Cloning a novel developmental regulating gene, Xotx5: its potential role in anterior formation in Xenopus laevis

The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann's organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2.     

Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.     

Xenopus Xotx2 and Drosophila otd share similar activities in anterior patterning of the frog embryo

Despite the obvious anatomical differences between the fly and the vertebrate body plans, several genes involved in their development are largely conserved. In this work we provide evidence that overexpression of the Drosophila orthodenticle (otd) gene in Xenopus laevis has a similar effect to that of its homolog Xotx2. Injections of otd mRNA in whole embryos lead to posterior truncations and to induction of ectopic cement glands, similar to Xotx2 injections. In animal cap assays, otd, like Xotx2, is able to activate the cement gland marker XAG and to suppress the expression of the epidermal marker XK81. Finally, as assayed by Einsteck transplantation assays, otd, like Xotx2, is able to respecify a tail/trunk organizer to a head organizer. In this work we also show that Xotx2 and otd share molecular functions that regulate early regional specification of the Xenopus anterior neural plate. Gain-of-function experiment targeting low doses of either otd or Xotx2 mRNAs in the neural plate promote reduction of Xrx1 and Xbf1 expression domain; no changes are observed for the anterior mesodermal marker Xgsc, the dorsal diencephalic marker Xbh1, and the midbrain/hindbrain marker Xen2. otd/Xotx2 inhibition activity of Xrx1 and Xbf1 expression is consistent with the strong inhibition of Xfgf8 expression in the anterior neural ridge observed upon otd/Xotx2 mRNA injection.     

The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann's organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain- and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes- and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.     

Early anteroposterior division of the presumptive neurectoderm in Xenopus

We analyze the timing of neural patterning in Xenopus and the mechanism by which the early pattern is generated. With regard to timing, we show that by early gastrula, two domains of the anteroposterior (A/P) pattern exist in the presumptive neurectoderm, since the opl gene is expressed throughout the future neural plate, while the fkh5 gene is expressed only in more posterior ectoderm. By mid-gastrula, this pattern has become more elaborate, with an anterior domain defined by expression of opl and otx2, a middle domain defined by expression of opl and fkh5, and a posterior domain defined by expression of opl, fkh5 and HoxD1. Explant assays indicate that the late blastula dorsal ectoderm is specified as the anterior domain, but is not yet specified as middle or posterior domains. With regard to the mechanism by which the A/P pattern is generated, gain and loss of function assays indicate that quantitatively and qualitatively different factors may be involved in inducing the early A/P neural pattern. These data show that neural patterning occurs early in Xenopus and suggest a molecular basis for initiating this pattern.