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The homeobox gene otx2 is a key regulator for specifying the rostral part of the vertebrate head. In Xenopus, otx2 directly controls the differentiation of the cement gland, the anterior-most organ formed in the tadpole. Since embryos of a direct developing frog, Eleutherodactylus coqui, lack a cement gland, we are interested in whether altered expression of the otx2 gene is involved in this evolutionary change. We have cloned the E. coqui homologue of otx2, Ecotx2. The homeodomain of the Ecotx2 protein is identical to the mouse and zebrafish Otx2 proteins and differs by a single amino acid from the Xenopus Otx2 protein. Study of the spatiotemporal expression pattern shows that Ecotx2 RNA is progressively restricted to the anterior region of the embryo during gastrulation and becomes further restricted to the future forebrain and midbrain during neural development. In Xenopus, in addition to the conserved expression in the anteriorneuroectoderm, the expression in ectoderm expands more anteriorly to the cement gland primordium. This anterior expansion of otx2 expression is not found in E. coqui, correlating with the loss of a cement gland. When misexpressed in Xenopus laevis ectoderm, Ecotx2 can activate expression of the cement-gland-specific genes XCG and XAG1, indicating that the function of activating the pathway of cement gland formation is retained by the Ecotx2 protein. Our results indicate that there are modifications in the pathway of cement gland formation, upstream of otx2 expression, in the development of E. coqui.
FIG. 1. Comparisons of the deduced amino acid sequences of the Ecotx2, Xotx2, and zotx2 gene products. The homeodomain is boxed.
Bold letters indicate an insertion of four amino acids (MFSA) located upstream of the Ecotx2 homeodomain and an amino acid change,
valine to isoleucine, found in the Xotx2 homeodomain. The black triangle indicates the position where a deletion of a single amino acid
was found in Ecotx2. PCR primers, P1 to P4, are also illustrated; they were used for PCR cloning of Ecotx2 cDNAs.
FIG. 2. Embryonic expression of Ecotx2. (AâC) Whole-mount in situ hybridization using a digoxygenin (DIG)-labeled probe specific for
Ecotx2. The entire yolk mass has been removed to increase accessibility of probes. In all panels, anterior is to the left. (A) Top view of an
early neurula (Sh13) with expression localized in a narrow band crossing the most anterior region of the neural plate (np). Arrowheads
indicate the lateral limits of Ecotx2 expression in the neural plate. b, blastopore. (B, C) Top views of mid- (Sh15) and late neurulae (Sh16).
Ecotx2 expression is confined to the future forebrain and midbrain. Arrows indicate the caudal limits of Ecotx2 expression. (D) Lateral view
of a late neurula of X. laevis, hybridized with a DIG-labeled Xotx2 probe. The expression of Xotx2 in the neuroectoderm is similar to Ecotx2
expression in E. coqui (C), with the caudal limit to the mid-/hindbrain junction (m/h; Blitz and Cho, 1995). However, an additional
expression domain, corresponding to the cement gland primordium (CG), is detected in X. laevis. This anterior-most expression of otx2 is
not found in E. coqui.
FIG. 3. Ecotx2 RNAs activate XCG gene expression in X. laevis animal caps. XCG expression was analyzed by whole-mount in situ
hybridization. (A) Animal cap explants from embryos injected with Ecotx2 RNAs. (B) Explants from embryos injected with Xotx2 RNAs.
(C) Explants from uninjected embryos. XCG transcripts were detected in both Ecotx2-injected and Xotx2-injected explants, but in neither
uninjected explants nor b-gal RNA-injected explants (not shown).
FIG. 4. Ecotx2 RNA injections activate expression of cement
gland marker XAG1. XAG1 expression was analyzed by RT-PCR
using EF-1a as a control. XAG1 expression was elevated in animal
cap explants injected with Ecotx2 RNAs (lane 3) or Xotx2 RNAs
(lane 4). A small amount of XAG1 transcripts was also detected in
uninjected explants (lane 2).
