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Development
1999 Oct 01;12620:4513-23. doi: 10.1242/dev.126.20.4513.
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The homeobox gene, Xanf-1, can control both neural differentiation and patterning in the presumptive anteriorneurectoderm of the Xenopus laevis embryo.
Ermakova GV
,
Alexandrova EM
,
Kazanskaya OV
,
Vasiliev OL
,
Smith MW
,
Zaraisky AG
.
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From the onset of neurectoderm differentiation, homeobox genes of the Anf class are expressed within a region corresponding to the presumptive telencephalic and rostral diencephalic primordia. Here we investigate functions of the Xenopus member of Anf, Xanf-1, in the differentiation of the anteriorneurectoderm. We demonstrate that ectopic Xanf-1 can expand the neural plate at expense of adjacent non-neural ectoderm. In tadpoles, the expanded regions of the plate developed into abnormal brain outgrowths. At the same time, Xanf-1 can inhibit terminal differentiation of primary neurones. We also show that, during gastrula/neurula stages, the exogenous Xanf-1 can downregulate four transcription regulators, XBF-1, Otx-2, Pax-6 and the endogenous Xanf-1, that are expressed in the anteriorneurectoderm. However, during further development, when the exogenous Xanf-1 was presumably degraded, re-activation of XBF-1, Otx-2 and Pax-6 was observed in the abnormal outgrowths developed from blastomeres microinjected with Xanf-1 mRNA. Other effects of the ectopic Xanf-1 include cyclopic phenotype and inhibition of the cement gland, both by Otx-2-dependent and -independent mechanisms. Using fusions of Xanf-1 with the repressor domain of Drosophila engrailed or activator domain of herpes virus VP16 protein, we showed that most of the observed effects of Xanf-1 were probably elicited by its functioning as a transcription repressor. Altogether, our data indicate that the repressor function of Xanf-1 may be necessary for regulation of both neural differentiation and patterning in the presumptive anteriorneurectoderm.
Fig. 1. (A) At the late gastrula stage, Xanf-1 is expressed in
the anterior neurectoderm. Xenopus embryo hybridized in
whole-mount is shown from the anterior, dorsal side up.
(B) Before DEX application, GFP-Xanf-1-BDGR is
uniformly distributed into the ectodermal cells of the early
gastrula embryo. (C) 30 minutes after DEX treatment of the
embryo shown in B, GFP-Xanf-1-BDGR is accumulated into
cell nuclei. (D) Expansion of the neural plate in cranial region
of the midneurula embryo microinjected with GFP-Xanf-1-
BDGR mRNA. The expanded area at the right half of the
neural plate (left to the viewer) is occupied by PGLC
containing the hybrid protein. Note lateral displacement of the
cranial neural fold (arrowheads) at the microinjected side.
Lateral borders of the neural plate are marked by dotted lines.
(E) Whole-mount in situ hybridization with NCAM probe.
The neural plate is expanded at the right side of the
midneurula embryo microinjected with GFP-Xanf-1-BDGR
mRNA. (F) The same embryo as in E, shown alive under epifluorescence.
(G) Transverse section of embryo shown in E
demonstrates enlargement of the right half of the neural plate.
(H) Inhibition of the expression of Xtwist in the cranial neural
fold (arrow) of the midneurula embryo microinjected with
GFP-Xanf-1-BDGR mRNA. (I) The dorsal limit of expression
of epidermal marker, Xep-1, is shifted ventrally at the right
side of the embryo microinjected in the right animal
blastomere with GFP-Xanf-1-BDGR mRNA and treated by
DEX. The position of the midline is marked by dashed line.
(J,K) Animal cap explants of embryos microinjected with
GFP-Xanf-1-BDGR mRNA or noggin mRNA and hybridized
with NCAM probe at the 32 stage equivalent. No signals were
observed in GFP-Xanf-1-BDGR-containing explants treated
with DEX (J), in comparison with those microinjected with
noggin mRNA (K). (L) In situ hybridization with type II b-
tubulin mRNA probe showing suppression of primary
neurones (arrowhead) on the microinjected side of the late
neurula embryo.
Fig. 2. (A) Brain of stage-47 normal embryo. Dorsal view.
Abbreviations: tel, telencephalon; di, diencephalon; mes,
mesencephalon; hind, hindbrain. The telencephalic (B) and
mesencephalic (C) outgrowths (arrowheads) developed in embryos
microinjected with GFP-Xanf-1-BDGR mRNA and treated by DEX.
(D,E) GFP label in the telencephalic outgrowth (arrowhead) of the
stage-47 embryo microinjected with the mixture of GFP and GFPXanf-
1-BDGR mRNAs and treated by DEX under white and UV
light, respectively. (F,G) No brain abnormalities were observed in the
embryo microinjected with the same mixture, but not treated by
DEX, despite high concentration of the exogenous proteins in brain
cells (arrowhead).
