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In a search for novel developmental genes expressed in a spatially restricted pattern in dorsal ectoderm of Xenopus we have identified XAG-2, a cement gland-specific gene with a putative role in ectodermal patterning. XAG-2 encodes a secreted protein, which is expressed in the anterior region of dorsal ectoderm from late gastrula stages onwards. Activation of XAG-2 transcription is observed in response to organizer-secreted molecules including the noggin, chordin, follistatin and cerberus gene products. Overexpression of XAG-2 but not of the related cement gland marker XAG-1 induces both cement gland differentiation and expression of anterior neural marker genes in the absence of mesoderm formation. Further, we show that XAG-2 signaling depends on an intact fibroblast growth factor (FGF) signal transduction pathway and that XAG-2-induced anterior neural fate of ectodermal cells can be transformed to a more posterior character by retinoic acid. Based on these findings we propose a role for XAG-2 in the specification of dorsoanterior ectodermal fate, i.e. in the formation of cement gland and induction of forebrain fate of Xenopus.
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9533957
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Fig. 2. XAG-2 encodes a secreted protein. Autoradiograph showing the
protein product of in vitro translated XAG-2 RNA (ivt XAG-2), of XAG-
2fs injected control oocytes (cont. oocyte) and XAG-2-injected oocytes
(oocyte + XAG-2). Further, the protein pattern of medium conditioned by
XAG-2fs-injected oocytes (cont. medium) and medium conditioned by
XAG-2-injected oocytes (XAG-2 cond. medium) is shown. Note that
both the high molecular weight form of XAG-2 protein (indicated by an
asterisk) and the low molecular weight form of XAG-2 (marked by an
arrowhead) are found in the XAG-2 conditioned medium, while the control
medium does not contain detectable amounts of these protein products.
Fig. 3. XAG-2 is expressed in anterior non-neural ectoderm, activated by LiCl treatment and reduced in ventralized embryos. (A) Whole-mount in situ
hybridization showing the localization of XAG-2 RNA during various developmental stages as well as in LiCl and UV-treated embryos (G). (A) XAG-2
expression during gastrulation and neurulation. XAG-2 transcripts are first detected at mid to late gastrula stage (stage 11 3/4) in the anterior part of the
prospective neural plate (albino embryo on the left). Inset shows at a higher magnification the spotted pattern of XAG-2 expression in anterior dorsal
ectoderm. At the end of gastrulation, XAG-2 transcripts are confined to the anterior border between dorsal and ventral ectoderm, a region that corresponds to
the cement gland anlage (albino embryo in the middle). Pigmented wildtype embryo on the right shows specific expression of XAG-2 mRNA in the
developing cement gland (white arrow) at the end of neurulation (stage 19). (B) Sagittal section through an early neurula stage embryo probed for XAG-2
mRNA. Cells highlighted by XAG-2 (blue) will give rise to the cement gland. Anterior is to the left; bar = 125 mm. (C) Double in situ hybridization of stage
12 albino embryos (anterior view) using XAG-2 and Xcpl-1 as probes. XAG-2 RNA (turquoise) is located to the anterior non-neural ectoderm while Xcpl-1
RNA cannot yet be detected. (D) Anterior view of an early neurula embryo (stage 13) stained for XAG-2 (turquoise) and Xcpl-1 (purple) RNA. Xcpl-1 RNA
is localized in the anterior neural ridge immediately adjacent to the XAG-2 expression domain. Although difficult to determine by whole-mount in situ
hybridization, an overlap between XAG-2 and Xcpl-1 expressing cells is not visible, suggesting that the anterior-most ectoderm is precisely patterned already
at the end of gastrulation. (E) Anterior view of a mid-neurula stage embryo (E) and an embryo at stage 17 of development (F) stained for XAG-2
(turquoise) and Xcpl-1 (purple). The expression domains of XAG-2 and Xcpl-1 are more clearly separated at these stages with XAG-2 labeling the cement
gland anlage and Xcpl-1 highlighting the anterior neural ridge. (G) Transcription of XAG-2 is enhanced by LiCl-treatment. Lateral view of early neurula
stage embryos, anterior to the right. LiCl-treatment leads to ectopic activation of XAG-2 expression such that the domain of XAG-2 expressing cells extends
posteriorly to the blastopore (wildtype embryo on the right), while in untreated embryos (albino embryo on the left) XAG-2 transcripts are restricted to the
anterior region, only. (H) Anterior view of an early neurula embryo ventralized by UV-irradiation (embryo on the right) and an untreated control embryo
(embryo on the left), each probed for XAG-2 mRNA. Note that the expression domain of XAG-2 is markedly reduced by UV-treatment when compared with
the expression domain in untreated control embryos. The average DAI (Kao and Elinson, 1988) of this batch of ventralized embryos was 2. Abbreviations:
ae, archenteron; ane, anterior neuroectoderm; bc, remnant of blastocoel; cga, cement gland anlage; ne, neuroectoderm; pcp, prechordal plate.
Fig. 4. RT-PCR analysis of XAG-2 expression in animal cap explants in
response to neural inducing molecules such as noggin, a dominant negative
BMP-receptor (tBR), chordin and cerberus. While in control explants
(control) only trace amounts of XAG-2 transcripts are present, injection
of RNA coding for noggin (125 pg), tBR (4 ng), chordin (200 pg) or
cerberus (200 pg) results in transcriptional activation of XAG-2 in animal
caps. Xbra expression was analyzed to demonstrate that no mesoderm was
present. EF1-a expression served as a loading control. -RT indicates that
the reverse transcriptase was omitted from this reaction; WE, RNA from
whole embryos was used in this reaction.
