Taira M et al. (1994), Expression of the LIM class homeobox gene Xlim-...

XB-ART-21217

Development 1994 Jun 01;1206:1525-36. doi: 10.1242/dev.120.6.1525.

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Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation.

Taira M , Otani H , Jamrich M , Dawid IB .

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The LIM class homeobox gene Xlim-1 is expressed in Xenopus embryos in the lineages leading to (i) the notochord, (ii) the pronephros, and (iii) certain cells of the central nervous system (CNS). In its first expression phase, Xlim-1 mRNA arises in the Spemann organizer region, accumulates in prechordal mesoderm and notochord during gastrulation, and decays in these tissues during neurula stages except that it persists in the posterior tip of the notochord. In the second phase, expression in lateral mesoderm begins at late gastrula, and converges to the pronephros at tailbud stages. Expression in a central location of the neural plate also initiates at late gastrula, expands anteriorly and posteriorly, and becomes established in the lateral regions of the spinal cord and hindbrain at tailbud stages. Thus Xlim-1 expression precedes morphogenesis, suggesting that it may be involved in cell specification in these lineages. Enhancement of Xlim-1 expression by retinoic acid (RA) was first detectable in the dorsal mesoderm at initial gastrula. During gastrulation and early neurulation, RA strongly enhanced Xlim-1 expression in all three lineages and also expanded its expressing domains; this overexpression correlated well with RA phenotypes such as enlarged pronephros and hindbrain-like structure. Exogastrulation reduced Xlim-1 expression in the lateral mesoderm and ectoderm but not in the notochord, suggesting that the second phase of Xlim-1 expression requires mesoderm/ectoderm interactions. RA treatment of exogastrulae did not revert this reduction.

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Species referenced: Xenopus laevis
Genes referenced: foxa4 gsc lhx1 mtor ncam1 tbxt
???displayArticle.antibodies??? B3gat1 Ab3 Ncam1 Ab1 Notochord Ab1

