Messenger NJ and Warner AE (2000), Primary neuronal differentiation in Xenopus emb...

XB-ART-9704

Dev Biol 2000 Apr 15;2202:168-82. doi: 10.1006/dbio.2000.9646.

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Primary neuronal differentiation in Xenopus embryos is linked to the beta(3) subunit of the sodium pump.

Messenger NJ , Warner AE .

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In amphibian embryos, activation of additional sodium pumps in neural plate cell membranes ensures that neural plate-derived neurons differentiate subsequently in the neural tube. When the sodium pump is inhibited during the mid-neural fold stages, neuronal differentiation fails. The effect is irreversible. We find that these events operate through the Na pump beta(3) subunit. When neural plate-specific Na pumps are activated, transcripts for beta(3) decline precipitately during the mid-neural fold stages, first in the neural plate and then in the dorsal mesoderm. As the neural tube closes, beta(3) returns, specifically in motor neurons and interneurons. Inhibition of the Na pump with the cardiac glycoside strophanthidin prevents the normal fall in beta(3) during neurulation: beta(3) is maintained in the neural plate until the neural tube closes, but lost from the dorsal mesoderm. Complete elimination of beta(3) transcripts from dorsal structures then occurs. Inhibiting the Na pump does not induce cell death (assessed by TUNEL staining) in the nervous system. Transcripts for X-Delta, NeuroD, and GSK3beta are not affected by inhibition of the Na pump. Xotch and N-tubulin transcripts fall to very low levels and Xotch disappears permanently from the nervous system. When beta(3) transcript expression is maintained throughout neurulation, by over expression of injected mRNAs, Xotch is eliminated from the neural tube and somites and switches to the ectoderm.

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Species referenced: Xenopus
Genes referenced: atp1b3 dll1 gsk3b igf2bp3 ncl neurod1 notch1 odc1 sst.1 tbx2 tubb2b

