Takagi S et al. (1987), Specific cell surface labels in the visual cent...

XB-ART-28081

Dev Biol 1987 Jul 01;1221:90-100.

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Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies.

Takagi S , Tsuji T , Amagai T , Takamatsu T , Fujisawa H .

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Monoclonal antibodies (MAbs) against the optic tectum of Xenopus tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-B2 stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eye-enucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers.

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Species referenced: Xenopus laevis
Genes referenced: nrp1 plxna1 prss1 tbx2 tecta.2
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	Fig.1 Indirect immunofluorescence of MAb-45 and MAb-B2 on cryostat sections of the optic tectum of Xenopus tadpoles (stage 53). (a) A coronal section of the mesencephalon stained with MAb-A5. Only the outer layers of the optic tectum (OT) show the fluorescence (arrow), whereas the tegmentum (TG) is negative (X100). (b) A section similar to (a) stained with MAb-BX. Both the optic tectum and tegmentum are stained. (c) Immunofluorescence with MAb-A5 in the optic tectum is restricted to the 8th and 9th plexiform layers (X160). (d) The section next to (c) is stained with MAb-B2. The 3rd, 5th, and 7th plexiform layers of the optic tectum show immunofluorescence with MAb-B2, but the 8th and 9th layers are practically negative. (e) The same section as (d) viewed with bright-field optics. The numbers indicate the 3rd, 5th, 7th, 8th, and 9th plexiform layers of the optic tectum, respectively.
	FIG. 2. Indirect immunofluorescence of MAb-A5 and MAb-B2 on cryostat sections of the diencephalon of Xenopus tadpoles (stage 53). (a) A coronal section stained with MAb-A5. The nucleus Belonci shows immunofluorescence (arrow) (X130). (b) The section next to (a) stained with MAb-B2. The nucleus Belonci is negative. (c) A coronal section of the diencephalic region that is located rostra1 to the region shown in (a). The corpus geniculatum thalamicum is stained with MAb-A5 (arrow). (d) The section next to (c) stained with MAb-B2. The corpus geniculatum thalamicum is negative.
	FIG. 3. Indirect immunofluorescence of MAb-A5 and MAb-B2 on cryostat sections of the mesencephalic region, located rostrally to the sections shown in Fig. 1. (a) A coronal section stained with MAb-A5. The pretectum shows immunofluorescence (arrow). A small region corresponding to the basal optic nucleus shows the faint fluorescence at the ventrolateral part (*) (X110). (b) The section next to (a) stained with MAb-B2. The pretectum and the basal optic nucleus are negative. (c-e) Enhanced image showing localization of MAb-A5 binding in a ventrolateral part of the mesencephalon processed by a color image analyzer: (c) integrated image (128 times) (X250), (d) specific object image extracted as the highest area which corresponds to the basal optic nucleus, and (e) image in (d) superimposed on (c).
	FIG. 4. Whole mount of the diencephalon and mesencephalon of tadpoles (stage 51). (a) Retinal central pathways in a Xenopus tadpole. All retina1 axons from the right eye were labeled with HRP to show the projection to the visual centers, i.e., the optic tectum (OT), nucleus Belonci (NB), corpus geniculatum thalamicum (CGT), and basal optic nucleus (BON). OC: optic chiasm. For the method of HRP labeling, see Fujisawa et ul. (1981). (b) The tadpole brain stained with MAb-A5 by the ABC method. The optic tectum, the nucleus Belonci, and the corpus geniculatum thalamicum are positively stained. The staining of the basal optic nucleus is obscure. (c) The tadpole brain stained with MAb-B2 by the ABC method. All visual centers are negative (X40).
	FIG. 5. Indirect immunofluorescent analysis of the expression of A5-antigen in the optic tectum of eye-enucleated tadpoles (stage 51) in which the right eye was enucleated at stage 29. The left tecta (*) was not innervated by optic nerves (X175).
	FIG. 6. Indirect immunofluorescence of MAb-A5 in the mesencephalic region in development. (a) A section of the rostra1 part of the optic tectum at stage 40 (X90). (b) A section caudal to the section in (a). (c) A section of the rostra1 part of the optic tectum at stage 45 (X70). (d) A section of the caudal part of the optic tectum at stage 45. Arrows indicate the stained regions.
	FIG. 7. Immunofluorescent staining of viable tadpole brains with MAb-A5 (a) and MAb-B2 (b) (stage 53). Numbers indicate the 3rd, 5th, 7th, 8th. and 9th plexiform layers of the optic tectum (X140)
	FIG. 8. MAb-A5 indirect immunofluorescent staining of trypsin-digested optic tectum (stage 53). (a) Intact optic tectum was treated with 0.05% trgpsin for 2 hr and then fixed and stained with MAb-A5 (b) Control nontrypsinized optic tectum stained with MAb-A5 (X100).
	FIG. 9. Fluorogram of SDS-PAGE of immunoadsorbed polypeptides. From left to right, lane 1: NP-40 extract of labeled brain. Numbers indicate the positions of molecular mass markers (kDa). Lane 2: polypeptides immunoadsorbed with MAb-B2. Arrowheads indicate the specific bands. Lane 3: polypeptides immunoadsorbed with MAb-A5. An arrowhead indicates the specific band. Lane 4: polypeptides adsorbed with control MAb which is nonreactive to Xenopus brain, as revealed by immunofluorescent staining.
	plxna1 (plexin A1) gene expression in a Xenopus laevis embryo, NF stage 53, as assayed by immunohistochemistry. Coronal sections of brain, dorsal up.