentiation of mesodermal tissues and on the agent with which they are induced. The data further show that “ventral mesoderm” induced by low concentrations of activin is distinct from that induced by b-FGF, and imply that activin induces ventral mesoderm of the trunk while b-FGF induces posterior mesoderm of the tailbud.

Cell migration during morphogenesis and organogenesis is under strict spatial and temporal control. Several lines of evidence suggest that migration is regulated by interactions of cells with the extracellular matrix (ECM) (see reviews by Thiery, 1985; Bronner-Fraser, 1990; Johnson et al., 1990). These interactions take place between cell surface receptors and their ECM ligands, among which fibronectin has been shown to be of major importance (Yamada, 1983). In vitro, fibronectin promotes cell migration (Ali and Hynes, 1978; Heasman et al., 1981; Rovasio et al., 1983; Shi et al., 1989) and it is required in vivo for mesodermal cell migration during amphibian gastrulation as well as foravian neural crest cell migration (Boucaut et al., 1984a,b; Bronner-Fraser, 1985, 1986; Darribère et al., 1988, 1990; Poole and Thiery, 1986). Because of the widespread distribution of fibronectin during development, attention has recently turned to ECM-components which interfere with cell-adhesion to fibronectin and therefore might act to guide migrating cells along the correct pathways (Perris and Johansson, 1987; Chiquet-Ehrismann et al., 1988; Hoffman et al., 1988).

Tenascin (TN, Chiquet and Fambrough, 1984 a,b; Chiquet-Ehrismann et al., 1986), also called cytotactin (Grumet et al., 1985), has been shown to inhibit adhesion in vitro to purified fibronectin-substrata (Tan et al., 1987; ChiquetEhrismann et al., 1988; Halfter et al., 1989) and to inhibit in vivo amphibian mesodermal cell migration (Riou et al., 1990). It is a large oligomeric molecule (for review, see Chiquet-Ehrismann, 1990; Chiquet et al., 1991) which has been purified from different vertebrates including human, mouse, chick and Xenopus (Chiquet and Fambrough, 1984b; Erickson and Taylor, 1988; Friedlander et al., 1988, Riou et al., 1991; Weller et al., 1991). The expression pattern of TN is highly restricted during development. In mammalian fetuses, TN is found at sites of mesenchyme condensation during interactions with epithelia and during mammary gland, kidney and tooth organogenesis (ChiquetEhrismann et al., 1986; Aufderheide et al., 1987), as well as in condensing cartilage (Mackie et al., 1987). In the chick embryo, TN is deposited along peripheral nerve growth pathways and it promotes neurite outgrowth in vitro (Wehrle and Chiquet, 1990; Wehrle-Haller et al., 1991). TN is also present at tips of growing bronchi during lung organogenesis (Koch et al., 1991). During embryogenesis, TN is found in the ECM closely associated with neural crest cell-migration pathways of mammal, avian and amphibian embryos (Crossin et al., 1986; Epperlein et al., 1988; Mackie et al., 1988; Riou et al., 1988).

Little is known about the mechanisms that control TN accumulation within the ECM of highly defined regions of the embryo. In amphibian embryos, TN expression in the ECM begins after completion of neurulation (Epperlein et al., 1988; Riou et al., 1988), and remains confined to the dorsal region of the embryo throughout tailbud stages. Analysis of TN-distribution in Spemann-graft experiments has already suggested that TN-patterning, at least at the trunk level, is under the control of dorsal mesoderm (Riou et al., 1988). The mesoderm of amphibian embryos arises from the action of early inductive signals from vegetal blastomeres on animal blastomeres (for review, see Gurdon et al., 1989; Smith et al., 1989; Slack, 1991a). Increasing evidence has shown that these inductive signals are triggered by growth factors which belong to fibroblastic growth factor (FGF) and transforming growth factor-β (TGF-β) families (for review, see Slack et al., 1989; Jessel and Melton, 1992). Amphibian homologues of basic FGF (b-FGF) and activin A have been purified (Slack and Isaacs, 1989; Smith et al., 1990). In in vitro experiments, activin can induce isolated blastula animal cap cells to form dorsal and ventral mesoderm whereas b-FGF induces predominantly ventral mesoderm (Green et al., 1990). b-FGF (Slack and Isaacs, 1989) and recently activin (Asashima et al., 1991) activities have been detected in early cleavage stage embryos of Xenopus. Both activities are present in unfertilized eggs indicating that the inductive process might be supported by a maternal store. However, mesoderm patterning probably involves additional mechanisms. Recent results show that dorsal and ventral animal cap cells do not respond to activin or b-FGF identically, indicating that animal hemispheres are prepatterned (Sokol and Melton, 1991; Kimelman and Maas, 1992). The nature of the prepatterning signal is not known but might involve molecules of the Wnt family (Sokol et al., 1991; Smith and Harland, 1991; Christian et al., 1992).