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	FIG. 2. The spatial expression of b3 transcripts between stages 10.5 (early gastrula) and 20 (neural tube closure). (A) Controls. (H, I) Strophanthidin treated. np, neural plate; nt, neural tube; som, somite; nc, notochord; yp, yolk plug. All transverse sections apart from A and B, which were taken through the yolk plug with dorsal ectoderm at the top. In B, the yolk plug is just visible at the bottom of the panel. In A, arrows indicate local dense expression in dorsal lip mesoderm. In G, arrow indicates position of future motor neuron that is already expressing b3. Strophanthidin treatment does not induce cell death in the nervous system. (J, K) Sections through control (J) and strophanthidin-treated (K) embryos at stage 26 that had been stained by the TUNEL procedure. In both embryos two TUNEL-positive cells are present in the nervous system either dorsolaterally (J, control) or in the roof plate (K, strophanthidin treated). TUNEL-positive cells arrowed. A, B, Bar 5 150 mm; E, F, H, I, bar 5 100 mm; C, D, G, bar 5 50 mm; J, K, bar 5 200 mm. FIG. 3. Spatial expression of b3 after neural tube closure. (A) Control embryos at stage 25 (A), 30 (B, C), and 35 (D). In C, the arrows indicate b3 expression in dorso-lateral regions of the otic vesicle (ov). In D, arrow indicates b3 in the retina. (E) Sibling embryo at stage 30 after treatment with strophanthidin between stages 14 and 20. Note that b3 is not expressed in any dorsal structure. (F, G, H, I) Comparison between the expression pattern of b3 (F, H) and N-tubulin (G, I) in the spinal cord (F, G) and brain (H, I). Note that only N-tubulin is expressed in Rohan Beard cells (arrowed F, G). nt, neural tube; nc, notochord; som, somite; ov, otic vesicle; fp, floor plate. Transverse sections. Bar 5 100 mm. Note that the strophanthidin-treated embryo in E has hypertrophy in the brain and eyes and is substantially larger in this region than its sibling, shown in C.
	FIG. 1. The temporal expression of b3 transcripts. (A) Northern blot of total RNAs probed with the b3 subunit probe. Stage 17. The RNA ladder is to the right. A single band is detected at 1.8 Kb. (B) RNAse protection assay to show temporal expression of b3 between the blastula stage and stage 25. Note fall in transcript levels at stage 17 and recovery by stage 23. By contrast, b1 transcripts increased slightly between the late blastula (stage 9) and early gastrula (stage 10.5) stages, but remained more or less constant for the rest of neurulation. (C) Plot of b3 transcripts relative to ODC for a single batch in which data was collected between the 64-cell stage and stage 25. RNAse protection assay. Ordinate, b3 transcript levels relative to ODC; abscissa, stage of development. Note that b3 transcripts fall at stage 17, but are recovering at stage 18. (D) Data collated from several batches showing the temporal changes in b3 mRNAs between the gastrula stage and stage 23. Ordinate, b3 transcripts relative to ODC; abscissa, developmental stage. The vertical bars give the standard error of the mean. n 5 at least 4 for each stage.
	FIG. 2. The spatial expression of b3 transcripts between stages 10.5 (early gastrula) and 20 (neural tube closure). (A–G) Controls. (H, I) Strophanthidin treated. np, neural plate; nt, neural tube; som, somite; nc, notochord; yp, yolk plug. All transverse sections apart from A and B, which were taken through the yolk plug with dorsal ectoderm at the top. In B, the yolk plug is just visible at the bottom of the panel. In A, arrows indicate local dense expression in dorsal lip mesoderm. In G, arrow indicates position of future motor neuron that is already expressing b3. Strophanthidin treatment does not induce cell death in the nervous system. (J, K) Sections through control (J) and strophanthidin-treated (K) embryos at stage 26 that had been stained by the TUNEL procedure. In both embryos two TUNEL-positive cells are present in the nervous system either dorsolaterally (J, control) or in the roof plate (K, strophanthidin treated). TUNEL-positive cells arrowed. A, B, Bar 5 150 mm; E, F, H, I, bar 5 100 mm; C, D, G, bar 5 50 mm; J, K, bar 5 200 mm.
	FIG. 3. Spatial expression of b3 after neural tube closure. (A–D) Control embryos at stage 25 (A), 30 (B, C), and 35 (D). In C, the arrows indicate b3 expression in dorso-lateral regions of the otic vesicle (ov). In D, arrow indicates b3 in the retina. (E) Sibling embryo at stage 30 after treatment with strophanthidin between stages 14 and 20. Note that b3 is not expressed in any dorsal structure. (F, G, H, I) Comparison between the expression pattern of b3 (F, H) and N-tubulin (G, I) in the spinal cord (F, G) and brain (H, I). Note that only N-tubulin is expressed in Rohan Beard cells (arrowed F, G). nt, neural tube; nc, notochord; som, somite; ov, otic vesicle; fp, floor plate. Transverse sections. Bar 5 100 mm. Note that the strophanthidin-treated embryo in E has hypertrophy in the brain and eyes and is substantially larger in this region than its sibling, shown in C.
	FIG. 4. Inhibiting the Na pump abolishes the transient fall in b3 during neurulation. RNAse protection assays comparing the temporal expression pattern of b3 in control embryos (A) and siblings treated with strophanthidin (1026 M) between stages 14 and 20 (B). (C) Quantitative comparison of b3 transcript levels. Ordinate, b3 mRNAs relative to ODC; abscissa, developmental stage. Note that control embryos show the normal transient suppression of b3 during the mid- to late neural fold stages while in strophanthidintreated embryos b3 is maintained until the neural tube closes and then falls.
