Levi G et al. (1990), Thyroxine-dependent modulations of the expressi...

XB-ART-25966

Development 1990 Apr 01;1084:681-92.

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Thyroxine-dependent modulations of the expression of the neural cell adhesion molecule N-CAM during Xenopus laevis metamorphosis.

Levi G , Broders F , Dunon D , Edelman GM , Thiery JP .

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During amphibian metamorphosis, a complete remodeling of the phenotype takes place under complex hormonal control whose final effectors are thyroid hormones. This process implies the activation of coordinated programs of cell death, proliferation, migration, adhesion and differentiation. Inasmuch as the neural cell adhesion molecule N-CAM is thought to play a central role in the control of morphogenetic processes, we have studied by immunohistofluorescence and immunoblots the patterns of expression of N-CAM at different stages of Xenopus laevis metamorphosis. A scan was made of all major organs and appendages. Before the metamorphic climax, all neuronal cell bodies and processes express high levels of N-CAM. During the metamorphic climax, N-CAM expression decreases sharply on the cell bodies and processes of the peripheral nervous system (PNS) but remains high in the central nervous system (CNS). Towards the end of metamorphosis, the PNS and spinal nerves are virtually negative for N-CAM while the CNS is still positive. The optic and olfactory nerves, although myelinated, are still strongly positive for N-CAM. The lens and olfactory epithelia express N-CAM throughout metamorphosis. In the brain. N-CAM is present at all times as three polypeptides of 180, 140, and 120 X 10(3) Mr; before metamorphosis some of the N-CAM is in its polysialylated form. During metamorphosis and the subsequent growth of the animal, the amount of N-CAM decreases gradually. In all polypeptides, the polysialylated form is the first to disappear. Cardiac muscle expresses high level of N-CAM from its first formation throughout metamorphosis; in contrast, the level of N-CAM in skeletal muscle is high in newly formed muscles, but decreases rapidly after myoblast fusion. The liver of adult Xenopus contains large amounts of a 160 X 10(3) polypeptide that is recognized by polyclonal and monoclonal antibodies against N-CAM. cDNA probes of Xenopus brain N-CAM recognize major transcripts of 9.2, 3.8 and 3.3 kb in Xenopus liver mRNA; these bands are different in size from those recognized in brain mRNA (9.5, 4.2 and 2.2 kb). Premetamorphic liver does not express the 160 X 10(3) form of N-CAM, which can be first detected at stage 59 and persists then through all the life of the animal. Expression of N-CAM in the liver can be induced in premetamorphic animals (stage 51-52) by a 48 h treatment with thyroxine. All hepatocytes are responsive.(ABSTRACT TRUNCATED AT 400 WORDS)

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Species referenced: Xenopus laevis
Genes referenced: drg1 ncam1
???displayArticle.antibodies??? Ncam1 Ab7 Ncam1 Ab8 Ncam1 Ab9 Neuronal Ab6

