Ferreiro B et al. (1994), XASH genes promote neurogenesis in Xenopus embr...

XB-ART-20488

Development 1994 Dec 01;12012:3649-55.

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XASH genes promote neurogenesis in Xenopus embryos.

Ferreiro B , Kintner C , Zimmerman K , Anderson D , Harris WA .

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Neural development in Drosophila is promoted by a family of basic helix-loop-helix (bHLH) transcription factors encoded within the Achaete Scute-Complex (AS-C). XASH-3, a Xenopus homolog of the Drosophila AS-C genes, is expressed during neural induction within a portion of the dorsal ectoderm that gives rise to the neural plate and tube. Here, we show that XASH-3, when expressed with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm, suggesting that XASH-3 promotes neural development. Moreover, XASH-3/XE12 RNA injections into embryos lead to hypertrophy of the neural tube. Interestingly, XASH-3 misexpression does not lead to the formation of ectopic neural tissue in ventral regions, suggesting that the domain of XASH proneural function is restricted in the embryo. In contrast to the neural inducer noggin, which permanently activates the NCAM gene, the activation of neural genes by XASH-3/XE12 is not stable in naive ectoderm, yet XASH-3/XE12 powerfully and stably activates NCAM, Neurofilament and type III beta-tubulin gene expression in noggin-treated ectoderm. These results show that the XASH-3 promotes neural development, and suggest that its activity depends on additional factors which are induced in ectoderm by factors such as noggin.

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Species referenced: Xenopus
Genes referenced: actl6a ascl1 ascl2 ncam1 nog tcf3 tubb3 twist1

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	Fig. 1. The specificity of expression of neural and muscle transcripts in animal caps from embryos injected with different bHLH encoding mRNAs. Top: 125 pg (high) but not 25 pg (low) of XE12 mRNA induces NCAM and neurofilament expression (lanes b and c). 833 pg of XASH-3 mRNA alone induces only slight NCAM expression, but when it is combined with 25 pg of XE12 mRNA, there is a dramatic increase in NCAM and NF expression (lanes d and e). Note that the level of neural transcripts in the XASH-3/XE12-injected condition are close to the levels in whole uninjected embryos (lane i). 833 pg of XMyoD mRNA induces both NF and NCAM expression, but when this is combined with 25 pg of XE12, the levels of NCAM and NF expression are not increased (lanes f and g). Bottom: 125 pg of XE12 mRNA induces some expression of muscle-specific actin (lane b), but neither XE12 at 25 pg orXASH-3 at 833 pg alone, or in combination, induce muscle actin expression (lanes c-e). However, XMyoD at 833 pg does activate muscle actin expression and this is dramatically enhanced by combination with 25 pg of XE12 mRNA (lanes f and g). These results were replicated twice in independent experiments. The asterisk shows cytoskeletal actin mRNA, which is recognized by the cardiac actin probe and acts as a loading control. These lanes were cut and rearranged from a single exposure of one experiment.
	Fig. 2. XASH3/XE12 transiently activates neural genes. Neurofilament and NCAM are induced strongly in naive ectoderm by the injection of XASH-3/XE12 when the caps are assayed 1 day after the injection (lane a). If the caps are cultured for a second or third day, the level of these neural genes is dramatically reduced (lane d). Control caps (lanes b and e) show no neural gene expression on day 1 or 2. This experiment was repeated at least three times, and although there was some variation in the extent of the decrease in NCAM and NF expression XASH-3/XE12 induced caps, the decrease was always dramatic by 3 days.
	Fig. 3. Phenotypes of XASH/XE12-injected embryos. (A). Direct dorsal view of a whole-mount embryo injected with 90 pg ofbXASH-3 mixed with XE12 in the right side (anterior to right), and stained for the expression of NCAM mRNA by in situ hybridization (Harland, 1991). The neural tissue on the injected side (brackets) is obviously much wider along its entire length including hindbrain (hb) and spinal cord (sc) than that on the uninjected side. (B) Direct dorsal view of a late neural plate embryo shows a striking reduction in ectodermal Xtwist expression (in white boxes) on the XASH-3/XE12-injected right side (brackets). Anterior (a) is to the right. (C) A section through the spinal region of an embryo like that in A. In this section, also labeled for NCAM expression, we note the lateral expansion (between arrowheads) of the neural tissue on the injected right side (brackets). The normally sized notochord (nc), marking the midline, is visible directly below the neural tube (nt). (D) Expansion of the neural tube (nt) in a XASH-1/XE12-injected embryo. Here the tube was photographed using Nomarski optics. The white vertical bar marks the midline of the neural tube.
	Fig. 4. Phenotypic penetrance of injected embryos. Percentages of embryos injected unilaterally that express the neural hypertrophy and the Xtwist reduction phenotypes in one experiment. XASH-3/XE12 strongly produced both phenotypes in a high fraction of the embryos studied. XASH-1/XE12 injections were less effective. Small numbers on top of the bars indicate numbers of embryos studied in each condition. For these experiments, embryos at the two- or four-cell stage were injected with the mRNAs listed, in combination with b- gal mRNA. They were fixed in paraformaldehyde when they reached neural plate through neural tube stages, stained with X-gal, and then paraffin sectioned. If the embryo showed clear unilateral blue staining in the region of the neural plate or tube, it was then determined whether there was an obvious expansion of this tissue on the injected side. For the Xtwist phenotype, similarly injected embryos were simultaneously stained for b-gal activity and Xtwist expression by in situ hybridization (Harland, 1991). A reduction of Xtwist staining on the b-gal expression side was counted as a positive. The experiment looking at enlargement of the neural tube with XASH-3/E12 was carried out at least four times with five or more embryos, and in each case all the misexpressing embryos showed neural hypertrophies.
	Fig. 5. XASH-3/XE12 efficiently induces neural genes in noggin caps. Animal caps from embryos injected with noggin RNA are induced to express NCAM, but not the more differentiated neural marker type III b-tubulin (lane b). Low doses (90 pg) of XASH-3 mixed with XE12 induce neither (lane c). When noggin RNA is combined with the low dose of XASH- 3/XE12, the result is the induction of NCAM and b- tubulin that mimic the levels in normal embryos (lanes d and e).