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Andreazzoli M
,
Gestri G
,
Angeloni D
,
Menna E
,
Barsacchi G
.
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The anteriormost part of the neural plate is fated to give rise to the retina and anteriorbrain regions. In Xenopus, this territory is initially included within the expression domain of the bicoid-class homeobox gene Xotx2 but very soon, at the beginning of neurulation, it becomes devoid of Xotx2 transcripts in spatiotemporal concomitance with the transcriptional activation of the paired-like homeobox gene Xrx1. By use of gain- and loss-of-function approaches, we have studied the role played by Xrx1 in the anterior neural plate and its interactions with other anterior homeobox genes. We find that, at early neurula stage Xrx1 is able to repress Xotx2 expression, thus first defining the retina-diencephalon territory in the anterior neural plate. Overexpression studies indicate that Xrx1 possesses a proliferative activity that is coupled with the specification of anterior fate. Expression of a Xrx1 dominant repressor construct (Xrx1-EnR) results in a severe impairment of eye and anteriorbrain development. Analysis of several brain markers in early Xrx1-EnR-injected embryos reveals that anterior deletions are preceded by a reduction of anterior gene expression domains in the neural plate. Accordingly, expression of anterior markers is abolished or decreased in animal caps coinjected with the neural inducer chordin and the Xrx1-EnR construct. The lack of expansion of mid-hindbrain markers, and the increase of apoptosis in the anterior neural plate after Xrx1-EnR injection, indicate that anterior deletions result from an early loss of anterior neural plate territories rather than posteriorization of the neuroectoderm. Altogether, these data suggest that Xrx1 plays a role in assigning anterior and proliferative properties to the rostralmost part of the neural plate, thus being required for eye and anteriorbrain development.
Fig. 1. Effects of Xrx1 overexpression. (A-C) Embryos microinjected
with Xrx1 RNA in the right dorsoanimal blastomere at 8-cell stage.
The arrows point to ectopic pigmented retina located between eye
and diencephalon. (B,C) Transverse sections of an embryo similar to
the one shown in A; (B) a section where the ectopic pigmented retina
is visible (arrow); (C) a more posterior section where the duplication
of the neural tube becomes evident (arrowhead). (D-O) Whole-mount
in situ hybridization analysis of embryos microinjected with Xrx1
RNA at 8-cell stage in one dorsoanimal blastomere. (D-K¢) The
staining pattern of the gene of interest (blue) on the injected side (i)
should be compared with that on the uninjected control side (c). In
the same panels, the distribution of Xrx1-injected RNA is visualized
by cohybridization with Xrx1 antisense RNA revealed by magenta
staining. (M-O) Nuclear b-gal RNA has been used as a tracer and the
b-galactosidase activity is represented by the red staining.
(D-F) Expression of Xpax6 (blue) in Xrx1-injected embryos.
(D) Xpax6 expression at stage 13 is not significantly affected by Xrx1
overexpression; (E,F) Xpax6 expression in the injected and control
side of a stage 23 embryo, respectively. Note that the normal Xpax6
gap of expression in the midbrain present in the control side (area
between lines) has been filled by ectopic Xpax6 expression in the
injected side of the embryo. (G-I) Expression of Xsix3 (blue) in
Xrx1-injected embryos. (G) Expression of Xsix3 at stage 13 is not
affected by Xrx1 RNA injection. (H,I) Xsix3 expression in the
injected and control side of a stage 23 embryo, respectively. The eye
expression of Xsix3 in the injected side appears expanded
dorsoposteriorly when compared to the control side.
(J,K,K¢) Expression of Xotx2 (blue) in Xrx1-injected embryos.
(J) Expression of Xotx2 at stage 13 is repressed by Xrx1
overexpression. (K,K¢) Examples of Xotx2 expression in stage 23
Xrx1-injected embryos where Xotx2 expression is extended laterally
(K) or posteriorly (K¢) in the injected side. The white line marks the
posterior boundary of Xotx2 expression in the control side.
(L) Double whole-mount in situ hybridization performed on a stage
13 normal embryo showing the complementarity of Xrx1 (magenta)
and Xotx2 (blue) expression domains. (M,N) Expression of XAG-1
(blue) in Xrx1-injected embryos. (M) Expression of XAG-1 is
repressed at stage 13 by Xrx1 overexpression. (N) XAG-1 is
ectopically activated in the injected side of a stage 23 embryo.
