September 1, 1993;
Expression of LIM class homeobox gene Xlim-3 in Xenopus development is limited to neural and neuroendocrine tissues.
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
. 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
, 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.
[+] show captions
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
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.