Turner DL and Weintraub H (1994), Expression of achaete-scute homolog 3 in Xenopu...

XB-ART-21149

Genes Dev 1994 Jun 15;812:1434-47. doi: 10.1101/gad.8.12.1434.

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Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.

Turner DL , Weintraub H .

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In Drosophila, the proneural genes of the achaete-scute complex encode transcriptional activators that can commit cells to a neural fate. We have isolated cDNAs for two Xenopus achaete-scute homologs, ASH3a and ASH3b, which are expressed in a subset of central nervous system (CNS) neuroblasts during early neurogenesis. After expressing either ASH3 protein in developing Xenopus embryos, we find enlargement of the CNS at the expense of adjacent non-neural ectoderm. Analysis of molecular markers for neural, epidermal, and neural crest cells indicates that CNS expansion occurs as early as neural plate formation. ASH3-dependent CNS enlargement appears to require neural induction, as it does not occur in animal cap explants. Inhibition of DNA synthesis shows that additional CNS tissue does not depend on cell division--rather it reflects conversion of prospective neural crest and epidermal cells to a neural fate. The differentiation of the early forming primary neurons also seems to be prevented by ASH3 expression. This may be secondary to the observed activation of Xotch transcription by ASH3.

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Species referenced: Xenopus
Genes referenced: ascl1 ascl2 elavl1 hes4 lsamp ncam1 nefm notch1 tbx2 twist1
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	Figure 1. Alignment of ASH3a and ASH3b proteins to each other and to other members of the achaete-scute bHLH subfamily. Regions of sequence similarity between the vertebrate ASH1/ASH3 proteins and the Drosophila AS-C proteins are shaded. The bHLH domain of ASH3a/b has 65-71% amino acid identity with the bHLH domains of the Drosophila AS-C proteins (excluding the size difference in the loops), and 78% {ASH3b) or 82% (ASH3a) identity with the Xenopus ASHla bHLH domain. In addition to the bHLH domain, all of these proteins have a related sequence at the carboxyl terminus, within a larger acidic region. ASH3b appears to have a second version of this sequence that is most similar to achaete (ac, underlined). The carboxyl terminus of the Drosophila AS-C proteins has been suggested to be a regulatory site (Villares and Cabrera 1987). Limited sequence similarity is also present aminoterminal to the basic region in the vertebrate proteins and lethal of scute (l'sc). (sc) Scute. Sources for sequences: ac and sc (Villares and Cabrera 1987); l'sc (Alonso and Cabrera 1988); MASH1 (Johnson et al. 1990); and ASHla (Ferreiro et al. 1992).
	Figure 2. Expression of molecular markers in wild-type and ASH3b RNA-injected embryos. (A-C) Expression of ASH3a mRNA in wild-type embryos, detected by whole-mount in situ hybridization at three representitive stages. (A) An early neural plate embryo (stage 13.5) showing longitudinal stripes of ASH3a expression within prospective hindbrain and spinal cord. A transverse stripe is also present in the anterior of the future hindbrain (arrow). (B) Expression persists as a continuous longitudinal stripe (s) on each side of the spinal cord and hindbrain in an early tail bud embryo (stage 28), and ASH3a is also expressed in the retina (r) and forebrain (f). A second stripe of expression within the hindbrain is also visible (open arrow). The approximate extent of the hindbrain is shown by the bar above the embryos in both B and C. (C) The head of a later tail bud embryo (stage 34) showing the loss of ASH3a expression as neurons differentiate. ASH3a expression is shown by purple staining; brown dots are pigmented cells. The pigment epithelium of the eye is also brown. Note that expression in the retina is reduced to a band at the margin, the location of the retinal progenitors at this stage. The extent of expression in the forebrain and hindbrain is reduced, but the longitudinal stripes remain in the hindbrain and spinal cord (s). Anterior is toward the right in B and C and toward the top in A and D-M. Embryos in B, C, I, J, and M are cleared. N-CAM mRNA expression in a wild-type (D) and ASH3b RNA-injected embryo (E). A vertical bar shows the position of the midline and the injected side is labeled inj in E. The N-CAM-positive CNS is expanded laterally on the injected side along its entire length. Arrows indicate the boundaries of the prospective spinal cord; a double-headed arrow denotes the midline. The neural tube has failed to close because of the extra tissue. The eye vesicle (e) is labeled in D but is hidden in E because of the open neural tube. The embryo shown in E and other injected embryos shown in Figures 2-4 are examples of a strong but reproducible level of effect arising from ASH3b injection (see Materials and methods). (F) twist mRNA is expressed in the cranial neural crest but is almost absent after ASH3b injection (G). The arrow points out a small number of twist-positive cells that remain. N-CAM protein expression in an ASH3b-injected embryo treated with HUA (see text) also shows an expansion of the CNS (H). Arrows denote the borders of the prospective spinal cord; a double-headed