November 1, 2013;
Identification and evolution of molecular domains involved in differentiating the cement gland-promoting activity of Otx proteins in Xenopus laevis.
Otx genes are a class of vertebrate homeobox genes, homologous to the orthodenticle gene of Drosophila melanogaster, that play a crucial role in anterior embryo
patterning and sensory organ formation. In the frog, Xenopus laevis, at least three members of this class have been isolated: otx1
); they are involved in regulating both shared and differential processes during frog development. In particular, while otx2
are both capable to promote cement gland
(CG) formation, otx1
is not. We performed a molecular dissection of Otx5
proteins to characterize the functional parts of the proteins that make them differently able to promote CG formation. We show that a CG promoting domain (CGPD) is localized at the Otx5
C-terminus, and is bipartite: CGPD1 (aa210-255) is the most effective domain, while CGPD2 (aa177-209) has a lower activity. A histidine stretch disrupts CGPD1 continuity in Otx1
determining its loss of CG promoting activity; this histidine-rich region acts as an actively CG repressing domain. Another Otx1
specific domain, a serine-rich stretch, may also be involved in repressing Otx1
potential to trigger CG formation, though at a much lower level. This is the first evidence that these domains, specific of the Otx1
orthology group, play a role during development in differentiating Otx1
action compared to other Otx family members. We discuss the potential implications of their appearance in light of the evolution of Otx functional activities.
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Fig. 1. Multi-alignment of Xenopus Otx1, Otx2 and Otx5. Multi-alignment of Otx sequences has been obtained using ClustaW. “∗”: identical residue; “:”: conserved substitution; “.”: semi-conserved substitution. Homeodomain: shaded dark grey box; Otx tail: shaded light grey box; Otx2 and Otx5 retinal specificity boxes (RS box): shaded yellow boxes; Otx1 Ser-rich region: open box; Otx1 His-rich region: light green box; cement gland promoting domain D2 (CGPD2): shaded light blue box; cement gland promoting domain D1 (CGPD1): shaded blue box.
Fig. 2. Schematics of the constructs used in this study. Light grey: Xotx1 constructs; dark grey: Xotx5 constructs. RS: retinal specificity box; Ser-rich: serine-rich region; His-rich: histidine-rich region; D1: CGPD1; D2: CGPD2.
Fig. 3. Results of embryos microinjections and ag1 in situ hybridization. (A and B) A total amount of 800 pg of capped mRNAs corresponding to the different constructs as shown was ventrally (A) or dorsally (B) bilaterally microinjected in Xenopus embryos at 4-cell stage; embryos were grown to tailbud stage and processed by whole mount in situ hybridization with an ag1 probe. When microinjected dorsally (B), otx1, otx2 and otx5 induce posterior defects in Xenopus embryos; this activity is maintained by all constructs, but Xotx5-87δC. LacZ was coinjected as a tracer. Microinjection of LacZ alone was used as negative control.
Fig. 4. Results of ag1 in situ hybridization on animal caps injected with the different constructs. Animal caps were dissected from embryos injected with 800 pg capped mRNAs as shown and ag1 expression was detected by in situ hybridization. GFP was used as microinjection tracer; microinjection of GFP alone was used as negative control.
Fig. 5. Results of RT-PCR for ag1 and for muc2 detection in animal caps injected with different constructs as indicated in A, B and C. Animal caps were dissected from stage 9 embryos injected with 800 pg capped mRNAs of the different constructs as shown and grown up to stage 25. ag1 and muc2 expression was detected by RT-PCR on extracted mRNA. odc was used as internal reference. GFP is used as injection tracer; caps injected with GFP alone were used as a negative control.