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Martynova NY
,
Eroshkin FM
,
Ermolina LV
,
Ermakova GV
,
Korotaeva AL
,
Smurova KM
,
Gyoeva FK
,
Zaraisky AG
.
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The question of how subdivision of embryo into cell territories acquiring different fates is coordinated with morphogenetic movements shaping the embryonic body still remains poorly resolved. In the present report, we demonstrate that a key regulator of anterior neural plate patterning, the homeodomain transcriptional repressor Xanf1/Hesx1, can bind to the LIM-domain protein Zyxin, which is known to regulate cell morphogenetic movements via influence on actin cytoskeleton dynamics. Using a set of deletion mutants, we found that the Engrailed-type repressor domain of Xanf1 and LIM2-domain of Zyxin are primarily responsible for interaction of these proteins. We also demonstrate that Zyxin overexpression in Xenopus embryos elicits effects similar to those observed in embryos with downregulated Xanf1. In contrast, when the repressor-fused variant of Zyxin is expressed, the forebrain enlargements typical for embryos overexpressing Xanf1 develop. These results are consistent with a possible role of Zyxin as a negative modulator of Xanf1 transcriptional repressing activity.
Figure 2. Expression of Zyxin during X. laevis development. A: Top: RT-PCR of total RNA from embryos at the indicated stages with primers to cDNAs of Zyxin and Ef1-alpha (Ef1) as a loading control. Bottom: Immunoblotting with Rabbit polyclonal anti-Zyxin (final dilution 1:300) and anti-tubulin antibodies (DM1A, Sigma, final dilution 1:10,000) as a loading control and detected with SuperSignal West Femto Trial Kit. B-E: Expression of Zyxin mRNA revealed by whole-mount in situ hybridization. B: Early gastrula. Lateral view, anterior to the right. C: Late gastrula. Anterior view, dorsal to the top. D: Late gastrula. Dorsal view, anterior to the top. E: Middle neurula. Dorsal and lateral view from the right side, anterior to the top. F: Region-specific RT-PCR (for Zyxin and Ef1) of total RNA from tissue explants extirpated at the middle neurula stage (stage 14) from regions indicated by red dotted lines: 1, anterior neural plate; 2, headendomesoderm; 3, trunk neural plate along with underlying axial mesoderm; 4, ventralectoderm; 5, endoderm. G: Late neurula. Dorsal view, anterior to the top. H: The end of neurulation. Lateral view from the left side. Anterior to the top. I: Tailbud stage. Dorsal to the top. Anterior to the right. J,K: Expression of Zyxin and Xanf1 mRNA, respectively, at the middle neurula stage. Anterior view, dorsal side to the top. L: Scheme of sections shown on L and M. Two halves of the same embryo were hybridized separately with Xanf1 and Zyxin probe and then compared. Red asterisk indicates anterior margin of the neural plate. M,N: Expression of Xanf1 and Zyxin mRNA at the early (stage 13) and middle (stage 15) neurula stage, as seen in two bilateral halves of the same embryo (see text for details). Neural plate margins on C, J, and K are marked with dotted line. aem, anteriorendomesoderm; anp, anterior neural plate; ap, animal pole; ba, branchial arches; bp, blastopore; cg, cement gland; fb, forebrain; fp, floor plate; gc, gastrocoel; hb, hindbrain; ey, eye; nt, neural tube; ov, otic vesicle; sm, somites.
