Taira M et al. (1993), Expression of LIM class homeobox gene Xlim-3 in...

XB-ART-22271

Dev Biol 1993 Sep 01;1591:245-56. doi: 10.1006/dbio.1993.1237.

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Expression of LIM class homeobox gene Xlim-3 in Xenopus development is limited to neural and neuroendocrine tissues.

Taira M , Hayes WP , Otani H , Dawid IB .

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The Xenopus LIM class homeobox gene Xlim-3 was identified initially as a fragment isolated by polymerase chain reaction cloning with an embryonic cDNA library as template (Taira et al., 1992, Genes Dev. 6, 356-366). cDNA clones representing most of the Xlim-3 mRNA were isolated from an adult brain library. The predicted Xlim-3 protein contains two copies of the LIM domain, a homeodomain, and a C-terminal region rich in proline, glycine, and serine. RNA blot hybridization showed that Xlim-3 mRNA is detected in dorsal regions at neural tube and tailbud stages and in adults predominantly in the pituitary gland and weakly in the eye and brain. Whole mount in situ hybridization revealed that Xlim-3 mRNA is first detectable at the neural plate stage in the stomodeal-hypophyseal (pituitary) anlage and in the neural plate where labeled cells were found adjacent to the forming floor plate. In situ hybridization analysis on serial sections at later stages showed that embryonic Xlim-3 expression persists in the pituitary and pineal, as well as in some cells of the retina, hindbrain, and spinal cord. In the retina, Xlim-3 mRNA was only detected in a distinct sublamina of the inner nuclear layer, but not in dividing cells of ciliary margin. This discrete manner of Xlim-3 expression, especially persistent expression in the pituitary (before morphogenesis of the gland to adult), supports a role in the specification and maintenance of differentiation of distinct neuronal and neuroendocrine tissues.

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Species referenced: Xenopus
Genes referenced: lhx1 lhx3 pomc prl.1