Fig. 3. (A-H). Whole-mount in situ hybridization with digoxigenin-labelled probes of Xanf-1
(A-C), Otx-2 (D-F), Pax-6 (G-I) and XBF-1 (J-L) mRNAs. Embryos shown from the anterior,
dorsal side up, are at the late gastrula (stage 12-12.5), midneurula (stage 14-15) and late
neurula (stage 18-19), respectively. External and internal borders of the neural folds of
embryos at the 15 stage are marked by dotted lines.
Fig. 4. (A,B) Expression of Xanf-1 at the late gastrula
and midneurula stages, respectively, as seen on
sagittal sections. Dorsal side up, anterior to the right.
At both stages, the Xanf-1 expression domain in the
outer layer of neurectoderm has a very sharp boundary
(arrowhead) with the Xanf-1 non-expression domain.
(C,D) Expression of Otx-2 at the late gastrula and
midneurula stages, respectively. Whereas at the end of
gastrulation Otx-2 is expressed throughout the anterior
neurectoderm (C), by the midneurula stage its
expression is ceased in the region superimposed with
the Xanf-1 expression domain. (E) At the end of
gastrulation, XBF-1 is expressed in a single band of
cells, just beneath the superficial layer of the anterior
neurectoderm. (F) During early neurula stage, the
expression of XBF-1 is extended posteriorly in the
superficial cell layer, where the second expression
band is formed. (G) Schematic diagram of the Xanf-1,
Otx-2 and XBF-1 expression patterns on sagittal
sections of the midneurula.
Fig. 5. (A,B) Inhibition of the Otx-2
expression in the early neurula embryo
microinjected with GFP-Xanf-1-BDGR
mRNA. The same embryo is shown
after whole-mount in situ hybridization
with Otx-2 mRNA probe (A), and alive
under epi-fluorescence (B). Note, that
the band-shaped area where Otx-2
expression is abnormally repressed
(arrowhead) corresponds to the
location of PGLC. (C-J) Ectodermal
grafts shown by arrowheads were
homotopically transplanted at the early
gastrula stage from the embryo
microinjected with mixture of GFPXanf-
1-BDGR and GFP mRNAs to the
non-injected embryo. (C) Note much
lower levels of Pax-6 expression in the
graft in comparison with the
symmetrical zone in the left side of
embryo. (D) The same embryo as in C
is shown alive under epi-fluorescence.
The contour of the embryo is shown by
dashed line. (E) In the embryo
untreated by DEX, cells of the graft
intensively express Pax-6. (F) The
same embryo as in E is shown under
epi-fluorescence. (G,H) Inhibition of
the endogenous Xanf-1 in the grafted
cells and epi-fluorescent view of the
graft, respectively. (I) No inhibition of endogenous Xanf-1 is seen in the graft of the embryo untreated by DEX. (J) Inhibition of XBF-1 in the
graft after DEX treatment. (K,L) Though XBF-1 is downregulated within the territories occupied by PGLC during neurulation, in further
development its expression is reactivated (L), to the level even higher than that of the untreated embryos (K). Embryos hybridized at stage 26
with XBF-1 probe are shown from the anterior, dorsal side up. In the DEX-treated embryo (L), the expression of XBF-1 is expanded ventrally,
over the cement gland territory. In this embryo, the cement gland differentiation was suppressed by exogenous Xanf-1 (see Fig. 8 for the
details), and only three small patches of cement gland cells remain.
Fig. 6. Exogenous Xanf-1 can impose telencephalic identity on cells
of the dorsal part of abnormal diencephalic outgrowths, but not on
cells of the outgrowths located in more posterior regions. All
embryos were microinjected at the 8-cell stage in the right dorsal
blastomere with mixture of GFP-Xanf-1-BDGR and GFP mRNA.
DEX was applied at the midgastrula stage. (A) In the DEX-untreated
embryo, XBF-1 is expressed exclusively in the telencephalon. (B) In
the DEX-treated embryo, an additional site of the XBF-1 expression
(arrowhead) is seen in the dorsal part of the abnormal diencephalic
outgrowth. (C) In contrast, no expression of XBF-1 is seen in the
mesencephalic outgrowth (arrowhead). (D-F) Hybridization with the
probe for another telencephalic marker, Emx-1 also demonstrates that
exogenous Xanf-1 is able to induce telencephalic differentiation in
the diencephalic outgrowth (E, arrowhead), but not in the
mesencephalic one (F, arrowhead). (G,H) The expression of Pax-6,
which is normally observed in the diencephalon and telencephalon
(G), but not in the mesencephalon, is not induced by exogenous
Xanf-1 in the mesencephalic outgrowth (H, arrowhead). (I-L) Otx-2,
which is normally expressed in the di- and mesencephalon (I), in
microinjected embryos is also intensively expressed in the
mesencephalic outgrowth (L, arrowhead). (J,K) The mesencephalic
outgrowth shown in L is composed of PGLC derived from the graft
containing GFP-Xanf-1-BDGR mRNA.