In this paper, we have examined the spatial expression of TN transcripts in Xenopus tailbud-stage embryos by in situ hybridization. We show that TN mRNA is very abundant in paraxial mesoderm but undetectable in lateral plate mesoderm. Cells dispersed around notochord and neural tube also appear to transcribe TN, as do cells underlying tail fin epidermis. We go on to study TN expression in response to mesodermal induction. Isolated animal cap tissue cultured alone differentiates into epidermis, which expresses neither TN protein nor TN mRNA. TN expression is, however, elicited in response to isolated dorsal vegetal blastomeres and to high concentrations of activin, both of which treatments lead to formation of muscle and/or notochord. Low concentrations of activin, and ventral vegetal blastomeres, which induce mesoderm of ventral character, are poor inducers of TN. However, b-FGF, which also induces ventral mesoderm, elicits strong expression. These results indicate that TN regionalization is a complex process, dependent both on the pattern of differentiation of mesodermal tissues and on the agent with which they are induced. The data show that “ventral mesoderm” induced by low concentrations of activin is distinct from that induced by b-FGF, and suggest that activin induces ventral mesoderm of the trunk while b-FGF induces posterior mesoderm of the tailbud.

Polymerase chain reaction (PCR) amplification and cloning of a cDNA probe for Xenopus TN

PCR amplification was performed using single-stranded cDNA from XTC cells as a template. Briefly, total RNA was purified from XTC cells by the guanidium isothiocyanate method (Chirgwin et al., 1979). Poly (A)+ RNA was isolated by oligo(dT)-cellulose affinity chromatography and first-strand cDNA was synthesized using the “cDNA synthesis system plus” (Amersham). Two degenerate oligonucleotides were used as primers for PCR: X4: 5′ GGTCTCAGYTTCATYTC 3′ and X9: 5′ GACTCCAT-GASNTACCA 3′ (N is A, T, C or G; S is G or C; Y is C or T). They were designed to flank a 230 bp fragment of chick-TN cDNA located in the region encoding the C-terminal fibrinogen-like domain (Spring et al., 1989) which contains high amino acid identity with fibrinogen-like sequences of several invertebrate and vertebrate species (J. Spring, unpublished results). PCR amplification was carried out with the “GeneAmp DNA amplification reagent kit” (Perkin-Elmer/Cetus) including X4 and X9 at 1 μM. The thermal cycler (Perkin-Elmer/Cetus) was programmed for 40 cycles as follows: 94°C, 1 min; 42°C, 2 min; 72°C, 3 min. A single 230 bp PCR product was purified from an agarose gel stained with ethidium bromide and it was cloned into the EcoRV site of pBluescript KS+ (Stratagene). Sequencing of the amplified DNA fragment (XTn-230) was performed with a “Sequenase version 2” kit (United States Biochemicals).

Embryos, combinations of blastomeres and mesoderm induction assay

Xenopus embryos were obtained after mating of adults stimulated with human chorionic gonadotropin (hCG, Sigma). Hormone (350 units for the female and 150 units for the male) was injected into the dorsal lymph sac. Alternatively, synchronous embryos were obtained by artificial fertilization as described by Newport and Kirschner (1982). In all cases, the jelly coat was removed with 2.5% cysteine (pH 8.0) and embryos were cultured in 10% normal amphibian medium (NAM) (Slack, 1984) at a constant temperature of 23°C. Stages were according to Nieuwkoop and Faber (1967).

Combinations of blastomeres from 32-cell stage embryos (stage 6) were carried out according to Dale and Slack (1987). The dorsal half of embryos was identified by the lighter pigmentation of animal blastomeres. Combinations were cultured in 75% NAM containing 50 μg/ml gentamycin until control embryos reached stage 29/30.

Mesoderm induction assays with purified growth factors were done as described by Green et al. (1990). Inductions were carried out with human activin A (Genetech), Xenopus activin A purified from XTC-conditioned medium (Smith et al., 1990) or bovine bFGF (R & D Systems). The source of activin A will not be specified in the following since similar results were obtained with human or Xenopus activin A. Animal caps were dissected at the mid-blastula stage (stage 8) and immediately transferred to drops of 75% NAM containing 1 mg/ml bovine serum albumin (BSA, Sigma) and the appropriate growth factor. Activin A was used at 10 ng/ml, 0.4 ng/ml or 0.2 ng/ml, and b-FGF at 30 ng/ml or 5 ng/ml. Explants were incubated for 1 hour and then cultured in 75% NAM containing 50 μg/ml of gentamycin until control embryos reached the desired stage.

Antibodies

The production and specificity of IgGs against Xenopus TN have been reported previously (Riou et al., 1991). Tissue specificities of the monoclonal antibodies 12/101 and MZ15 for muscle and notochord respectively have been described elsewhere (Kintner and Brockes, 1984; Smith and Watt, 1985). Goat anti-rabbit IgGs conjugated to fluorescein isothiocyanate (FITC), and goat antimouse IgGs conjugated to tetramethylrhodamine isothiocyanate (TRITC), were purchased from Biosys.

