XB-ART-13096Development June 1, 1999; 126 (11): 2451-60.
The anteriormost part of the neural plate is fated to give rise to the retina and anterior brain 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 anterior brain 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 anterior brain development.
PubMed ID: 10226004
Article link: Development
Genes referenced: ag1 chrd.1 egr2 en2 foxg1 gsc otx2 pax2 pax6 rax six3
Morpholinos referenced: rax MO2
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|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.|