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Retinoid receptors, which are members of the nuclear hormone receptor superfamily, act as ligand-dependent transcription factors. They mediate the effects of retinoic acid primarily as heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs). To analyse their function, xRXR beta synthetic mRNA was injected into Xenopus embryos in combination with normal and mutated xRAR alpha transcripts. Two informative phenotypes are reported here. Firstly, over-expression of xRXR beta with xRAR alpha results in the formation of ectopic primary neurons. Secondly, blocking retinoid signalling with a mutated xRAR alpha results in a lack of primary neurons. These two phenotypes, from contra-acting manipulations, indicate a role for retinoid signalling during neurogenesis.
Fig. 2. xRXRβ transcripts are found predominantly in the anteriorneurectoderm and adjacent neural crest. A transcript-specific probe derived form the 5′ part of the cDNA clone was used to detect xRXRβ transcripts in the neurula and tailbudembryo. The embryos were rendered transparent in Murray�s clear. (A) Dorsal view at the neurula stage shows an accumulation of transcripts in the anteriorneurectoderm (arrowhead) and in adjacent neural crest (open arrow), though essentially all tissues express a low level of xRXRβ when determined by RNase protection assay (data not shown). (B) At the tailbud stage transcripts are localised to the anterior neural tube (arrowhead), the dorso-frontal segment of the developing eye (e) and migrating cranial neural crest (bracketed).
Fig. 3 Co-injection of RNA encoding heterodimeric xRXRβ and xRARα2 results in the formation of ectopic neurons at the tailbud stage. Dorsal views of embryos stained by wholemount in situ hybridisation for XIF3 at the tailbud stage, (anterior to the right). Embryos were injected with: (A) xRARα2/xRXRβ, (B) xRARα2, (C) xRXRβ, (D) xRARα2∆h*d, (E) xRARα2∆h*d/RXRβ; (F) was uninjected. Co-injection of xRARα2/xRXRβ results in embryos that have abnormal patterns of XIF3 expression with positive regions adjacent to the neural tube (e.g. arrow in A). However injection of individual receptors (B,C) or a combination of control transcript xRARα2∆h*d and xRXRβ (E) has no effect on XIF3 expression. Embryos injected in an identical manner were assayed with the anti- HNK1 monoclonal antibody to demonstrate the formation of ectopic neurons. (G) xRARα2/xRXRβ-injected and (H) uninjected embryos rendered transparent in Murray�s clear to see neural cell bodies. Axons (arrows) are apparent in both embryos but ectopic clusters of anti-HNK1 positive cell bodies (arrowheads) are only seen adjacent to the neural tube in xRARα2/xRXRβ injected embryos (G) and not in controls (H).
Fig. 5. Ectopic neurons form even when cell proliferation is inhibited in neural precursors. Embryos at the early gastrula stage were treated with hydroxyurea and aphidicolin (HUA) to inhibit cell division (Harris and Hartenstein, 1991) and assayed for XIF3 at the tailbud stage. XIF3 staining is somewhat punctate along the neural tube of uninjected and control xRARα2∆h*d/xRXRβ- coinjected embryos, probably reflecting the HUA treatment. Coinjection of xRARα2/xRXRβ, however, generates ectopic neurons both in untreated and HUA-treated embryos (arrowheads). It is therefore likely that the ectopic neurons do not form through increased proliferation of neural precursors.
Fig. 7. Injection of transcripts encoding the dominant negative xRARα2 result in a lack of sensory neurons. (A,B) Embryos injected with the dominant negative xRARα2∆393 into both cells at the two- cell stage have decreased numbers of sensory neuron axons, detected by the anti-HNK-1 antibody. (A) Uninjected embryo at the tailbud stage. Examples of sensory neuron axons are marked by arrowheads. (B) Embryo injected with control transcript xRARα2∆h*d has the normal complement of axons. (C) Embryos injected with 400 pg of xRARα2∆393 have very few sensory axons. Staining within the neural tube represents the cell bodies and axon tracts of anti-HNK-1 positive cells that are unaffected by xRARα2∆393 transcript injection. (D) Transverse section through an uninjected embryo showing bilateral primary sensory neurons (large arrowhead) and axons. (E) Transverse sections through a tailbud stage embryo co- injected into one cell at the two-cell stage with xRARα2∆393 and synthetic RNA encoding β-galactosidase. At the tailbud stage the embryos were assayed for both β-galactosidase activity (blue stain) and with the anti HNK-1 antibody to detect sensory neurons (brown). β-galactosidase activity acts as a lineage tracer to identify those parts of the embryo that received the coinjected synthetic transcripts. Only regions that did not receive injected transcripts developed primary sensory neurons (large arrowhead), although anti-HNK-1 staining is still seen in axon tracts and in some interneurons (small arrowhead). In addition, the ventralneurocoel is consistently displaced towards the uninjected side of the embryo.