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	Fig. 1. Distribution of N-CAM and NC-1 immunoreactivity in the spinal cord and dorsal root ganglia. Section of premetamorphic Xenopus tadpoles (stage 54 NF) (A,B) and of animals toward the end of the metamorphic climax (stage 64 NF) (C,D) were double stained with polyclonal antibodies against Xenopus N-CAM (A,C) and with NC-1 monoclonal antibody (B,D). Before metamorphosis all neuronal cell bodies and processes of the CNS and peripheral ganglia including the neural epithelium lining the ependymal canal were labeled by anti-N-CAM antibodies; NC-1 recognized mostly the fibers in the CNS and cell bodies and fibers in the PNS. After metamorphosis the staining of anti-N-CAM antibodies persisted in the CNS but disappeared on the cell bodies and fibers of the PNS; intense staining with anti-NC-1 persisted both in the CNS and in the PNS. drg, dorsal root ganglia; sc, spinal cord. Bar: 100um.
	Fig. 2. Distribution of N-CAM and NC-1 immunoreactivity in the nerves. Section of premetamorphic Xenopus tadpoles (stage 52 NF) (A,B) and of animals toward the end of the metamorphic climax (stage 64 NF) (C-F) were double stained with polyclonal antibodies against Xenopus N-CAM (A,C,E) and with NC-1 monoclonal antibody (B,D,F). Before metamorphosis all nerves were intensely stained by anti-N-CAM and NC-1 antibodies. During metamorphosis the staining of anti-N-CAM antibodies was greatly reduced in peripheral nerves (C) but remained high in central nerves such as the optic nerve (E); NC-1 staining persisted in the nerves throughout metamorphosis, dm, dorsal muscle; m, muscle; on, optic nerve; sn, sciatic nerve. Bar: 100um.
	Fig. 3. Staining pattern of anti-N-CAM antibodies on Xenopus liver at different stages through metamorphosis. (A) Stage 57 NF; (B) stage 59 NF; (C) stage 61 NF; (D) stage 64 NF. N-CAM reactivity is first detectable on hepatocytes at stage 59 and persists then throughout the life of the animal. Only hepatocytes and no other cell types were labeled by anti-N-CAM antibodies. The staining was clearly a cell surface staining particularly intense in regions of cell-cell contact. Bar: 50um.
	Fig. 4. Induction of N-CAM expression in the liver of premetamorphic Xenopus. Premetamorphic tadpoles (stage 54 NF) were treated for 48 h with 3 10 M thyroxine added directly in the rearing water. The reactivity of anti-N-CAM antibodies on livers of control (A) and treated animals (B) was then compared. All hepatocytes respond to thyroxine treatment synthesizing N-CAM that is then rapidly incorporated in the membrane and localized in areas of cell-cell contact. Bar: 60um.
	Fig. 5. Western and Northern blot analysis of N-CAM expression in Xenopus liver and brain. (A) Western blot analysis of N-CAM expression in Xenopus liver and brain. 100 /.ig of proteins extracted from different tissues were resolved on 7 % polyacrylamide gels in the presence of SDS and immunoblotted with anti-N-CAM polyclonal antibodies. Lane 1, premetamorphic liver (stage 54 NF). Lane 2, as lane 1 after 48 h treatment of the animal with 3x10 M thyroxine. Lane 3, postmetamorphic liver (stage 64 NF). Lane 4, adult liver. Lane 5, premetamorphic brain. Lane 6, as lane 5 after 48 h thyroxine treatment. Lane 7, postmetamorphic brain (stage 64). Lane 8, adult brain. Mr markers are at 205, 116 and 97X103, respectively. (B) Northern blot analysis of RNA from adult Xenopus liver (lane 1) and adult Xenopus brain (lane 2), with a Xenopus N-CAM random primed probe. Size markers are at 9.9, 3.8 and 2.2 kb respectively.
	Fig. 6. Staining pattern of anti-N-CAM antibodies in developing muscles during Xenopus metamorphosis. (A) Section through a stage 52-53 NF Xenopus hindlimb. The nerves and the condensing muscles are strongly N-CAM-positive. Condensing cartilage and skin are negative. (B) Section through the abdominal wall of a stage 54 NF Xenopus. Muscle bundles are strongly N-CAM-positive, the skin is negative, c, cartilage; m, muscle; n, nerve; sk, skin. Bar: 100um.
	Fig. 7. Expression of N-CAM in Xenopus heart during metamorphosis. Cardiac muscle was brightly stained by anti-N-CAM antibodies from its first formation throughout metamorphosis. (A) Stage 48 heart; (B) stage 64 cardiac muscle, a, atrium; v, ventricle. Bar: 100/.an.
	Fig. 8. Staining, pattern of anti-N-CAM antibodies on Xenopus lens and olfactory epithelium. (A) The lens epithelium of stage 52 Xenopus is strongly labeled by anti- N-CAM antibodies; lens fibers are not stained. (B) Tangential section through the same lens; the cell surface of the epithelial cells is strongly stained in areas of cell-cell contact. (C) Transverse section through the same epithelium, the apical aspect of the epithelial cells is not stained. (D) Section through the olfactory organ of a stage 64 NF Xenopus tadpole. The olfactory nerve and the sensory part of the olfactory epithelium are strongly stained by anti-N-CAM antibodies. If, lens fiber; oe, olfactory epithelium; oln, olfactory nerve. Bar=50um.