Moody SA et al. (1996), Developmental expression of a neuron-specific b...

XB-ART-18661

J Comp Neurol 1996 Jan 08;3642:219-30. doi: 10.1002/(SICI)1096-9861(19960108)364:2<219::AID-CNE3>3.0.CO;2-8.

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Developmental expression of a neuron-specific beta-tubulin in frog (Xenopus laevis): a marker for growing axons during the embryonic period.

Moody SA , Miller V , Spanos A , Frankfurter A .

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In mammals, there are seven classes of beta-tubulin genes, one of which, class III, is neuron specific. Using class-specific monoclonal antibodies, class III beta-tubulin protein could not be detected in frog embryos or in adults with either Western blotting or immunohistochemical techniques. In contrast, the class II beta-tubulin protein, which is predominant in mammalian brain but is also expressed in other tissues, is expressed only in neurons in frog embryos. Protein was detected only in neurons from late stages of neural tube closure through premetamorphic stages. At stages 21-28, the pioneering axons of Rohon-Beard, commissural, primary motor, and trigeminal ganglion neurons were distinctly stained in the axon scaffolds that they formed in the embryonic brain and the peripheral mesenchyme. Nonneuronal cells, both outside the nervous system and within it (e.g., radial glia, Müller glia, roof plate, and floor plate cells) were not immunoreactive. Throughout swimming and premetamorphic stages, neuronal cells in all brain regions became immunoreactive as they differentiated and extended axons. Whereas many embryonic neurons became postmitotic during gastrulation stages, neurons expressed detectable levels of class II beta-tubulin protein only beginning at the onset of overt axon outgrowth. These observations demonstrate that the neuron-specific beta-tubulin in frog is a different gene from that in mammals, and its protein product is detectable at the time of axonogenesis rather than neurogenesis.

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Species referenced: Xenopus laevis
Genes referenced: mtnr1al not ret tbx2 zic1