Immunofluorescence and histology

Immunofluorescence was carried out after cryofixation in methanol and sectioning in PEG-400 distearate as previously described (Riou et al., 1988). Anti-Xenopus-TN IgGs were used at 20 μg/ml, MZ15 and 12/101 ascites were diluted at 1/200. FITC and TRITC conjugates were used at 5 μg/ml.

For histological analysis, explants were fixed in Smith’s fixative for 1 hour at room temperature and embedded in paraplast. Sections (10 μm) were stained with methyl green and pyronine.

RNAase protection assay

RNAase protection assays were carried out according to Krieg and Melton (1987). Total RNA from batches of 20 embryos or 30 animal cap explants was extracted by the “proteinase K/Lithium chloride method” (Kintner and Melton, 1987). Antisense RNA probes labelled with [α32P]UTP were obtained as follows: Plasmids pSP21 containing the cardiac actin probe (Mohun et al., 1984) and pSP72 containing the EF-1α probe (Krieg et al., 1989) were transcribed with SP6 polymerase after having been linearized with EcoRI and HinfI respectively. pXTn-230 was linearized with HindIII and transcribed with T7 polymerase. Embryonic RNA was hybridized with 2×105 counts/minute of XTn-230 and 2×104 counts/minute of EF-1α probes or with 2×105 counts/minute of cardiac actin probe alone. Hybridization was carried out for 36 hours at 45°C in 80% formamide, 0.4 M NaCl, 1 mM EDTA, 0.04 M Pipes. RNAase-protected fragments were analyzed on 6% acrylamide sequencing gels and revealed by autoradiography with Kodak X-Omat AR films.

In situ hybridization

Embryos were fixed overnight at 4°C in 4% paraformaldehyde in phosphate-buffered saline. After fixation, they were dehydrated in ethanol and embedded in paraplast. 10 μm sections were mounted on gelatin-coated slides. In situ hybridization was carried out according to Wilkinson and Green, (1990). 35S-antisense riboprobes were generated by T7 RNA polymerase using pXTn230 linearized with HindIII as template. The sense probe was obtained by T3 transcription of pXTn230 linearized with EcoRI. Hybridization was at 50°C. Hybridized sections were exposed to fresh NTB2 emulsion (Kodak) for 2 weeks at 4°C. Sections were counterstained with methyl green and pyronine. Silver grains were visualized using dark-field optics on a Leitz Dialux 20 microscope.

Expression of TN transcripts in mesodermal tissues of Xenopus embryos

A cDNA probe for Xenopus-TN, XTn-230, was generated by PCR amplification using cDNA from the XTC cell line as template and degenerate oligonucleotides as primers. The nucleotide sequence of XTn-230 is shown in Fig. 1A. It exhibits a strong homology (86%) with the published sequence of chick-TN cDNA (Spring et al., 1989). Conservation of the predicted amino acid sequence (Fig. 1B) is even higher (93%). XTn-230 was hybridized to northern blots of total RNA from both XTC cells and Xenopus embryos at the tailbud stage 35/36. A strong signal was observed at the level of a band corresponding to a transcript size of 8.5 kb (data not shown). This is similar to that reported for chick-TN and mouse-TN mRNA (Pearson et al., 1988; Jones et al., 1989; Weller et al., 1991).

Fig. 1.

Sequence comparison between XTn-230 (X) and chick TN (C). (A) Nucleotide sequence. Identical nucleotides are underlined. Sequences corresponding to the primers used for PCR amplification are indicated in lower case. XTn-230 nucleotide sequence has 86% homology with chick-TN cDNA sequence. (B) Deduced amino acid sequence. Identical amino acids are underlined. Amino acid identity is 93%.

Fig. 1.

Sequence comparison between XTn-230 (X) and chick TN (C). (A) Nucleotide sequence. Identical nucleotides are underlined. Sequences corresponding to the primers used for PCR amplification are indicated in lower case. XTn-230 nucleotide sequence has 86% homology with chick-TN cDNA sequence. (B) Deduced amino acid sequence. Identical amino acids are underlined. Amino acid identity is 93%.

Detection of mRNA encoding TN was carried out by RNAase protection analysis. A 311 base T7-promoted antisense RNA probe was transcribed from XTn230. Fig. 2 (lanes 1,10) shows that hybridization with total RNA from XTC cells led to the protection of about 230 bases of the probe whereas this fragment is absent from controls with tRNA alone. Analysis of embryonic total RNA for TN transcripts was performed at different stages including blastula (stage 9), gastrula (stage 12), neurula (stages 14, 15, 17,19) and tailbud (stages 25, 28) (Fig. 2, lanes 2-9). An EF1α antisense RNA probe was hybridized simultaneously to confirm that intact mRNA was present in every sample. Results from two separate experiments show that TN mRNA is first detectable at the neurula stage 14. The 230 base-protected fragment of the TN-probe is absent at the earlier stages studied although the 94 base EF-1α band indicates that comparable quantities of mRNA were present during hybridization. A strong RNAase-resistant band for TN is produced at stages when TN-polypeptides are integrated into the ECM (Epperlein et al., 1988).

