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Abstract
A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrulaembryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann''s organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.
Figure 1. [A] Nucleotide sequences of PCR products obtained as described in Materials and methods. The predicted amino acid
sequence of Xlim-l and Xlim-2B, which is identical, is shown at the top-, that of Xlini-3 is at the bottom. Underlining indicates amino
acids that are different from Xlim-l. Xlim-ZA has a single-amino-acid difference, isoleucine (ATT codon), which is also underHned.
Dots indicate identical nucleotides. The GenBank/EMBL accession numbers for sequences of PCR clones 30, 152, and 171 are Zl 1587,
Z11588, and Z11589, respectively. [B] Nucleotide and predicted amino acid sequence of Xlim-l. Amino acids are in the single-letter
code. Asterisks denote in-frame stop codons. Below the sequence the general structure of the cDNA clone, pXH32, is illustrated. The
GenBank/EMBL accession number is X63889. (C) Comparison of LIMl, LIM2, and homeo box domains of Xlim-l, lin-11 (Freyd et al.
1990), mec-3 (Way and Chalfie 1988), lsl-1 (Karlsson et al. 1990), and rhombotin (McGuire et al. 1989; Boehm et al. 1990). Identities
are indicated by dots; spaces inserted for alignment, by dashes. The consensus LIM motif is shown by vertical bars. The positions of
helix 1-3 and the turn are indicated in the homeo domain (Scott et al. 1989). PCR primers A and B are shown by arrows.
Figure 2. [A] Developmental expression of Xlim-1. Total RNA
samples (7.5 |xg) from stages 0 (unfertilized egg), 8 (early blastula),
9 (late blastula), 10 (initial gastrula), 11/12 (late gastrula),
14/15 (neural plate stage), 18/19 (neural tube stage), 23/24 (tail
bud stage), 33/34 (early tadpole), and 37/38 (tadpole) were separated
on a gel, blotted, and hybridized with an Xlim-l probe
(see Materials and methods). The positions of 18S and 28S
rRNAs are indicated at right. [B] Tissue distribution of Xlim-1
mRNA in adult Xenopus. Ten micrograms of total RNA was
loaded onto each lane. Staining of the gel with ethidium bromide
verified that closely similar amounts of RNA were loaded
onto each lane.
Figure 3. Spatial distribution of Xlim-1 mRNA in gastrula embryos.
Stage 11 Xenopus embryos were dissected into ectoderm
(Ec) and endoderm plus mesoderm (En -I- M), or into ventral (Ven)
and dorsal (Dor) halves. Ten micrograms of total RNA was applied
on each lane. Mix.l and EF-la were analyzed for comparison
(see text).
Figure 4. Spatial distribution of Xlim-1
mRNA in the gastrula as visualized by
whole-mount in situ hybridization (see
Materials and methods). A and B show the
same stage 11 embryo. A is photographed
from a lateral viewpoint, with the focal
plane at the center of the embryo; animal
at the top and dorsal on the right. The blastocoel
has largely collapsed during preparation.
B shows a vegetal view tilted
slightly dor sally; dorsal is at the top. In this
early midgastrula embryo the mesoderm
has involuted only a short distance from
the dorsal lip. Xlim-1 is expressed at the
dorsal lip and in a fan-like widening at the
anterior margin of the mesoderm. C and D
show a slightly older embryo at stage 11.5,
with animal at the top. The mesoderm has
migrated farther toward the animal pole, as
seen in the lateral view in C; dorsal is at
the right. The dorsal view in D shows that
the highest Xhm-1 expression is now located
in the anterior region of the dorsal
mesoderm, which will shortly form the
prechordal plate.
Figure 5. Induction of Xlim-l expression in animal explants. [A] Stage 8-8.5 animal explants were cultured in the presence of different
factors for 4 hr. (B) Stage 8-8.5 explants were cultured for 5 hr until siblings reached stage 10.5. (C) Stage 9-9.5 animal explants were
cultured for 30 min in CHX, 30 min in CHX plus activin A, and 2 hr in activin A; siblings reached stage 10-10.5. Five micrograms of
total RNA from each sample was analyzed for expression of different genes, as indicated. In A, hybridization with EF-la, and in B and
C, staining with ethidium bromide (not shown) verified that closely similar levels of RNA had been loaded. The following factor
concentrations were used: activin A, 50 pM (1.4 ng/ml); RA, 10 JJLM; TGF-p2, 200 ng/ml; TGF-p3, 50 ng/ml; bFGF, 100 ng/ml;
cycloheximide (CHX), 5 |xg/ml.
Figure 6. [A] Kinetics of induction of
Xlim-l and Mix.l in animal explants by 50
pM activin A. {B] Kinetics of induction of
Xlim-1 by 10 ixM RA and activin (50 pM)
plus RA; two data points with activin
alone show that the peak expression of
Xlim-1 in this experiment occurred at 4 hr,
as shown in A. A and B are different experiments.
The expression of the marker gene
EF-la increases continuously with incubation
time.
Figure 7. Expression of Xlim-1 in RA-treated embryos. Embryos
at stage 8.5-9 were exposed to 10 |JLM RA for 30 min, as
described (Durston et al. 1989). Total RNA was extracted at
various stages and analyzed for Xlim-1 RNA. At stage 11 some
of the embryos were dissected into dorsal and ventral halves and
5 |xg of total RNA was analyzed. Blotting with EF-la probe
showed that equal amounts of dorsal and ventral RNA were
loaded onto the gel.