(O) Expression of XBF-1 (blue) in Xrx1-injected embryos at stage
13. XBF-1 expression is expanded laterally on the injected side.
Fig. 2. Effects of Xrx1 RNA microinjection in one blastomere at 8-
cell stage on Xpax2, En2 and Krox20 expression. The staining
pattern of the gene of interest (blue) on the injected side (i) should be
compared with that on the uninjected control side (c). The
distribution of Xrx1-injected RNA is visualized by cohybridization
with Xrx1 antisense RNA (magenta staining). (A,B) Expression of
Xpax2 in stage 23 Xrx1-injected embryos. (A) Frontal view showing
repression of Xpax2 expression in the midbrain-hindbrain boundary
but not in ventral optic vesicles; the dorsal side of the embryo is on
the top. (B) Dorsal view of another embryo showing strong
repression in the Xpax2 midbrain-hindbrain expression domain and a
weak reduction of the otic vesicle domain. (C) Repression of En2
expression in a stage 24 Xrx1-injected embryo. (D) Expression of
Krox20 in a stage 24 Xrx1-injected embryo. Krox20 expression at the
level of rhombomere 5 appears to be reduced while rhombomere 3
expression domain is almost completely abolished. (B-D) The
anterior part of the embryo is oriented to the left.
Fig. 3. Effects of Xrx1-EnR RNA microinjection on eye and anterior
head development. (A) Schematic diagrams of Xrx1 constructs used
in microinjection experiments. The homeodomain (Hd), OAR
domain (OAR) and engrailed repressor domain (EnR) are indicated
as well as the amino- and carboxy-termini of the proteins (N and C,
respectively). (B) Stage 41 embryos resulting from microinjection of
400 pg Xrx1-EnR RNA in both dorsoanimal blastomeres at 8-cell
stage. The top embryo is an uninjected control. (C) Xrx1 rescues the
Xrx1-EnR phenotypes. Embryos were coinjected with 400 pg of
Xrx1-EnR RNA and 600 pg of Xrx1 RNA per blastomere in both
dorsoanimal blastomeres at 8-cell stage and analyzed at stage 41.
(D,E) Expression of Xrx1 in presumptive forebrain regions of stage
12.5 normal embryos. (D) Double whole-mount in situ hybridization
with Xrx1 (magenta) and the forebrain-specific marker XBF-1 (blue).
Xrx1 expression partially overlaps with that of XBF-1. (E) Control
embryo hybridized with Xrx1 alone (blue).
Fig. 4. Effects of Xrx1-EnR RNA microinjection in both
dorsoanterior blastomeres at 8-cell stage on the expression
of anterior genes. (A-D) Embryos analyzed for Xotx2
expression. (A,C) Dorsoanterior and lateral views of the
normal expression in stage 13 and stage 24 embryos,
respectively. (B,D) Dorsoanterior and lateral views of
embryos at stage 13 and 24, respectively, injected with
Xrx1-EnR RNA. (E-H) Embryos analyzed for Xpax6
expression. (E,G) Dorsoanterior and lateral views of the
normal expression in stage 13 and stage 24 embryos,
respectively. (F,H) Dorsoanterior and lateral views of
embryos at stage 13 and 24, respectively, injected with
Xrx1-EnR RNA. (I-L) Embryos analyzed for Xsix3
expression. (I,K) Dorsoanterior and frontal views of the
normal expression in stage 13 and stage 24 embryos,
respectively. (J,L) Dorsoanterior and frontal views of
embryos at stage 13 and 24, respectively, injected with
Xrx1-EnR RNA. (M,N) Embryos analyzed at stage 13 for
XBF-1 expression (dorsoanterior views). (M) Control
uninjected embryo. (N) Embryo injected with Xrx1-EnR
RNA. (O,P) Embryos analyzed at stage 24 for Xpax2
expression (lateral views). (O) Control uninjected embryo.
(P) Embryo injected with Xrx1-EnR RNA. Arrows indicate
Xpax2 expression domain corresponding to the midbrainhindbrain
boundary. (Q,R) Embryos analyzed at stage 12.5
for gsc expression (dorsal views). (Q) Control uninjected
embryo. (R) Embryo injected with Xrx1-EnR RNA.
(S,T) Embryos analyzed at stage 24 for Xotx-b expression
(lateral views). (S) Control uninjected embryo. (T) Embryo
injected with Xrx1-EnR RNA. Arrowheads indicate Xotx-b
expression in the pineal gland.
Fig. 5. Coinjection of chordin and Xrx1-EnR in
animal caps. (A-C) Animal caps dissected from
embryos microinjected with chordin RNA.
(D-F) Animal caps dissected from embryos
coinjected with chordin and Xrx1-EnR RNAs. The
probes used were XBF-1 (A,D), Xpax6 (B,E) and
Xotx2 (C,F).
Fig. 6. (A-D) En2 (A,B) and Krox20 (C,D) expression in
uninjected (A,C) and Xrx1-EnR-injected embryos (B,D),
as observed at stage 28. (E-G) TUNEL staining in stage
12 embryos. Dorsal-anterior (E) and lateral (F) views of
Xrx1-EnR-injected embryos are shown. (G) Dorsalanterior
view of a control uninjected embryo. In E and
G, posterior is to the top and anterior to the bottom. In F,
dorsal is to the top and anterior to the left.
