Messenger NJ and Warner AE (1989), The appearance of neural and glial cell markers...

XB-ART-26542

Development 1989 Sep 01;1071:43-54.

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The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo.

Messenger NJ , Warner AE .

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Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.

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Species referenced: Xenopus laevis
Genes referenced: prss1 slc4a1 vim
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	Fig. 1. Comparison of nitrocellulose blots of extracts from fish optic nerve and Xenopus stage 35/36 embryos. (A) Anti-70K neurofilament staining of Xenopus; (B) anti- Band 2 staining of Xenopus; (C) anti-Band 2 staining of fish optic nerve. The blots were 125I labelled and run against a series of Mr markers: lactalbumen, 14-2K; trypsin inhibitor, 20-1K; trypsinogen, 24K; carbonic anhydrase, 29K; glyceraldehyde-3-phosphate dehydrogenase, 36K; egg albumen, 45K; bovine albumen, 66K. In Xenopus, the anti- 70K recognises a single band at 70K and the anti-Band 2 antibody recognized two bands at 98K and 76K, in addition to the low molecular weight neurofilament protein.
	Fig. 2. The pattern of staining with antibodies to neurofilament protein in Xenopus neural tube. (A,C,D) Stage 36. (A) Side view diagram to show orientation of horizontal sections in C and D; (C) section stained with anti-70K antibodies; (D) a similar region stained with anti-Band 2 antibodies. Both antibodies reveal filamentous staining of axons running in the developing white matter of the spinal cord. (B,E,F) Stage 23. (B) Side view diagram to show orientation and location of sections in E and F; (E) transverse section through caudal region of the brain stained with anti-70K showing early axons in the lateral margin; (F) horizontal section through the neural tube stained with anti-Band 2 antibodies showing the first developing axons in the marginal zone. Bars = 100^m for A and B, 25 j.im for remainder.
	Fig. 3. (A-F) The distribution of glial cells in the Xenopus nervous system at stage 36. (A) Diagram showing location of sections in B and C. (B) Horizontal section through the neural tube stained with anti-GFAP showing radial glial cells stretching across the complete thickness of the neural tube, with endfeet in both ependymal and marginal zones. (C) Horizontal section through the caudal end of the brain stem stained with anti-GFAP. Note extensive staining of radial glial cells and profuse, overlapping end feet in the marginal zone. (D) Diagram showing location of sections in E and F. (E) Transverse section through the developing forebrain showing radial glial cells. (F) High power of lateral wall of brain taken from the adjacent section to that shown in C. Note GFAP staining apparent laterally and ventrally, but not dorsally and restricted to mantle and marginal zones. (G) Diagram to show location of section in H. (H) Neurofilament staining in caudal end of brain revealed with anti-70K antibodies. Note rostrocaudal axons running in the ventrolateral tracts. Bars = 100//m for A and D, 25 (tmi for remainder.
	Fig. 4. Comparison of staining with three antibodies that recognize glial cells in the neural tube of Xenopus stage 23 larvae, visualized using indirect immunofluorescence. (A,B) Diagrams showing location of sections C-F. (C) Horizontal section showing anti- Band 3 staining. (D) Transverse and (E) horizontal sections showing anti-GFAP staining. (F) Horizontal section stained with anti-vimentin antibodies. Note similar pattern of staining with all three antibodies, n, notochord; nt, neural tube. Bars = 100 j/m for A and B, 25 /.an for C-F.
	Fig. 5. Developing glial and neuronal cells in the brain of the axolotl (Amblystoma mexicanum) using indirect immunofluorescence. (A) Diagram of a transverse section through an axolotl stage 23 brainstem showing the location of the regions shown in B and C; (B) glial cell (GFAP), (C) neuronal (Band 2) staining in the stage 23 brainstem. Dotted line in C marks the lateral margin of the brain. Note clear recognition of developing radial cells in B, and absence of neurofilament staining in C. D (GFAP) and E (70K) similar comparison on the axolotl stage 35/36 brain, b, brainstem; n, notochord. Bar = 50 fim for A and 25 /xm for B-E.