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	Fig. 1. Whole-mount in situ hybridization of Xlim-1 RNA. (A) Dorsal view of a stage 12 embryo (late gastrula). Anterior side (An) is top. Staining is detected in the prechordal mesoderm (pm) and forming notochord (n). Dark staining at the lateral to ventral edge is due to accumulated background. (B) Stage 12.5. The notochord is stained. Signal also appears in lateral mesoderm (black arrows) and in neural ectoderm (white arrows); the distinction between these regions is more clearly seen in Fig. 3G. (C) Ventrolateral view of stage 14 embryo showing that the staining in the lateral mesoderm forms a broad stripe. (D) Dorsal view of stage 14 (neural plate) embryo. Two lines of neural expression (white arrow, right only) are visible, which are now separated from the underlying mesoderm staining (black arrow). Notochord staining has diminished. (E) Dorsal view of stage 15/16 (neural fold) embryo. The two lines of neural expression expand anteriorly, and notochord staining disappears from the anterior part. (F) Stage 17/18. Neural expression expands posteriorly, while notochord staining disappears, except in the posterior tip. Lateral staining concentrates in the dorsal region (black arrow). (G) Anterior views with the plane of focus in the middle of the embryos at stages 14 (top panel), 15 (middle), and 16 (bottom). During the formation of the neural tube, staining is restricted to the deep layer. (H) Anterodorsal view of a stage 22 embryo. Pronephros (p) is stained. Neural expression is located in the hindbrain and spinal cord. Hindbrain staining is interrupted at the level of the otic vesicle, corresponding to rhombomere 4 (small white arrow). (I) Lateral view of head region of the same embryo as in H. A small white arrow indicates the gap of expression in the hindbrain. Weak staining occurs in the midbrain (open arrow). (J) Cross section of embryo at stage 28 (stained before sectioning) showing staining of the pronephros and lateral region of spinal cord. (K) Lateral view of stage 26 embryo. The expression in the midbrain is enhanced compared to that in I (open arrow). The gap of expression in the hindbrain becomes narrow (white arrow). The pronephros and pronephric duct on both sides are strongly positive. The posterior tip of notochord is still stained (arrow). (L) Lateral view of the tail bud region at stage 31, showing staining in the posterior tip of the notochord (arrow). An, anterior; n, notochord; p, pronephros; pm, prechordal mesoderm; s, somite.
	Fig. 2. Morphological appearance and mRNA levels of regulatory genes in RA-treated embryos.(A) Typical phenotype of an RA-treated embryo (upper panel), and normal control (lower panel), at the tailbud stage (stage 39/40). (B) Cross-section through the trunk of the embryo shown in A, stained with haematoxylin and eosin. (C) RNA blot analysis of RA-treated (RA) and untreated (C) embryos, at stage 11. The embryos were dissected into dorsal (D) and ventral (V) halves. The data on Xlim-1 and EF-1a (loading control) are from Taira et al. (1992); the same filter was rehybridized with XFKH1, Xbra, and gsc probes.
	Fig. 3. Effects of RA on Xlim-1 expression as visualized by wholemount in situ hybridization. (A) Vegetal view of stage 10.25 embryo (initial gastrula). Dorsal at the top. Xlim-1 expression in the dorsal marginal zone is enhanced in the RA-treated embryo (RA; right panel) compared to control (left). (B) Stage 11 (mid-gastrula). Enhancement of Xlim-1 expression by RA is pronounced. (C) Lateral view of stage 10.5/11 embryo. Dorsal to the right. In the untreated embryo (left), dorsal mesoderm is stained but ventral mesoderm and ectoderm are not; in the RA-treated embryo, ventral mesoderm and dorsal ectoderm are also stained. The embryos were bisected for photography. (D) Dorsal view of an RA-treated embryo at stage 12 (late gastrula). Forming notochord and anterior-lateral region are stained, but presumptive somite region is not. (E) RA-treated embryo at stage 14. Assignment of staining to neural plate (white arrow) and lateral mesoderm (black arrow) was made on the basis of anterior views (see panel G). The neural staining appears to be shifted anteriorly compared to the control (Fig. 1B,D). (F) Lateral view at stage 13. Anterior (An) is left and dorsal is up. The superficial ectodermal layer is not obvious due to light printing. Filled triangles indicate the anterior edge of staining. The stained region expands more anteroventrally in the RA-treated embryo, and the ventroposterior mesoderm is also stained. (G) Anterior views with the plane of focus on the middle of embryo. Left, control embryo at stage 13; right, RA-treated embryo at stage 12 (upper and lower parts are from the same embryo at different exposures; embryo was dissected for photography). The ectoderm region between the two arrowheads is stained. (H) Dorsal view of stage 22 embryo; anterior (An) is top. The pronephros (p) in the RA-treated embryo (right) is enlarged compared with that in the control (left). (I) Ventral view of RA-treated embryo; anterior is left. Xlim-1-positive tissue connects left and right pronephroi at their anterior edge. (J) Stage 33/34. The entire hindbrain-like region in the RA-treated embryo is stained (left). In the control (right), staining is seen in the midbrain, hindbrain, spinal cord, and more weakly, in the forebrain. Apparent staining in the eyes is due to natural pigmentation. An, anterior; p, pronephros; RA, retinoic acid-treated embryos.
	Fig. 4. Exogastrulae were stained by whole-mount in situ hybridization for Xlim-1 (blue), or by immunocytochemistry (brown) with Tor 70 (notochord marker), and with anti-NCAM and anti- HNK-1 antibodies (neural markers). (A) Exogastrulae at equivalent stage 13. Ectoderm and endomesoderm separated during processing. Xlim-1 stains the notochord, with the distal (anterior) tip being more intense. (B) Stage 17 equivalent; ectoderm and endomesoderm have separated. Thick arrows indicate apparent remnants of lateral mesoderm expression; thin arrows, staining in the posterior tip of notochord. (C) Stage 23/24 equivalent; ectoderm and endomesoderm have separated. Letters a-d identify ectodermal halves, which correspond to those in the lower panel. Weak Xlim-1 staining occurs in only two of five ectodermal parts (open triangles); the posterior tip of the notochord also stains (thin arrow; compare Tor 70 staining); lateral staining (thick arrow), which appears in one of the five endomesodermal halves, is substantially reduced compared with the pronephros staining in the control (N). Tor 70 staining (brown) shows well developed notochord in the endomesoderm (lower panel). (D) Stage 26 equivalent; exogastrulae have remained intact. Comparatively weak Xlim-1 staining in the ectoderm is seen in all samples (open triangles); the tip of the notochord (thin arrow) is also positive. Only one embryo shows weak staining in the endomesoderm (thick arrow). The posterior tip of the notochord projects into or close to the ectoderm (lower panel) where it is adjacent to the Xlim-1-positive regions, implying that these exogastrulae were incomplete. (E) Stage 23/24 equivalent. Arrows indicate anti-NCAM staining. (F) Stage 23/24 equivalent. Arrows indicate anti-HNK-1 staining. N, normal embryos.