	FIG. 5. Inhibiting the Na pump suppresses expression of Xotch and N-tubulin, but not X-Delta, NeuroD, or GSK3b. (A) RNAse protection assays to show the decline of N-tubulin and Xotch transcripts in embryos treated with strophanthidin between stages 14 and 20. In both cases transcript levels have declined substantially by stage 20 and are still very low at stage 30. (B) Time course of expression of Xotch and X-Delta between stages 14 and 25 in control embryos and embryos treated with strophanthidin between stages 14 and 20. A fall in Xotch transcripts is apparent at stage 17 and remains low after strophanthidin is removed. X-Delta transcripts do not change. (C) Temporal expression of NeuroD and GSK3b between stages 14 and 23 in control and strophanthidintreated embryos. Inhibiting the Na pump does not modify transcript expression.
	FIG. 6. Inhibiting the Na pump suppresses the expression of Xotch but not X-Delta. In all cases the Na pump was blocked with strophanthidin between stages 14 and 20. Comparisons between sibling embryos. Transverse sections. np, neural plate; nt, neural tube; nc, notochord; ncl, neurocoel; som, somite; ov, otic vesicle. (A–E) X-Delta. (A) Stage 17 control. Note expression in deep neural plate in regions where neurons will differentiate (arrows) and substantial expression in the somites. (B) Stage 21 control. Expression in the neural tube is limited to the future sites of motor and lateral neurons (arrows). This section passes through the somites at a level where X-Delta is not visible because of the segmental expression pattern. (C) Stage 35 control at level of the otic vesicle. X-Delta expression is limited to the mantle zone and forms pronounced dorso-ventral stripes (arrowed). (D) Stage 21 strophanthidin treated. The expression pattern of X-Delta matches the control (arrows). (E) Stage 35. Strophanthidin-treated embryo. This embryo was severely affected by Na pump inhibition during neurulation, which has lead to substantial distortion of neural tube morphology. Nevertheless, X-Delta continues to be expressed in a similar pattern to the control. Bands of X-Delta expression arrowed in the otic vesicle may have been reduced. (F–L) Xotch. (F) Stage 17 control. Xotch is excluded from the floor plate, but is widely expressed elsewhere in the neural plate. Xotch, like X-Delta, is segmentally expressed in the somites and this section passes through a region of expression. (G) Stage 21 control. Xotch expression in the neural tube is more restricted than at stage 17, but there is diffuse expression in the outermost layer where neurons will differentiate. This section passes through the intersegmental regions of the somites where Xotch is not expressed. (H) Stage 30. Xotch expression in the neural tube has shifted and cells in the neuro-epithelial (germinal) layer express Xotch. Ventrally Xotch is excluded from the marginal and mantle zones. Xotch is excluded from the floor plate. Dorsally, there is patchy expression of Xotch in the mantle and marginal zones of the neural tube. (I) Stage 35. Xotch has become restricted to cells lining the neurocoel, some of which spread into the mantle zone, with weak and patchy expression elsewhere. Xotch continues to be excluded from the roof and floor plates. (J) Stage 21, strophanthidin treated. Xotch has been eliminated from all dorsal structures. (K) Stage 30. Strophanthidin treated. Xotch is still absent from the neural tube, although there is now weak, patchy expression in the notochord. Pigmented cells lining the neurocoel are prominent in this embryo. (L) Stage 35. Strophanthidin treated. Xotch is not detected in neural tube cells, although there may be weak expression in some cells in the otic vesicle. The stain has precipitated within the otic vesicles and there is precipitate in some intercellular spaces. Pigment cells are visible in the ectoderm.
	FIG. 7. Overexpression of b3 suppresses Xotch. (A) The time course of b3 transcript expression in control embryos and sibling embryos that had been injected with a CMV1. b3 RNA sense construct at the one-cell stage. A single probe was used to detect both exogenous (440 nt) and endogenous (140 nt) b3 transcripts). Control embryos reproduce the normal transient fall in b3 during the mid- to late neural fold stages. Embryos injected with b3 sense RNAs express both exogenous and endogenous transcripts. Note that the characteristic transient fall in b3 transcripts at stage 17 is overridden when b3 is overexpressed. (B, C, D, E) In situ hybridization of sibling embryos at stage 22. (B, C) b3 (B) and Xotch (C) in control embryos. b3 is confined to the developing motor neurons, while Xotch is widely expressed in outer cells of the neural tube, with expression also in the somites, but little in the ectoderm. In embryos that overexpress b3 (D, E), b3 transcripts are detected throughout the neural tube, the lateral mesoderm, somites, and notochord (D), while Xotch is not detectable. However, intense expression of Xotch is now apparent in the ectoderm. Bar 5 100 mm. nt, neural tube; nc, notochord; som, somite.
	FIG. 8. Diagram summarizing the alterations in Na pump activity in neural plate cells (A) and the concomitant alterations in transcript levels for the a1, b1, and b3 subunits of the Na pump between the mid-gastrula stage (10.5) and closure of the neural tube (stage 23) (B). While additional Na pumps are activated in the neural plate the membrane potentials of neural plate cells increase and intracellular sodium falls (A). Functional activation begins at stage 14 and neural plate sodium pumps reach a new steady state at about stage 17. Note that b3 transcript levels fall when the Na pump is activated and return once the Na pump reaches a new steady state. Membrane potential data taken from Blackshaw and Warner (1976a); intracellular Na from Breckenridge and Warner (1982); a1 from Davies et al., (1996); b3 and b1 from the present paper.