XB-ART-17479Development November 1, 1996; 122 (11): 3409-18.
A posteriorising factor, retinoic acid, reveals that anteroposterior patterning controls the timing of neuronal differentiation in Xenopus neuroectoderm.
During early development of the Xenopus central nervous system (CNS), neuronal differentiation can be detected posteriorly at neural plate stages but is delayed anteriorly until after neural tube closure. A similar delay in neuronal differentiation also occurs in the anterior neural tissue that forms in vitro when isolated ectoderm is treated with the neural inducer noggin. Here we examine the factors that control the timing of neuronal differentiation both in embryos and in neural tissue induced by noggin (noggin caps). We show that the delay in neuronal differentiation that occurs in noggin caps cannot be overcome by inhibiting the activity of the neurogenic gene, X-Delta-1, which normally inhibits neuronal differentiation, suggesting that it represents a novel level of regulation. Conversely, we show that the timing of neuronal differentiation can be changed from late to early after treating noggin caps or embryos with retinoic acid (RA), a putative posteriorising agent. Concommittal with changes in the timing of neuronal differentiation, RA suppresses the expression of anterior neural genes and promotes the expression of posterior neural genes. The level of early neuronal differentiation induced by RA alone is greatly increased by the additional expression of the proneural gene, XASH3. These results indicate that early neuronal differentiation in neuralised ectoderm requires posteriorising signals, as well as signals that promote the activity of proneural genes such as XASH3. In addition, these result suggest that neuronal differentiation is controlled by anteroposterior (A-P) patterning, which exerts a temporal control on the onset of neuronal differentiation.
PubMed ID: 8951057
Article link: Development
Species referenced: Xenopus
Genes referenced: ascl2 dll1 en2 foxg1 nog otx2 tubb2b
Article Images: [+] show captions
|Fig. 1. Expression of the neuronal differentiation marker N-tubulin, in stage 16 neural plate (A), stage 27 (B,C,D) and stage 31 (E,F) tadpole Xenopus embryos. (A-D) Embryos analysed for N-tubulin expression shown in whole mount; (E,F) N-tubulin staining in sections. (A,C) Hybridised with En-2, which is a marker for the midbrain-hindbrain boundary and is shown with an arrow. (A-C) Frontal views; (D) side view of the head. Neuronal differentiation takes place at the neural plate stage and is confined to three stripes on either side of the dorsal midline. Note that the expression of N-tubulin is not detected anterior to En-2 at the neural plate stage. In contrast, N-tubulin expression can be detected in the forebrain, starting at later tadpole stages (stage 27) when it is localised to the epiphysis (black arrow), ventral postoptic diencephalon (white arrow) and olfactory placodes (white arrowhead; see also Hartenstein, 1993). At stage 31, N-tubulin expression is abundant posteriorly (F) as well as anteriorly (E). (E) A section through the forebrain (fb), including olfactory placodes (op); (F) a section through the hindbrain (hb) at the level of the otocysts (ot).|
|Fig. 3. Whole-mount in situ hybridisation showing that XASH3 promotes neuronal differentiation in noggin caps, but not until the tadpole stage. Xenopus embryos were injected bilaterally at the 2-cell stage, with noggin (A,D) or noggin plus XASH3 RNA (B,E), animal caps were dissected at blastula stage, cultured either until the neural plate stage (stage 16; A,B) or the tadpole stage (stage 27; D,E) and hybridised with a probe for N-tubulin. (C,F) Control embryos at stage 16 and stage 27, respectively. Note that noggin alone does not promote the formation of N-tubulin-expressing cells either at stage 16 (A) or at stage 27 (D). Noggin plus XASH3-injected animal caps undergo neuronal differentiation, but only if cultured until stage 27 (E), not at stage 16 (B). In contrast, N- tubulin is expressed abundantly at stage 16 (C) in the embryo, suggesting that noggin plus XASH3-injected animal caps need additional signals in order for neuronal differentiation to occur at the neural plate stage.|
|Fig. 4. Blocking lateral inhibition by X-Delta-1stu does not promote early neurogenesis in response to XASH3. Animal caps expressing noggin (A) or noggin and XASH3/X-Delta-1 stu (B) do not express N-tubulin at the neural plate stage. In contrast, when animal caps that express noggin (C) or noggin and XASH3/X-Delta-1stu (D), are treated with RA at stage 9 and analysed at the same way as in A and B, they express N-tubulin. Highest levels of N-tubulin is seen in those co-injected with noggin and XASH3 /X-Delta-1stu and treated with RA (D). (C,D) Examples of sites of N-tubulin hybridisation are shown by arrowheads. In the whole embryo (E), injection of XASH3/X-Delta-1stu increases the width and density of N-tubulin cells, as previously described (Chitnis and Kintner, 1996). (E) The injected side is shown with an arrow. C, is a control embryo.|
|Fig. 6. XBF-1 expression marks the developing telencephalon in Xenopus embryos. Whole-mount in situ hybridisation shows that XBF-1 is expressed at the anterior end of the neural plate at the late gastrula (stage 12.5 ; A) and the neural plate stages (stage 16; B). At the tadpole stage (stage 27), XBF-1 is expressed in neuroepithelial cells of the telencephalon (C).|
|Fig. 8. RA applied at blastula stages but not at late gastrula stages, posteriorises the anterior neural plate and simultaneously induces premature anterior N-tubulin expression. Xenopus embryos were either left untreated (A,D) or treated with RA when sibling embryos reached stage 9 (B,E) or stage 12. 5 (C,F), and analysed simultaneously for the expression of Xotx2 and N-tubulin. Xotx2 staining is light blue while N-tubulin staining is magenta in color. Embryos shown in A-C have been cleared in benzyl benzoate in order to reveal possible N-tubulin staining in the deep regions of the neural tissue and are shown in D-F, respectively. Uninjected embryos expresses Xotx2 but no N-tubulin in the anterior neural plate (A,D). In embryos that have been treated with RA at stage 9, the anterior markers Xotx2 is suppressed and N-tubulin is massively induced in the anterior end of the neural ectoderm (B,E). In embryos that have been treated with RA at stage 12.5 the anterior marker Xotx2 is suppressed to a lesser degree than in embryos treated with RA at stage 9 (C,F). When cleared, these embryos show few N- tubulin cells anteriorly (F).|
|tubb2b ( tubulin, beta 2B class IIb ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior right, dorsal up.|