Umbhauer M et al. (1992), Expression of tenascin mRNA in mesoderm during ...

XB-ART-23441

Development 1992 Sep 01;1161:147-57.

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Expression of tenascin mRNA in mesoderm during Xenopus laevis embryogenesis: the potential role of mesoderm patterning in tenascin regionalization.

Umbhauer M , Riou JF , Spring J , Smith JC , Boucaut JC .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a inhba mt-tr tnc trna
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	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. 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-1a. 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-1a 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-1a mRNA shows that comparable quantities of mRNA were included in each experiment.
	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 mm.
	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 actin 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. 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 anti- TN 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 mm.
	Fig. 6. Expression of TN in the ECM of explants where mesoderm of ventral character was induced. Double immunostainings with anti- TN 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 mm, (B, D, F) = 100 mm.