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???displayArticle.abstract??? Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.
Fig. 1. Sequence alignment of Shisa family members
and temporal expression of Xshisa-2 during Xenopus
development. (A) Comparison of the predicted
amino acid sequence of X. laevis Shisa-2 with X. laevis
Shisa and mouse Shisa. Xshisa-2 shares 76.9% of identity
(positives - 84.4%) with mShisa-2 and 30.7% identity
(positives - 46.3%) with Xshisa. Identical amino acids
among all are shaded red while identical amino acids in
only two sequences are shaded blue. The absence of
residues at the corresponding region is indicated by
dashes. The two conserved cysteine-rich domains (CRD)
are shown in green. The GenBank accession number for
X. laevis Shisa-2 is DQ342341. (B) Temporal expression
pattern of Xenopus Shisa-2 by RT-PCR analysis. RT-PCR
was performed with total RNA from different developmental
stages. Xshisa-2 transcripts are present maternally
at very low levels, increase in the beginning of
neurulation and continue to be expressed during early
development. Stages are indicated on top. ODC was
used as a loading control.
Fig. 2. Xshisa-2 expression in the end of
gastrulation and beginning of neurulation.
Whole mount in situ hybridization with a
Shisa-2 DIG-labeled antisense RNA probe
was performed on embryos at the end of
gastrulation and beginning of neurulation. (A)
Xshisa-2 expression by the end of gastrulation
(st 13) is restricted to two narrow stripes
on the dorsal side of the embryo, but not in
the notochord. Posterior dorsal view. (B-C)
Hemisections of stage 12 and 14 embryos show that Xshisa2 mRNA is restricted to the posterior portion of the paraxial mesoderm. Hemisections with
dorsal to the right. A, anterior; P, posterior; D, dorsal; V, ventral; ar, archenteron; bc, blastocoel; pm, paraxial mesoderm; sm, somitic mesoderm.
Fig. 3. Expression pattern of Xshisa-2 during tailbud stages. From early tailbud stage onward, a dynamic pattern is observed in the forming
somites. (A-B) At stage 18, Xshisa-2 becomes progressively reduced in the anterior paraxial mesoderm. (B’) Transverse section showing Xshisa-2
expression in the entire somite region. (C) Parasagittal section of a stage 18 embryo. (D) Double whole mount in situ hybridization with Xshisa-2 DIGlabeled
antisense RNA probe and XmyoD fluo-labelled antisense RNA probe. The expression domain of XmyoD extends more posteriorly than the
Xshisa-2 expression domain. (E-H,J-K) Expression of Xshisa-2 is stronger in the presomitic mesoderm and decreases as somites form. Transverse
sections through the trunk region of stage 27 (G’’) and 30 (J’) embryos show Xshisa-2 expression in the entire somite. (H,H’) Double whole mount in
situ hybridization with Xshisa-2 and PAPC shows that Xshisa-2 is not expressed in the unsegmented region of the presomitic mesoderm. (I) Whole mount
in situ hybridization with Xshisa shows expression restricted to the head region, a distinct expression pattern than the one observed for Xshisa-2. (K’)
A complex expression pattern is also observed in the head, including the lens (ln) and branchial arches (ba). (A,C,E-G,H-J,K,K’) Lateral view. (B,D,G’). Dorsal
view. All embryos are oriented with anterior to the right.