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	Fig. 2. Characterization of the class I1 p-tubulin antibodies. A The binding of the mouse monoclonal antibody 7B9 to adult rat brain tubulin in the presence of carboxyl terminal p-tubulin peptides (identified in box). Only the p-I1 peptide blocks 7B9 binding. Each data point represents the mean 2 S.E. (n = 3). B Western blot to detect class I1 p-tubulin at different developmental stages and in adult tissues. Three embryo equivalents of protein were loaded into each lane for each developmental stage. For the adult adrenal (adren) and brain, 20 yg of total protein were loaded per lane. Molecular weight markers are indicated on the right. A single 53 kD band at tadpole stages and in brain represents p-tubulin. This blot was performed with JDR 3B8 (Banerjee et al., 1958); the same results were obtained with 7B9.
	Fig. 3. A monoclonal antibody (Tu27), which recognizes a conserved sequence in all 6-tubulins, stains all tissues and cell types in frog embryos. A: Radial processes and mitotic figures (arrows) in the neural plate (np), notochord (noto), and adjacent somite (s) are immunoreactive. Dark staining in surface cells (top) is due to pigment granules. Inset: High magnification of a mitotic spindle. B: The dorsolateral (dlt) and ventrolateral (vlt) axon tracts and numerous radial glial fibers extending to the central canal (outlined) are darkly stained in the stage 24 neural tube (nt). Cells in the somite (s) and notochord (noto) also are stained diffusely. C: At the hindbrainimidbrain junction of a stage 38 embryo, Tu27 staining is intense in axon tracts (at), peripheral nerves, and ganglia (in this case, the ophthalmic lobe of the trigeminal ganglion; Vg), in radial embryonic glial fibers extending to the ventricle (arrows), and in cells in the roof plate (rp) and floor plate (fp). Top inset: High magnification of radial glial endfeet (arrows) at ventricle (ve) and a mitotic spindle (arrowhead). Bottom inset: High magnification of labelled cells in the floor plate (brace) and commissural axons (Ca). D: In the rostra1 midbrain of a stage 45 embryo, Tu27 staining is intense in these same regions. Also note the intense staining in surrounding nonneural tissues (n, notochord; ph, pharyngeal epithelium). Inset: High magnification of embryonic radial glial endfeet at the ventricular surface (dashed lines). Scale bars = 32 pm in A, 35 ym in B, 33 pm in C, 46 pm in D, 3.6 pm in A inset, 6 pm in C and D insets.
	Fig. 4. Transverse sections of stagt: 45 retinas stained with Tu27 (A), TuJl (B), and 7B9 (C). All cellular elements including Muller glia (arrows), ciliary margin (cm, between brackets), and lens stain with Tu27; none stains with TuJ1. Class I1 P-tubulin is detected in cells in the ganglion layer (gl) and in fibers in the inner plexiform layer (ipl) and the outer plexiform layer (opl). Pigmented epithelium (pe) of retina i, not immunoreactive. Scale bars = 60 um.
	Fig. 5. Monoclonal antibodies that recognize the isotype-defining domain of class I1 8-tubulin stain only neurons in frog embryos. A-C: Transverse sections of caudal (A,B) and rostra1 (C) spinal cord from stage 22 embryos. Class I1 p-tubulin antibodies stain Rohon-Beard neuron cell bodies (RBI, peripheral RB axon sprouts (open arrow), the dorsolateral axon tract (dlt), and commissural neurons (Co) and their axons (solid arrows). D: Transverse section of stage 26 embryo. Class I1 6-tubulin antibodies stain the cell bodies of trigeminal ganglia (Vg), their central axons in the ventrolateral tract (small brackets in the brainstem), and their peripheral axons (arrows) coursing toward the cement gland (cg). Melanin granules account for the dark color in the cement gland, skin, and pigmented epithelium of the retina (ret). Scale bars = 37 pm in A-C, 150 bm in D.
	Fig. 6. A: Transverse section of stage 28 spinal cord showing intense class I1 p-tubulin staining in Rohon-Beard (RBI, commissural (Co), and primary motoneurons (PM) and their pioneering axons. Large arrowheads indicate RB and PM axons, and small arrowheads indicate Co axons. B: Wholemount preparation of stage 32 spinal cord showing class I1 P-tubulin immunostaining in the dlt and vlt and a motor axon (arrowheads) as it crosses the notochord (noto). Orientation is according to arrows in lower left corner (A, anterior; D, dorsal; P, posterior; V, ventral). C: Wholemount preparation of stage 32 head showing class I1 p-tubulin immunostaining of forebrain tracts and nuclear groups (arrows). The olfactory epithelium (OLF) is dark due to intracellular melanin granules, but, at later stages, olfactory neurons and their axons stain with 7B9. DVDT, dorsoventral diencephalic tract; TPC, tract of the posterior commissure; TPOC, tract of the postoptic commissure; Vn, trigeminal nerve; RET, retina. D Wholemount preparation of a stage 45 brain. Note the abundant staining of cells (arrows) and axon tracts, e.g., the posterior commissure (PC). OB, olfactory bulb; TE, telencephalon. Scale bars = 25 pm in A, 87 pm in B, 38 pm in C, 50 pm in D.
	Fig. 7. Class I1 p-tubulin immunostaining at stage 45. A Transverse section of the spinal cord. Several neurons (arrowheads) and all of the axon tracts on the periphery of the cord are intensely stained with class I1 p-tubulin antibodies. Note that floor plate (fp) and roof plate (rp) cells are not stained. The dark structures just dorsal to the spinal cord are melanocytes (mel). B: Transverse section of rostra1 midbrain. All neuronal clusters and axon tracts are intensely stained. The only stained elements in the region of the floor plate (fp) are commissural axom (ca). Note the absence of staining in the roof plate (rp) and in processes near the ventricle (outlined by dashed lines). Scale bars = 19 um in A, 36 um in B.