Fig. 2.

Appearance of TN mRNA during embryogenesis. RNAase protection analysis. For each embryonic stage, total RNA from 20 embryos is hybridized with the antisense RNA probes XTn-230 and EF-1α. A high background is present because low concentrations of RNAase were used to obtain maximal detection of TN transcripts. Hybridization with XTn-230 generates a 230 base protected fragment which is not present on control with tRNA alone. EF–1α mRNA protects 94 bases of the antisense probe. Lane1: negative control with tRNA alone. Lanes 2–9: embryo RNA. Lane 2: mid-blastula stage (stage 8). Lane 3: late gastrula stage (stage 12). Lanes 4–7: neurula stages 14 (lane 4), 15 (lane 5), 17 (lane 6) and 19 (lane 7). Lanes 8, 9: middle (stage 25) and late (stage 28) tailbud stages. Lane 10: positive control including total RNA from XTC cells (this sample was only hybridized with XTn-230). TN transcripts begin to be present at the early neurula stage-14 at a low level. They are strongly revealed at tailbud stages. Detection of EF-1α mRNA shows that comparable quantities of mRNA were included in each experiment.

Fig. 2.

Appearance of TN mRNA during embryogenesis. RNAase protection analysis. For each embryonic stage, total RNA from 20 embryos is hybridized with the antisense RNA probes XTn-230 and EF-1α. A high background is present because low concentrations of RNAase were used to obtain maximal detection of TN transcripts. Hybridization with XTn-230 generates a 230 base protected fragment which is not present on control with tRNA alone. EF–1α mRNA protects 94 bases of the antisense probe. Lane1: negative control with tRNA alone. Lanes 2–9: embryo RNA. Lane 2: mid-blastula stage (stage 8). Lane 3: late gastrula stage (stage 12). Lanes 4–7: neurula stages 14 (lane 4), 15 (lane 5), 17 (lane 6) and 19 (lane 7). Lanes 8, 9: middle (stage 25) and late (stage 28) tailbud stages. Lane 10: positive control including total RNA from XTC cells (this sample was only hybridized with XTn-230). TN transcripts begin to be present at the early neurula stage-14 at a low level. They are strongly revealed at tailbud stages. Detection of EF-1α mRNA shows that comparable quantities of mRNA were included in each experiment.

In situ hybridization analyses of the tissue distribution of TN mRNA in tailbud stage 29/30 embryos are shown in Fig. 3. TN transcripts are present throughout the somitic mesoderm. Comparing the antisense probe with the sense, it is clear that TN mRNA is detected in high amounts in myotome and sclerotome cells (Fig. 3A, B, C). TN is also transcribed in cells dispersed around the neural tube and the notochord. This is clearly seen in truncal and posterior transverse sections (Fig. 3D, E, F). Although TN transcripts are not apparent in the lateral mesoderm, signals could be observed in the pericardial mesoderm (Fig. 3A). In posterior sections, TN transcripts are detectable in cells close to the postanal gut and in the inner layer of the fin epidermis (Fig. 3F).

Fig. 3.

Expression of TN transcripts in tailbud stage-29/30 embryos. Transverse sections. (A, B, D, E, F) Hybridizations with the antisense probe. (C) Control hybridization with the sense probe. (A, B, C) Anterior truncal level; (A) heart primordium region; (B, C) level posterior to A. TN transcripts are principally detected in the somitic mesoderm. Transcription of TN mRNA also occurs at a lower level in the pericardial mesoderm (arrowheads). No hybridization is observed in the sense control. (D, E, F) Posterior levels : high levels of TN mRNA are expressed in somites and in cells dispersed around neural tube and notochord (arrowheads). TN transcripts are also detected in the basal layer of the tailfin epidermis (arrows) and in cells close to the postanal gut (large arrowhead). No hybridization can be seen in lateral plate mesoderm. Scale bar = 250 μm.

Fig. 3.

Expression of TN transcripts in tailbud stage-29/30 embryos. Transverse sections. (A, B, D, E, F) Hybridizations with the antisense probe. (C) Control hybridization with the sense probe. (A, B, C) Anterior truncal level; (A) heart primordium region; (B, C) level posterior to A. TN transcripts are principally detected in the somitic mesoderm. Transcription of TN mRNA also occurs at a lower level in the pericardial mesoderm (arrowheads). No hybridization is observed in the sense control. (D, E, F) Posterior levels : high levels of TN mRNA are expressed in somites and in cells dispersed around neural tube and notochord (arrowheads). TN transcripts are also detected in the basal layer of the tailfin epidermis (arrows) and in cells close to the postanal gut (large arrowhead). No hybridization can be seen in lateral plate mesoderm. Scale bar = 250 μm.