Fig. 7. (A,B). The cyclopic phenotype, characterised by eye fusion
and reduction of the forebrain, is observed in the tadpoles when
PGLC containing GFP-Xanf-1-BDGR mRNA cross the anterior
neural fold (A). (C) The control embryo in which PGLC did not
cross the anterior neural fold. (D-F) Transplantations of small
ectodermal grafts from microinjected to non-injected embryos reveal
that the cyclopic phenotype develops only when grafts occupy the
anterior margin of the anterior neural fold (E), but not if they are
located more posteriorly (D) or anteriorly (F).
Fig. 8. Exogenous Xanf-1 can
inhibit differentiation of the
cement gland. All embryos at the
tailbud stage are shown from
ventral side, anterior to the top.
(A-E) Inhibition of the cement
gland in the embryos
microinjected at the 8-cell stage
with GFP-Xanf-1-BDGR mRNA
in both dorsal blastomeres.
(A) Almost complete suppression
of the cement gland differentiation
in the embryo whose head area is
occupied by PGLC. (B) A very
small patch of the cement gland
cells is still formed just in the
place where GFP labelling is
absent (arrow). (C,D) In the
control embryo microinjected
with GFP-Xanf-1-BDGR mRNA
but untreated by DEX, no signs of
the cement gland inhibition are
seen (C) despite wide expansion
of PGLC in head area (D).
(E) Hybridization with cement
gland marker XAG-1 probe shows
that several small patches of the
cement gland cells differentiated
in the embryo microinjected with
GFP-Xanf-1-BDGR mRNA and
treated by DEX (left), instead of a big single cement gland in the DEX-untreated embryo (right). (F,G) Transplantations of small ectodermal
grafts from embryos microinjected with GFP-Xanf-1-BDGR mRNA to non-injected embryos. (F) The cement gland differentiation is inhibited
in the central part of the normal cement gland area occupied by the DEX-treated graft. Two small cement glands (arrowheads) develop from
those parts of the presumptive cement gland territory that are not occupied by the graft. (G) Epi-fluorescent view of the embryo shown on F
reveals grafted cells. (H) Hybridization with the epidermal marker Xep-1 probe demonstrates that in the embryo shown in F and G, cells of the
graft acquire epidermal fate. (I-L) Xanf-1 can suppress the cement gland differentiation induced by Otx-2. (I). The cement gland differentiation
in the control embryo is revealed by XAG-1 probe. (J) Activation of GFP-Xanf-1-BDGR in embryos microinjected with the mixture of Otx-2
and GFP-Xanf-1-BDGR mRNAs results in severe reduction of the XAG-1 expression, despite the presence of Otx-2. (K,L) In animal caps
isolated at the late blastula stage from embryos microinjected with the mixture of Otx-2 and GFP-Xanf-1-BDGR mRNAs, exogenous Xanf-1
demonstrates the same epistatic effect over Otx-2 as observed in the whole embryos. (K) An intense expression of XAG-1 under the influence of
Otx-2 in the animal caps not treated by DEX. (L) Activation of GFP-Xanf-1-BDGR in treated caps represses the XAG-1 expression.
Fig. 9. (A-F) Microinjections
of EnR-Xanf-1-BDGR
mRNA (mixed with GFP
mRNA) elicit the effects
similar to those caused by
exogenous Xanf-1.
(A) Hybridization with
NCAM probe demonstrates
expansion of the neural plate
on the microinjection side.
(B) The abnormal
diencephalic outgrowth in
the microinjected embryo
contains cells expressing the
telencephalic marker, XBF-1
(arrow). (C,D) Inhibition of
the Otx-2 expression (C)
within the territory occupied
by PGLC (D). (E,F) Inhibition of the cement gland differentiation in the medial region of cement gland territory occupied by PGLC, results in
splitting into two separate cement glands. (G-L) In contrast to EnR-Xanf-1-BDGR, VP16-Xanf-1-BDGR injected into the right side of the
embryo produces some effects opposite to those elicited by Xanf-1 alone: downregulation of NCAM (arrowhead G), expansion of the right
Xtwist expression domain (arrowhead H), the ectopic expression of Otx-2 (arrowheads I,J), cement gland differentiation (K,L) observed in the
regions occupied by cells containing the activated VP16-Xanf-1-BDGR.