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	FIG. 1. eDNA clones and nucleotide sequence of Xlim-8. (A) Schematic representation of the cDNAs. Top: thin line, noncoding region; box, coding region; shaded box, LIM domains; filled box, homeodomain. Below: eDNA clones that were sequenced. Thin line, extent of Xlirn-S sequences; thick line, unrelated sequence; vertical bar, EcoRl site. (B) Nucleotide and deduced amino acid sequence of Xlim-9. LIMl and LIM2 domains and homeodomain are indicated by arrows above the amino acid sequence . .Asterisks, stop codons. eDNA and PCR clones and an oligonucleotide probe used for in situ hybridizat ion of sections are shown under the nucleotide sequence. The nucleotide sequence of eDNA pXH23-1, shown in the figu re, is identical to that of PCR clone #189; in PCR clone #171, one nucleotide is G instead of A, changing the amino acid f rom Asn (N) to Asp (D) as shown in parentheses. This difference may represent the t.wo expected copies of the Xlim-3 gene in the pseudotetr aploid genome of Xenopus laevis (Graf and Kobel, 1991). Sequence data from this article have been deposited with EMBL/ GenBank Data Libraries under Accession No. Z2270'2.
	F IG. 2. Comparison oi deduced amino acid sequence of Xlim-3 with other LIM proteins. (A) Alignment of Xlim-3 and Xlim-1 sequences Dot, identical residues; dash, gaps. (B) Diagram of LIM class homeodomain proteins. Shaded box, LIM domains (LlMl and 11M2); filled box, horneodomain (HD); percentage sequence identity to Xlim-3 is indicated in each doma in.
	FIG. 3. RNA blot hybridi;t;aiion. (A) Embryonic expression. Stage 18/19 embryos (neural tube stage) were dissected into anter ior and posterior, or dorsal and ventral parts (left). Stage 33/34 embryos (tailbud) were dissected into head, anterior-dorsal, posterior-dorsa\, a nterior- ventral, and posterior-ventral parts (right). The blot was probed with the antisense Xlim-3 RNA probe and exposed for 23 days (upper panels). Trans lation in it iat ion factor EF- la was used as a measure of total RNA loaded on each lane (lower panels). (B) Tissue distribution of )l."li'm-3 RNA in adult X enopus. Two separate expP.riments are shown. (Left) the blot used for Xlim-1 (Taira et al., 1992} was reprobed with Xlim..- 3 a nd exposed for 15 days (left); t he upper band of 3.4 kb in the brain is a remnant of Xlim-1 hybridization. (Right) a different blot was exposed to a phosphor screen for 5 days and analyzed using the Phosphorlmager. Short lines represent pos itions of 18S and 28S rRNAs.
	FIG. 4. In situ whole moun\t hybridization of Xlim-3 RNA. (A) Lateral view o! neural plate stage embryo (stage 14). (B) Anterior view of the same embryo as in (A). The plane of focus is on the middle portion of embryo. (C) Lateral view of neural tube stage embryo (stage 23). (D) Anterodorsal view of the embryo shown in (C). (E) Stage 31 embryo. (F-K) Head regions of (F) stage 24, (G) stage 26, (H) stage 28 (dorsal view), (I) stage 33/34, (J) stage 37/38, (K) stage 37/38 (dorsal view). Open triangle, hybridization to spinal cord and hindbrain solid triangle, anterior site of xlim-3 expression that corresponds to the stomodeal-hypophyseal anlage at earlier stages (C, D, F, G) and to the pituitary rudiment at later stages (E, l); asterisk, the pineal anlage (F, G, H) and the pineal gland (E, I, J); OV, otic vesicle.
	FIG. 5. Coronal sections through the head of a stage 39/ 40 embryo, viewed in brightfield and darkfield. (A) Section showing Xlim-3 labeling over the entire pineal body (P), a ball of cells derived from the dorsomedial roof of the diencephalon, anterior to the level of the retina. (B) Low-power micrograph showing bilateral Xlim-3 gene expression in retina and more medially in the anterior pituitary (ap), dorsal to pharynx (Px), ventral to the infundibulum (inf) of posterior diencephalon (Di). (C) At a higher magnification, Xlim-3 mRNA is seen in sublamina-specic retinal cells in the outermost part of the inner nuclear layer (inl), but not in cells of the ganglion cell (gel), outer nuclear ( onl), pigmented retinal epithelial (pre) layers, or lens (L); note also the abrupt boundary (thick arrows) between the labeled and the unlabeled prollferatmg cells of the ciliary margin (cm). Calibration bars are (A) 100 um , (B) 200 um, (C) 50 um.
	FIG. 6. Coronal sections from just posterior to the otic vesicle showing the highly restricted Xlim-3 expression pattern at low (A) and high (B) magnification in the thin-roofed stage 39/40 hindbrain. (.A) Brightfleld and clarkfield views of the same section. Xlim-3-expressing cells were found similarly in other serial sections later al to the floor plate (fp) of ventral rhombencephalon (Rh), dorsolateral to the notochord (Nc). (B) Labeled cells are peripheral to, but not within the ventricular zone (vz) from which t hey arc thought to arise, indicating they may be postmitotic. Calibration bars are 100 urn.
	FIG. 7. Brightfield and darkfield photomicrographs of serial coronal sections from late larval stage 63 midbrain (mb) and the more ventral anterior (AL) and intermediate (IL) pituitary lobes, showing the relationship of .Xlim-3 gene expression (A) to that of neuropeptide genes for prolactin and growth hormone (B) and proopiomelanocortin (C) (Hayes and Loh, 1990). Xlim-3 mRNA is expressed in both lobes but not midbrain and is not correlated with the expression of a particular neuropeptide; furthermore, Xli»t-3 mRNA abundance appears to be much lower than that of the neuropeptide mRNAs. Calibration bar is 200 um.