Fig. 5. XAG-2 but not XAG-1 induces cement gland formation. (A) Anterior-ventral view of embryos injected with 2 ng XAG-2 RNA (upper row), control
XAG-2fs RNA (embryo in the lower left corner) or 2 ng XAG-1 RNA. Solely embryos injected with XAG-2 RNA show either enlarged cement glands (upper
left embryo) or ectopic cement gland structures (indicated by arrowheads). Neither injection of XAG-2fs RNA nor overexpression of XAG-1 RNA influences
the development of the cement gland. (B) Anterior-lateral view of XAG-2 injected embryos. Ectopic cement gland structures are pointed out by black
arrowheads. (C) Animal caps injected with XAG-2 RNA develop a morphologically differentiated cement gland (white arrows), while control explants (three
lower explants) injected with XAG-2fs RNA form atypical epidermis. (D) Histological analysis of animal caps injected either with XAG-2 or with XAG-2fs
RNA. Control-injected animal caps form atypical epidermis (cont), while animal caps injected with XAG-2 mRNA (+XAG-2) develop large cement gland
structures (cg) as judged by the formation of columnar epithelial cells. Note that differentiated neural tissue is not present in XAG-2-injected animal cap
explants. Bar = 100 mm.
Fig. 6. XAG-2 but not XAG-1 induces expression of anterior neural marker genes in Xenopus animal cap explants. (A) RT-PCR analysis of animal caps
injected with non-functional XAG-2fs RNA or XAG-2 RNA. Animal caps were cultured until siblings reached stage 24 of development. Explants were
assayed for the expression of the panneural marker N-CAM, the anterior neural markers otx2 and XIF3, the midbrainindbrain marker en-2, a marker for
rhombomeres 3 and 5, Krox-20, the spinal cord-specific marker Xlhbox6 and the mesodermal marker muscle actin. EF1-a was used as an internal loading
control. WE, RNA from whole embryos isolated at stage 24 was used in this reaction. (B) RT-PCR analysis of otx2 expression in animal caps injected with
the amount of XAG-2 RNA as indicated. Significant activation of otx2 expression in response to XAG-2 occurs at 200 pg reaching a maximum level at 1 ng
of injected XAG-2 RNA. Lack of Xbra expression in explants indicates that mesoderm was not present in animal caps. (C) RT-PCR analysis of ectodermal
explants isolated either from XAG-2- or XAG-1-injected embryos. Activation of the anterior neural marker genes otx2 and XANF-2 is only observed in
response to XAG-2 expression, while explants from XAG-1-injected embryos do not differ from controls. In (B) and (C) ornithine decarboxylase (ODC)
expression was used as a loading control. -RT, reverse transcriptase was omitted from the reaction: WE, RNA from whole embryos was used in this reaction
as a positive control.
Fig. 7. Overexpression of XAG-2 results in ectopic expression of anterior neural marker genes in whole embryos. (A) Anterior view of an uninjected stage 14
albino embryo (left embryo) and an embryo injected with XAG-2 RNA both probed for otx2. Ectopic otx2-expressing cells in ventral ectoderm are indicated
by arrowheads. Note that ectopic otx2 expressing cells (purple) are in close proximity to nuclear b-galactosidase expressing cells (turquoise). (B) Anteriorventral
view of an XAG-2 injected embryo stained for otx2 expression. Ectopic otx2 expressing cells (arrowheads) are found in ventrolateral ectoderm which
normally would form epidermis. Note that ectopic otx2-positive cells are located to the injected side (indicated by +), only. (C) XAG-2 can induce ectopic
expression of the anterior neural marker Xcpl-1. Anterior-lateral view of an early neurula embryo injected with XAG-2 RNA and subsequently probed for
Xcpl-1 expression. Again, ectopic Xcpl-1 expressing cells (marked by arrowheads) are localized to the injected side. Endogenous Xcpl-1 expression is
indicated by a black arrow. (D) Lateral view of an embryo at stage 13 injected with XAG-2 RNA and probed for Xcpl-1. Arrowheads indicate ectopic Xcpl-1
expressing cells (purple) in ventral ectoderm. Turquoise cells represent cells with nuclear b-galactosidase activity. The white arrow points at endogenous
Xcpl-1 expression domain in the anterior neural ridge.
Fig. 8. (A) XAG-2 signaling requires an intact FGF signal transduction pathway. RT-PCR analysis of ectodermal explants from embryos co-injected either
with 2 ng XAG-2 and 2 ng HAV RNA or with 2 ng XAG-2 and 2 ng XFD RNA. Anterior neural markers such as XANF-2 and otx2 are only activated in
XAG-2/HAV expressing animal caps, while co-expression of XAG-2/XFD results in a reduction of otx2 and XANF-2 expression to background levels. (B)
Treatment of XAG-2 expressing animal caps with all-trans retinoic acid results in posterior transformation. RT-PCR analysis of uninjected animal caps
treated with retinoic acid (+RA), of XAG-2 injected animal caps (+XAG-2) or of XAG-2 injected animal caps treated with retinoic acid (XAG-2 + RA).
Activation of the posterior neural markers Krox20 and Xlhbox6 concomitant with reduction of XANF-2 expression demonstrates transformation of XAG-2
expressing explants in response to retinoic acid treatment. Expression of ODC was used as a loading control in (A) and (B). -RT, reverse transcriptase was
omitted from the reaction; WE, RNA from sibling embryos (stages 123) was used for RT-PCR.