arrow shows the midline. The injected sides of the embryos shown in H and I are marked by nuclear B-galactosidase expression (fine reddish dots). en-2 mRNA is expressed at the midbrain-hindbrain boundary (I). Like N-CAM, en-2 expression in expanded after ASH3b RNA injection (l). K and L show embryos injected with a DNA expression vector encoding an epitope-tagged ASH3b protein. The embryos have been stained with an antibody to the epitope tag (see Materials and methods). (K) ASH3b expression is outside of the dorsal ectoderm so the CNS does not show any enlargement. (L) ASH3b expression is present in the dorsal ectoderm and the future brain is expanded (arrows; vertical bar indicates midline). Expression in the embryo in (L) is restricted to the ectoderm (not shown). (M) An embryo injected with an ASH3b DNA expression vector showing the reduction of myosin heavy chain protein expression. Note that the blastopore is displaced toward the injected side (arrow). The curved axis is indicated. Similar effects were also seen after ASH3b RNA injection (not shown).
	Figure 3. Expression of molecular markers at neural plate and gastrula stages in ASH3b RNA-injected embryos. (A-D, F, G, I, J) The dorsal side of stage 13.5-14 embryos. Anterior is at the top. (A,F) Schematic diagrams showing the expression patterns of N-CAM, twist, hairy2a, and EpA molecular markers in a wild-type embryo (A) and an injected embryo (F) (see text for references). (B) CNS tissue, identified by N-CAM mRNA expression, is expanded on the injected side (inj). The arrows show the lateral limits of N-CAM expression; the position of the midline is marked. (C) twist mRNA is present in the prospective neural crest on the control side but not on the injected side (arrows). Midline staining in C is twist expression in the notochord and head mesoderm (unaffected by ASH3b). The dorsal limit of expression of the epidermal antigen EpA (brown staining) is moved away from the midline after ASH3b RNA injection (D). (E) The anterior end of a different EpA-stained embryo. In D, E, and G the injected side is marked by nuclear 13-galactosidase staining (blue dots). (G) hairy2a mRNA expression at the border of the neural plate is broader, at a lower level, and farther laterally from the midline on the injected side (compare location of arrows), but midline expression appears unaffected. (H) Dorsal view of a gastrula embryo (stage 11.5) showing the expansion of the area surrounded by hairy2a (purple staining) on the injected side (arrows). The injected side is marked by red staining for nuclear ~-galactosidase. The position of the blastopore lip is shown (white arrows). (I) Expression of Xotch mRNA is elevated on the ASH3b-injected side and wider (compare distance of arrows from the unstained midline). The trigeminal placode (t) is separate from the neural plate on the uninjected control side but contained within the expanded Xotch staining on the injected side. (l) myoDb mRNA is expressed at normal levels on both sides of this injected embryo, but the expressing cells do not extend as far anteriorly and spread farther laterally on the injected side.
	Figure 4. Persistence and differentiation of the enlarged CNS after ASH3b RNA injection. (A-D) N-CAM mRNA detected in swimming tadpoles (stage 45) by whole-mount in situ hybridization; staining is purple. Some brown pigmentation is present in the pigment epithelium of the eye, but these animals are otherwise albino. (A) A wild-type animal. (B-D) A tadpole arising from an injected embryo (inj: injected side). On the uninjected control side {B), the eye (e) is normally positioned within the mostly neural crest-derived eye socket, and the gill arches (g) and vagus complex (v) are normal sized. However, on the injected side {D), the eye is adjacent to the brain because of the reduced eye socket, the vagus complex is smaller, and the gill arches are absent (large open arrow). A dorsal view (C) shows the massive enlargement of the brain and anterior spinal cord on the injected side. Small open arrows in B and D indicate the olfactory organs. The olfactory organ on the injected side is enlarged. (E) Dorsal view of a tadpole (stage 45) stained for the 2G9 neural epitope (purple). The 2G9 epitope stains differentiated neurons. The brain and anterior spinal cord are expanded laterally on the injected side (black line shows midline), but the anterior-posterior patterning of the brain, revealed by staining intensity variations, appears normal. Some lateral line organs (o) are disorganized or missing on the affected side, whereas the gill arches are smaller (open arrow). The eye is also positioned closer to the brain on the injected side, reflecting a slightly reduced eye socket. (F) A dorsal view of tailbud embryos (stage 31) stained with an antibody to NF-M. The lower embryo is an uninjected control; the upper embryo was injected with ASH3b RNA on the marked side. At this age, NF-M is present in early differentiated neurons (primary neurons) and their processes. On the affected side of the upper embryo, the more anterior NF-Mexpressing cells in the hindbrain are disorganized (arrow), whereas NF-M-expressing cells are almost absent from the spinal cord (posterior to arrow). The embryos in F are cleared. Anterior is to the left in A-C, E, and F and to the right in D.