N transcripts are expressed in Xenopus animal caps in response to mesodermal induction

The above results show that TN transcripts are mainly expressed in somitic mesoderm and in presumptive axial mesenchyme of tailbud stage 29/30 Xenopus embryos. We have gone on to do RNAase protection analyses to test whether transcription of the TN-gene occurs in vitro, in response to mesodermal induction by purified growth factors. Animal caps isolated from blastula stage-8 embryos were incubated with activin A (10 ng/ml or 0.2 ng/ml) or b-FGF (30 or 5 ng/ml) and cultured until control embryos reached stage 30. For each sample, one sixth of the RNA was processed for a parallel RNAase protection with the cardiac actin probe for the presence of muscle. This probe hybridizes with both cardiac actin mRNA and with cytoskeletal mRNA, thus generating two different protected fragments of 250 bases and 130 bases respectively (Mohun et al., 1984). The presence of the muscle-specific 250 base fragment provides information about the type of the mesoderm induced. Comparison of the intensity of the 130 base fragment specific for cytoskeletal actin allows one to check that similar quantities of total RNA were included in each sample.

TN gene activation occurs as a response to induction by activin A and b-FGF. Fig. 4A shows that the TN-specific 230 base RNAase-resistant fragment is produced in explants induced with activin A at 10 ng/ml as well as in those treated with b-FGF at 30 or 5 ng/ml. Conversely, no TN transcript is detectable in control explants or in explants incubated with activin A at 0.2 ng/ml. Identical results were obtained in three independent experiments. Parallel analyses with the cardiac actin probe (Fig. 4B) show that 130 base bands of similar intensities were generated for each sample indicating that comparable amounts of mRNA were analyzed. As expected, a strong 250 base band is observed for explants induced with activin A at 10 ng/ml which is absent when explants were treated with activin A at 0.2 ng/ml. Cardiac actin mRNA was detected once in three analyses after induction by b-FGF at 30 ng/ml (not shown) but was never present in explants induced with b-FGF at 5 ng/ml.

Fig. 4.

Expression of TN transcripts in response to mesodermal induction by purified growth factors. RNAase protection analysis. In each case, total RNA from 30 animal cap explants was extracted. Five sixths were hybridized with XTn-230 antisense RNA probe and one sixth with the cardiac actin antisense probe. Cardiac actin mRNA protects a 250 base fragment of the probe while cytoskeletal actin mRNA generates a 130 base band. The former shows the induction of muscle in explants.(A) Expression of TN transcripts;(B) Expression of TN transcripts;Lanes 1, 2: inductions with activin A at 10 ng/ml (lane 1) or 0.2 ng/ml (lane 2). Lanes 3, 4: inductions with b-FGF at 30 ng/ml (lane 3) or 5 ng/ml (lane 4). Lane 5: control uninduced animal caps. Although the activation of the TN gene in response to activin A is observed in parallel to the induction of muscle, TN transcripts are revealed in explants induced by b-FGF where cardiac actin mRNA is not detected. The intensity of the 130 base bands specific for cytoskeletal actin indicate that comparable quantities of mRNA were present for each experiment.

Fig. 4.

Expression of TN transcripts in response to mesodermal induction by purified growth factors. RNAase protection analysis. In each case, total RNA from 30 animal cap explants was extracted. Five sixths were hybridized with XTn-230 antisense RNA probe and one sixth with the cardiac actin antisense probe. Cardiac actin mRNA protects a 250 base fragment of the probe while cytoskeletal actin mRNA generates a 130 base band. The former shows the induction of muscle in explants.(A) Expression of TN transcripts;(B) Expression of TN transcripts;Lanes 1, 2: inductions with activin A at 10 ng/ml (lane 1) or 0.2 ng/ml (lane 2). Lanes 3, 4: inductions with b-FGF at 30 ng/ml (lane 3) or 5 ng/ml (lane 4). Lane 5: control uninduced animal caps. Although the activation of the TN gene in response to activin A is observed in parallel to the induction of muscle, TN transcripts are revealed in explants induced by b-FGF where cardiac actin mRNA is not detected. The intensity of the 130 base bands specific for cytoskeletal actin indicate that comparable quantities of mRNA were present for each experiment.

Accumulaton of TN in animal cap ECM after mesodermal induction

The above results show that activation of the TN gene occurs in animal cap cells as a response to mesodermal induction. In caps treated with activin, high levels of TN mRNA coincide with the expression of muscle actin transcripts, in agreement with the high levels of TN RNA in the somitic mesoderm of stage 29/30 embryos. However, in caps treated with b-FGF, strong induction of TN mRNA was observed without detectable activation of the muscle actin gene. To investigate this further, we have studied the distribution of TN protein in animal cap tissue in response to mesodermal induction by purified growth factors or vegetal blastomeres.

