Epperlein HH et al. (1988), The distribution of fibronectin and tenascin al...

XB-ART-27386

Development 1988 Aug 01;1034:743-56.

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The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos.

Epperlein HH , Halfter W , Tucker RP .

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It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)

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Species referenced: Xenopus laevis
Genes referenced: fn1 sst.1 tnc

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	Fig. 1. Western blots of purified chicken tenascin (lane A) and a crude Xenopus extract (lane B) with the polyclonal anti- TN. The anti-TN specifically recognizes a broad band that comigrates with chicken TN and a slightly lower band in the Xenopus preparation. Preimmune serum did not stain the Xenopus extracts (lane C).
	Fig. 2. Timing and routes of trunk neural crest cell migration in Xenopus using the X. laevis-X. borealis marking system. (A) Labelled {=borealis) cells (arrows) were found at stage 23 in the wedge between dorsal somites and neural tube. Note that the neural tube contains exclusively labelled cells. (B,C) At stage 32, labelled cells (arrows) were present on the ventromedial pathway between somites and neural tube (B) or between somites and notochord or endoderm (C). The inset in B shows a labelled nucleus at high magnification adjacent to laevis nuclei. (D-F) At stage 41, many more labelled cells were found on the ventromedial pathway. (D) borealis cells (arrows) between myotomes and notochord in a partial enlargement of the sagittal section shown in F. Notice labelled cells in the neural tube. (E) borealis cells at the ventral edge of the somites on a transverse trunk section of a different stage-41 larva. som, somites; nt, neural tube; not, notochord; en, endoderm; epi, epidermis; my, myotomes; pd, pronephric duct. Bars, A-C: 50um; D,E: 50um; F: 200um
	TNFig. 3. Anti-fibronectin and anti-tenascin staining of transverse sections through the anterior trunk region of X. laevis embryos before neural crest cell migration (stage 22). (A,B (enlarged)) Anti-fibronectin stains the extracellular spaces separating the major tissue blocks. It is particularly intense in the dorsal part of the embryo. (C) Anti-tenascin staining is limited to the borders of the somite mesoderm in the anterior trunk, nt, neural tube; not, notochord; som, somites; en, endoderm; vise and par, visceral and parietal layer of the lateral plate mesoderm. Bars, A: 200um; B,C: 50um.
	OSFig. 4. Anti-fibronectin and anti-tenascin staining of transverse sections through the anterior trunk region of axoloti embryos before neural crest cell migration (stage 25). (A,B (enlarged)) As in Xenopus, anti-FN staining is found in all of the major extracellular spaces. (C) No staining was observed with anti-TN. Abbreviations as in Fig. 3. Bars, A: 200um; B: 50um; C: 200um.
	Fig. 5. Anti-fibronectin and anti-tenascin staining of frontal and transverse sections (trunk region) of stage 29/30 Xenopus embryos during early neural crest migration. (A) Frontal section: FN is present subepidermally, in the intersomitic furrows and adjacent to the notochord. (B) Frontal section: at the same level of the notochord TN is present mainly in the intersomitic furrows and very weakly at the notochord itself. Anti-TN staining is more intense anteriorly. (C) Transverse section: anti-FN stains the ECM throughout the embryo; nt, neural tube. (D) Transverse section: anti-TN stains mainly the ECM in the myosepta. There is only very weak staining around the neural tube (nt) and notochord. (E,F) Frontal sections at the level of the neural tube, nt: FN is present in all extracellular spaces (E), whereas TN is limited to intersomitic and anterior somite areas (F). cr, cranial; ca, caudal. Bars, A-F: 50 um.
	Fig. 6. Anti-fibronectin and anti-tenascin staining of transverse sections through the midtrunk region of X. laevis during late neural crest cell migration (stage 35). (A,B (enlarged)) Anti-FN staining is found in the ECM throughout the embryo including subepidermal spaces. Staining is especially intense in the dorsal fin (df), around the neural tube (nt), notochord {not), dorsal aorta (da) and in the myosepta (ms). (C,D (enlarged, different embryo)) Anti-TN staining resembles the staining with anti-FN except for the entire ventral region, where no staining is observed. Bars, A,C: 100um;B,D: 50um.
	Fig. 7. Anti-fibronectin and anti-tenascin staining of midtrunk transverse sections in the axolotl (stage 35). (A,B (enlarged, different embryo)) Each of the neural crest cell migratory routes are intensely stained with anti-FN. (C,D (enlarged)) Anti-TN stains only areas in the medial portion of the embryo, i.e. the dorsal fin ECM, the medial surface of the somites and spaces around the neural tube, notochord and dorsal aorta, df, dorsal fin; som, somites; nt, neural tube; not, notochord; da, dorsal aorta; ms, myosepta; epi, epidermis. Bars, A,C: 200um; B,D: 100um.
	Fig. 8. Anti-tenascin and anti-fibronectin staining of transverse sections through the tail of stage 40 Xenopus larvae. (A,B (enlarged)) With anti-TN an intense staining reaction is observed around the neural tube, notochord and throughout the ECM including intersomitic furrows and spaces around notochord and dorsal aorta. (C) Anti-FN stains the ECM surrounding the notochord intensely, but elsewhere the ECM is stained relatively faintly, nt, neural tube; not, notochord; ao, aorta; df and vf, dorsal and ventral fin. Bars, A: 100um; B,C: 50um.
	Fig. 9. Cultures of axolotl neural crest cells on fibronectin- and tenascin-coated substrata. (A) Neural crest cells spread from neural fold cultures onto FN-coated substrata, forming a sheet of cells (28 h). (B) Parallel neural fold culture (28 h); crest cells have not yet spread onto TN-coated substrata. (C,D) 3 days after explantation, neural crest cells have spread onto both FN- and TN-coated substrata, though the cells on FN are more flattened than the cells on TN. Bar, A-D: 100 um.