	Fig. 6. 5-HT-containing cell bodies in the brainstem of a Xenopus embryo at stage 28, visualized with peroxidase. (A) Longitudinal, diagrammatic representation showing the location of the cell bodies and their axons drawn from serial sections of 6 embryos. (B) Transverse section at location indicated in A showing a pair of cell bodies on either side of the midline. (C) Horizontal section showing two groups of ventrally located cell bodies. (D) Horizontal section at a more dorsal level showing the axons and growth cones extending towards the forebrain. nt, neural tube; nc, neural canal. Bar = 100 ^on for A and 25 fan for B-D.
	Fig. 7. The developing 5-HT cell populations and their axons in the brain stem and rostral spinal cord of Xenopus stage 35/36 larvae. (A) Reconstruction showing the location of the cell bodies and their axons. The location of sections shown in B, C and D are indicated. (B) Horizontal view of 5-HT cell bodies located in brainstem. All cell bodies have a single lateral axon making contact with axons running caudally in the lateral tract. (C) Horizontal view of ascending dendrites. Note a more extensive network than at stage 27/28 and clearly visible varicosities on most dendrites. (D) Transverse section through brainstem showing 5HT cell bodies and a lateral axon running towards marginal zone. E and F show horizontal sections through the spinal cord. (E) Rostrocaudal axons in the lateral tract. (F) Similar section from embryo treated with 5,7-dihydroxytryptamine to prevent synthesis of 5-HT. nt, neural tube. Bars = 100 pm in A and 25 ;jm for B-F.
	Fig. 8. Series of horizontal sections through the brain of a Xenopus stage 35/36 embryo. A population of dorsoventral 5-HT fibres are shown, labelled with peroxidase, which cross the brain midline in the developing optic chiasma. Diagrammatic representation showing the pathway of the axons from a longitudinal (A) and horizontal (B) perspective are shown. C,D,E and F show sections taken from a series at the levels indicated in A to show density of the 5-HT-containing axons. Bar = 100jum (A), 50pun (B), 25 ^m (C-F).
	Fig. 9. The appearance of dopaminergic axons (stage 35/36) (A,C,E) and cell bodies (stage 37/38) (B,D,F) in the developing brain of Xenopus laevis embryos. (A) Composite diagram showing location of dopaminergic fibres at stage 35/36. (C) Transverse section at the level of the eyes showing dorsoventral axons running through the lateral tracts. (E) Transverse section further back in rostral neural tube showing rostrocaudal axons in lateral tract. (B) Composite diagram showing location of dopaminergic cell bodies and fibres at stage 37/38. (D) Transverse section at level of optic cups showing cell bodies in ventrolateral region of the brain. (F) Transverse section through rostral neural tube showing rostrocaudal axons in lateral tracts of white matter. NOTE: neural canal has collapsed in C and D. nc, neural canal; e, eye. Bars = 100 jum.
	Fig. 10. The developing dopaminergic system in a Xenopus laevis stage 39 tadpole, visualised by direct immunofluorescence. (A) Diagrammatic representation of Xenopus stage 39 brain and rostral neural tube to show location of axons and cell bodies. (B) Cell bodies and dorsoventral axons in midbrain transverse section. (C) Transverse section through rostral neural tube snowing rostrocaudal and dorsoventral axons in lateral tract. (D) Horizontal section through ventral hindbrain showing rostrocaudal axons, containing numerous varicosities, running along lateral tracts. (E) Horizontal section showing a few rostrocaudal axons running through lateral tracts in caudal neural tube, nc, neural canal; nt, neural tube; s, somites. Bars = 25 jum (100 pm for A).
	Fig. 11. The distribution of dopaminergic cell bodies and axons in the developing nervous system of a Xenopus stage 48 tadpole visualised by indirect immunofluorescence. (A) Longitudinal and (B) transverse diagrams of Xenopus stage 48 brain and rostral neural tube. The boxed area indicates the region illustrated in G. C-I show sections taken from a series through the same tadpole. (C) Transverse section through ventral forebrain showing lateral axons running through marginal zone. (D and E) Anterior populations of dopaminergic cell bodies close to midline showing axonal connections between adjacent cell bodies. (F and G) Two distinct populations of dopaminergic cell bodies in the 4th ventricle of the developing brain. (H and I) Rostrocaudal axons running through lateral tracts in the hindbrain. (J) Horizontal section through caudal neural tube showing two rostrocaudal axons running along the lateral tract. *, neural canal; s, somites; nt, neural tube; ant, anterior. Bars = 25;Â«m (100 f.im for A).