In the first series of experiments, we concentrated on inductions where high levels of somitic mesoderm were expected to form: combinations of tier A blastomeres from 32-cell-stage embryos with the dorsovegetal blastomere D1, or treatment of blastula stage-8 animal caps with activin A at 10 ng/ml. Table 1 summarizes the results obtained in double immunostaining experiments with anti-TN IgGs and marker antibodies for muscle (12/101) or notochord (MZ15) differentiation. Strong TN reactivity was present in 12 of the 25 combinations of tier A blastomeres with D1. Eleven of these contained muscle tissue (Fig. 5A) while none of the 13 TN-negative explants reacted with 12/101. As expected from RNAase protection experiments, TN expression was not detected in tier A blastomeres cultured alone or in untreated animal caps (Fig. 5B). Expression of TN was induced in all animal cap explants treated with activin A at 10 ng/ml. Reactivity for TN was limited to the extracellular matrix (Fig. 5C,E). It was observed in the ECM surrounding notochord (Fig. 5D) as well as at the periphery of blocks of striated muscle (Fig. 5F). ECM fibrils containing TN were also present under epidermis and in mesenchyme. Analysis of MZ15 reactivity shows that TN expression does not require the induction of notochord. Only 9 of the 27 explants studied contained notochord while all of these expressed TN. Conversely, induction of TN expression by activin A was always observed when muscle differentiation occurred. Forty explants induced with activin A at 10 ng/ml were processed for double immunostaining with anti-TN IgGs and 12/101. All were positive for both TN and muscle.

Table 1.

Expression of TN in animal cap tissue after induction of dorsal mesoderm

Expression of TN in animal cap tissue after induction of dorsal mesoderm
Expression of TN in animal cap tissue after induction of dorsal mesoderm
Fig. 5.

Expression of TN in animal cap tissue ECM after induction of dorsal mesoderm. (A) Combination of tier A blastomeres with the dorsovegetal blastomere D1. Double immunostaining with anti-TN IgGs and 12/101. TN (green staining) and muscle (red staining) patterns are superimposed. TN is revealed in ECM fibrils deposited in the vicinity of muscle cells reactive to 12/101. Reactivity to antiTN IgGs is also observed in the ECM (arrowhead) lining a vacuolated tissue which is probably notochord. (B) Control with tier A blastomeres cultured alone. No reactivity for the anti-TN IgGs is observed. (C-F) Induction with activin A at 10 ng/ml. (C,D) Double immunostaining with anti-TN IgGs (C) and MZ15 (D). TN is strongly revealed in the ECM of the explant. The detection of notochord tissue by MZ15 shows the induction of dorsal mesoderm. (E, F) Double immunostaining with anti-TN IgGs (E) and 12/101 (F). TN is present in the ECM deposited at the periphery and in the septa of a muscle block revealed by 12/101. Mus, muscle; Nt, notochord. Scale bar = 50 μm.

Fig. 5.

Expression of TN in animal cap tissue ECM after induction of dorsal mesoderm. (A) Combination of tier A blastomeres with the dorsovegetal blastomere D1. Double immunostaining with anti-TN IgGs and 12/101. TN (green staining) and muscle (red staining) patterns are superimposed. TN is revealed in ECM fibrils deposited in the vicinity of muscle cells reactive to 12/101. Reactivity to antiTN IgGs is also observed in the ECM (arrowhead) lining a vacuolated tissue which is probably notochord. (B) Control with tier A blastomeres cultured alone. No reactivity for the anti-TN IgGs is observed. (C-F) Induction with activin A at 10 ng/ml. (C,D) Double immunostaining with anti-TN IgGs (C) and MZ15 (D). TN is strongly revealed in the ECM of the explant. The detection of notochord tissue by MZ15 shows the induction of dorsal mesoderm. (E, F) Double immunostaining with anti-TN IgGs (E) and 12/101 (F). TN is present in the ECM deposited at the periphery and in the septa of a muscle block revealed by 12/101. Mus, muscle; Nt, notochord. Scale bar = 50 μm.

In a second set of experiments, we studied expression of TN in cases where ventral mesoderm was induced: combinations of tier A blastomeres with the ventrovegetal blastomere D4, and treatment of animal caps with low concentrations of activin (0.2 or 0.4 ng/ml) or with b-FGF (5 or 30 ng/ml). Because there are few markers of ventral mesoderm differentiation, a parallel histological analysis of differentiated tissues was carried out. For each batch of explants, one half was fixed when controls were at stage 29/30 while the remainder were cultured until controls were at stage 40 and then processed for histology. Differentiated explants were classified according to the histological grading of Slack (1991b). Only series containing a high frequency of inductions of ventral character (grade 1 or 2) were selected for immunohistochemical analysis. Examples of the most frequent cases encountered for each kind of induction are shown in Fig. 6. Accumulation of TN in the ECM was observed in each experimental series. In explants negative for 12/101, TN was principally observed in the subepidermal ECM. Expression of TN in fibrils organized around blocks of cells reacting with 12/101 only occurred occasionally. Interestingly, the frequency of TN expression in the absence of 12/101 staining was much higher in explants induced with b-FGF than in A/D4 combinations or activin inductions. These observations are summarized in Table 2. They agree well with the results of RNAase protection experiments. The absence of detection of TN transcripts in explants induced with activin at 0.2 ng/ml by RNAase protection is probably a result of the low frequency of TN induction and may also be due to low levels of TN mRNA expression. Conversely, the expression of TN mRNA without detectable cardiac actin mRNA in response to b-FGF is corroborated to the high frequency of TN expression observed in explants that do not contain 12/101positive tissue.The difference observed is not due simply to the higher proportion of inductions seen in response to bFGF compared with activin or D4 blastomeres. If the numbers of explants expressing TN at stage 29/30 are expressed as a proportion of the explants scored by histological criteria as induced, TN expression is seen to occur in 75% of explants treated with b-FGF and in only 36% and 44% in response to activin and D4 blastomeres, respectively.

