Wright CV et al. (1990), The Xenopus XIHbox 6 homeo protein, a marker of...

XB-ART-25919

Development 1990 May 01;1091:225-34.

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The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons.

Wright CV , Morita EA , Wilkin DJ , De Robertis EM .

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XIHbox 6 is an early spatially restricted marker for molecular studies of neural induction. The sequence of the full-length XIHbox 6 protein is reported. An antibody raised against a beta-galactosidase/XIHbox 6 fusion protein was used to analyze the expression of XIHbox 6 proteins during frog embryogenesis. The anterior border of XIHbox 6 expression lies just posterior of the hindbrain/spinal cord junction. Immunostaining extends the entire length of the spinal cord. A much weaker transient expression with a similar anterior border is observed in mesoderm. Almost all nuclei in the newly closed spinal cord contain XIHbox 6. The number of positive nuclei decreases over the next stages of development, until in later embryos XIHbox 6 is restricted to nuclei of the dividing neuroepithelium, and not the mantle or marginal zones of the spinal cord. When the limb buds begin to grow, there is a second burst of XIHbox 6 expression in proliferating neurons of the cervical and lumbar enlargements, where nerves arise that supply the limbs. The data suggest that XIHbox 6 expression is spatially and temporally restricted to immature neurons of the spinal cord, before their differentiation into mature neurons.

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Species referenced: Xenopus laevis
Genes referenced: hoxb9

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	hoxb9 ( homeobox B9) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry, NF stage 32, lateral view, anterior left, dorsal up.
	Fig. 1. Construction of XlHbox 6//?-galactosidase fusion proteins. The full-length XlHbox 6 protein is represented as a box with the homeodomain filled in black. FP1 was made from a filled-in EcoRl cDNA fragment (Sharpe et al. 1987) cloned into filled-in SamHI-cut pTRBO. FP2 was made by cloning a Smal fragment from this same cDNA fragment into the same recipient vector. As stated in the text, all immunostaining patterns described in this paper are identical with antibodies purified on affinity matrices containing either FP1 or FP2.
	Fig. 2. Sequence of the XlHbox 6 homeodomain protein. (A) The nucleotide and deduced amino acid sequence of the XlHbox 6 homeodomain protein. The 61 amino acid homeodomain is shown in bold and amino acids are numbered in intervals of 10. The position of a 1.59 kb intron is indicated. Six glutamine residues start at Gln70. (B) Comparison of the XlHbox 6 homeodomain sequence with those of other genes with Abd-B-like homeodomains. Sequence sources are: Hox- 2.5 (Bogarad et al. 1989); Hox-1.7 (Rubin et al. 1987); Hox-3.2 (Breier et al. 1988); Hox-5.2 (Dolle and Duboule, 1989); HB4 (Dolecki et al. 1988); AbdB (Celniker et al. 1989); Antp (McGinnis et al. 1984; Scott and Weiner, 1984). A dash indicates identity with the XlHbox 6 amino acid residue above it. The number in brackets indicates the percentage similarity allowing for conservative amino acid replacements. The conservative changes allowed in this estimate were: T,S; F,Y; E,D; Q,N; K,R; L,V,I,M.
	Fig. 3. Expression of XlHbox 6 antigens during Xenopus embryogenesis. Albino embryos were fixed at various stages and subjected to whole-mount immunostaining with XlHbox 6 antibodies. (A and A') Lateral and dorsal views of stage 23-24; lateral views in all subsequent panels. (B) Stage 26; (C) stage 29; (D) stage 32. Positive staining is at all times nuclear. The anterior border, which is constant throughout all of these stages, is marked by an arrowhead. The apparent staining in the cement gland (eg) and in the somite nuclei (myo) is artefactual as explained in the text. Abbreviations: arch, archenteron; endo, endoderm; eg, cement gland; myo, myotome; LPM, lateral plate mesoderm.
	Fig. 4. Expression of XlHbox 6 antigens is limited to posterior central nervous system. (A) A tailbud embryo (stage 25) was paraffin sectioned and immunostained for XlHbox 6 antigens. Black areas in the spinal cord represent immunostaining. This embryo was curled so that an anterior section through the brain is at left and a section through the spinal cord is at right. (B) Expression of XlHbox 6 in a stage 38 spinal cord. This is a slightly skewed, or oblique, lateral section. The periodic row of black dots (arrowheads) represents staining in a very few nuclei at the ependymal surface of the spinal cord. Abbreviations: ec, ependymal canal; sc, spinal cord; myo, myotomes; hb, hindbrain; no, notochord; endo, endoderm; LPM, lateral plate mesoderm; df, dorsal fin.
	Fig. 5. Most neuronal precursor cells express XlHbox 6 in stage 24 spinal cord. Dorsal is uppermost in all panels. (A) Section at high power magnification immunostained with anti-XlHbox 6 antibodies. (B) Same section counterstained with Hoechst 33258 viewed under dark field illumination. Note that nearly all nuclei appear to be darkly stained in panel A and that the Hoechst fluorescence in these nuclei is quenched by the immunostaining reaction product. (C) XlHbox 6 expression in lateral plate mesoderm. Transverse section of stage 25-26 tadpole decorated with XlHbox 6 antibodies and overstained to show more clearly the less intense staining in the lateral plate mesoderm nuclei. Abbreviations: ecto, ectoderm; ec, ependymal canal; sc, spinal cord; myo, myotome; LPM, lateral plate mesoderm; endo, endoderm; arch, archenteron; no, notochord, remains of blastopore.
	Fig. 6. Distribution of XlHbox 6 antigens in the spinal cord of stage 27 tadpole. A transverse section was (A) immunostained with XlHbox 6 antibodies and (B) counterstained with Hoechst 33258. At this stage of development, the spinal cord starts to display a degree of differentiation into defined layers. Now, not all of the spinal cord nuclei express XlHbox 6 antigens. The more peripheral nuclei which probably correspond to differentiated primary neurons are no longer stained. Immunostaining is confined to the entire inner layer (ependymal layer) and a few cells that have migrated laterally away from this mitotic zone. Note the nuclei that are much less intensely immunostained in the lateral plate mesoderm indicated by arrowheads. Abbreviations: ec, ependymal canal; sc, spinal cord; no, notochord; endo, endoderm; myo, myotome; ecto, ectoderm; LPM, lateral plate mesoderm.
	Fig. 7. Expression of XlHbox 6 antigens at early metamorphosis. A stage 49 albino tadpole was immunostained in whole-mount with anti-XlHbox 6 antibodies and then dissected under clearing solution to remove the central nervous system and tightly associated structures. (A) Low power magnification showing two condensations of intensely stained nuclei in the spinal cord (CE and LE). The arrow labelled Ant.B shows that the same anterior border of XlHbox 6 expression (Ant.B.) is observed as in earlier stages. (B) Dorsal view at higher power showing the cervical and lumbar condensations in more detail. Note the sympathetic ganglia which represent a possible additional site of XlHbox 6 expression. (C) Lateral view of the same tissue. Note that there are many immunostained nuclei between CE and LE, and also posteriorly of LE (arrowheads). Abbreviations; Ant.B., anterior boundary of XlHbox 6 expression; hb, hindbrain; sc, spinal cord; myo, myotome; sg, sympathetic ganglia; no, notochord; CE, cervical enlargement; LE, lumbar enlargement.