Table 2.

Expression of TN in animal cap tissues after induction of mesoderm of ventral character

Expression of TN in animal cap tissues after induction of mesoderm of ventral character
Expression of TN in animal cap tissues after induction of mesoderm of ventral character
Fig. 6.

Expression of TN in the ECM of explants where mesoderm of ventral character was induced. Double immunostainings with antiTN IgGs and 12/101. (A, C, E) Phase contrast pictures of the explants. Boxes indicate the areas shown in B, D, F respectively. (B, D, F) Reactivity to anti-TN IgGs. 12/101 was negative in all cases (not shown). (A, B) Stage-6 blastomere combination A/D4. (C, D): Blastula stage-8 animal cap explant treated with activin A at 0.2 ng/ml. (E, F) Stage-8 animal cap treated with b-FGF at 5 ng/ml. TN is strongly revealed in the subepidermal ECM of the explant treated with b-FGF (arrowhead) but is absent in the two other explants. Epi, doublelayered epidermis. Scale bars (A, C, E) = 100 μm, (B, D, F) = 100 μm.

Fig. 6.

Expression of TN in the ECM of explants where mesoderm of ventral character was induced. Double immunostainings with antiTN IgGs and 12/101. (A, C, E) Phase contrast pictures of the explants. Boxes indicate the areas shown in B, D, F respectively. (B, D, F) Reactivity to anti-TN IgGs. 12/101 was negative in all cases (not shown). (A, B) Stage-6 blastomere combination A/D4. (C, D): Blastula stage-8 animal cap explant treated with activin A at 0.2 ng/ml. (E, F) Stage-8 animal cap treated with b-FGF at 5 ng/ml. TN is strongly revealed in the subepidermal ECM of the explant treated with b-FGF (arrowhead) but is absent in the two other explants. Epi, doublelayered epidermis. Scale bars (A, C, E) = 100 μm, (B, D, F) = 100 μm.

The present work was undertaken to provide data about processes that determine the deposition of TN in the ECM of highly defined regions of the Xenopus tailbud stage embryo. In order to identify which embryonic tissues are synthesizing TN, we have produced a cDNA probe for Xenopus TN and have studied the distribution of TN transcripts in stage-30 embryos by in situ hybridization. We report that transcription of TN mRNA is prominent in cells dispersed around the neural tube and notochord as well as in myotome and sclerotome cells, but that no TN transcripts are detectable in lateral plate mesoderm. We have therefore gone on to investigate whether TN transcripts or protein can be induced in vitro in response to mesodermal induction, in order to provide information about TN expression in the context of defined subsets of differentiating mesodermal tissues. We show that strong expression of TN transcripts and protein is detected in animal/vegetal blastomere combinations as well as in animal caps treated with activin A or b-FGF, while animal pole tissue cultured in the absence of inducer never expresses TN. Interestingly, strong expression of TN is not only observed in situations where high amounts of somitic mesoderm are induced but also when the induced mesoderm is mostly of ventral character. In such cases, TN expression is poorly induced in animal/vegetal combinations and in response to activin, but is much more strongly induced by b-FGF.

It is striking that the distribution of TN transcripts in the tailbud stage embryo coincides with the distribution of the protein in the ECM (Epperlein et al., 1988). This is obvious at the level of somites. The densest TN-containing ECM is deposited in intersomitic furrows and transcription of TN mRNA is strong in the somitic mesoderm. However, we failed to detect TN transcripts in the developing central nervous system although TN is present in the ECM around the neural tube. This observation agrees well with the reported absence of TN transcripts in the neural tube of chicken embryos before day 8 (Prieto et al., 1990). We cannot rule out that low levels of TN mRNA are present in the neural primordium but it seems more likely that TN in the surrounding ECM is produced by neighbouring tissues. A comparable situation has been reported for mouse mammary tumors where stromal TN is synthesized by the surrounding mesenchyme (Inaguma et al., 1988). TN deposited in the vicinity of the neural tube might be synthesized by somites, as well as by dispersed cells that were strongly labelled by the antisense TN probe. The identity of these latter cells is unclear, and it is possible that they represent a heterogeneous population. Some may be axial mesenchyme liberated from the sclerotomic portion of somites. Others may derive from the neural crest. In this respect, it is noteworthy that neural crest cells are a major source of TN in the chicken embryo (Mackie et al., 1988; Tucker and McKay, 1991).

Besides putative neural crest cells and cells forming the basal layer of the tailfin epidermis, in situ hybridization shows that mesodermal derivatives make a significant contribution to TN synthesis. It is therefore likely that TN regionalization in the tailbud stage embryo relies to a large extent on the spatiotemporal pattern of mesoderm differentiation. Results obtained in in vitro mesoderm induction assays agree well with this hypothesis. It is striking that activin A inductions mimic the dorsoventral distribution of TN observed in the trunk region of tailbud stage embryos. Depending on the concentration of activin, we can observe the presence of TN in the ECM of explants where notochord and somitic muscle are differentiating whereas explants induced with low concentrations of activin A, which mostly contained mesoderm of ventral character, expressed TN only weakly. The situation is quite different for similar experiments performed with b-FGF since high levels of TN expression occurred in explants where no muscle differentiation could be observed. It is striking that TN was principally distributed in the subepidermal ECM of these explants. In the embryo, such a situation is observed under the tailfin epidermis. Our interpretation of the differences observed between activin A and b-FGF induction experiments is that animal cap tissue is induced by b-FGF to form posterior mesoderm while it responds to low concentrations of activin by differentiating into ventral mesodermal cell types of the trunk. In support of this are results showing that animal cap cells treated with these growth factors possess the properties of Spemann’s organizer, but those incubated with activin A induce the formation of secondary heads and trunks while those treated with b-FGF only produce additional tails (Ruiz i Altaba and Melton, 1989a). Moreover, b-FGF preferentially activates the genes Xhox 3, XlHbox 6 and Xpo which are normally expressed in the posterior region of Xenopus embryos (Ruiz i Altaba and Melton, 1989b; Cho and De Robertis, 1990; Sato and Sargent, 1991). Finally, inhibition of endogenous FGF receptor function in early Xenopus embryos has recently been shown to reduce posterior development (Amaya et al., 1991). It is therefore tempting to propose that mesodermal induction might specify the regionalization of TN expression in the embryo along both dorsoventral and anteroposterior axes.

The expression of fibronectin (FN) during amphibian gastrulation has been extensively studied (for review, see Boucaut et al., 1991; DeSimone et al., 1991). In early amphibian embryos, fibronectin provides an example of the regionalized deposition of an ECM component whose control differs from that of TN. FN synthesis relies on the activation of maternally-stored mRNA; it is secreted both by animal and vegetal cells of the blastula but its ECM distribution is restricted to the blastocœl roof (Boucaut and Darribère, 1983; Lee et al., 1984; Nakatsuji et al., 1985; Darribère et al., 1988; 1990). By contrast, our results suggest that the mechanisms regulating TN regionalization are likely to involve TN gene activation since synthesis of TN in the embryo is regionalized and is elicited in vitro by mesodermal induction. With respect to this, it is noteworthy that the 5′ end of chicken TN gene contains different putative regulatory sequences including six engrailed-like regulatory elements (Jones et al., 1990). Although the product of the Xenopus gene En-2 (which is related to Drosophila engrailed) is only expressed in the developing anterior nervous system (Hemmati-Brivanlou et al., 1990; 1991), regulation of TN expression by homeoproteins is possible. However, the strong expression of TN mRNA at the level of myotomes, along with the close correlation observed between the expressions of TN and muscle differentiation markers in response to mesodermal induction in vitro, raise the question of whether myogenic factors like XMyoD or XMyf-5 might activate the TN gene on the same way as they do for the cardiac actin gene (Hopwood and Gurdon, 1990; Hopwood et al., 1991).

In conclusion, we have shown that the distribution of TN protein in the ECM of tailbud stage embryos coincides with that of TN mRNA. This indicates that expression of TN in defined regions of the embryo is regulated, at least in part, by transcriptional control and not exclusively by the regionalized expression of an ECM ligand(s), as occurs with FN. This transcriptional control is related to the differentiation pattern of mesodermal tissues. The study of TN expression in animal cap cells in response to various mesodermal inducers indicates that the coordinated synthesis of TN by several mesodermal tissues at specific sites along the dorsoventral and anteroposterior axes of the embryo might be determined by early cellular interactions that specify the basic body plan. These expression domains might later be involved in defining migration routes of neural crest cells (Epperlein et al., 1988).

We would like to thank Dr D. W. DeSimone for invaluable advice concerning RNAase protection and Drs Shi, Delarue and Darribère for stimulating discussion. We are grateful to Dr H. Condamine for help with dark-field microscopy. We thank P. Groué, J. Desrosiers and C. Arnoult for excellent technical assistance as well as M. M. Trân Kim Lan for illustrations and typing the manuscript. This research has been supported by grants from CNRS, MEN, AFM and ARC.

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