Xenopus hindbrain patterning requires retinoid signaling.

We have asked how posterior neural tissue is patterned in Xenopus by assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARalpha2.2 receptor (RARDelta) to dominantly interfere with retinoid signaling. In embryos injected with RARDelta expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-20 in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARalpha2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARDelta. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and fibroblast growth factor (FGF) to posteriorize isolated anterior neurectoderm and show that both factors can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither factor is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

Efficient hormone-inducible protein function in Xenopus laevis.

A major problem in analyzing gene function during Xenopus development has been the inability to induce gene expression in a temporally controlled manner. We have attempted to solve this problem with a system of hormone-activated protein function, using the myogenic gene MyoD as a paradigm. We show that microinjection of RNA for MyoD fused to the ligand-binding domain of either the estrogen or glucocorticoid receptor results in hormone-dependent activation of MyoD function, as assayed by ectopic induction of muscle-specific actin mRNA. Induction is tightly regulated in both isolated animal caps and intact embryos, with ectopic muscle-specific actin expression inducible after 2 hr of hormone treatment. Higher levels of MyoD-receptor fusion proteins that native MyoD protein are present in embryos, apparently a result of increased fusion protein stability. This is the first demonstration that hormone-inducible fusion proteins can work effectively in a complex embryo.

Regulation of the Xenopus labial homeodomain genes, HoxA1 and HoxD1: activation by retinoids and peptide growth factors.

Vertebrate homologues of Drosophila labial are likely to play key roles in anteroposterior axis formation. However, little is known about the regulation of these genes during vertebrate development. Here we examine the expression and regulation of the Xenopus labial homeodomain genes, HoxA1 and HoxD1. HoxA1 was expressed around the dorsoventral circumference of the trunk in neurula embryos, with later expression in spinal cord, midbrain, hindbrain, and endolymphatic duct. By mid gastrula, HoxD1 was predominantly expressed in dorsolateral and ventral ectoderm, with a gap in expression at the dorsal midline. By neurula, ventral expression had declined with most expression restricted to dorsolateral mesoderm and ectoderm. Retinoic acid strongly induced HoxA1 and HoxD1 throughout the ectoderm and mesendoderm of gastrula stages, while in older embryos retinoids induced ectopic expression of these genes in more limited regions. Induction by retinoids was independent of protein synthesis. Surprisingly, HoxA1 was expressed at high levels in isolated animal caps in the absence of retinoic acid. The peptide growth factors bFGF and activin A strongly induced expression of HoxD1, but not HoxA1, in animal caps; however, RNA accumulated only many hours after the application of these factors. Overexpression of thyroid hormone receptor (c-erbA) prevented induction of HoxD1 by retinoic acid in animal caps. c-erbA also ablated expression of HoxD1 in whole embryos, suggesting a role for endogenous retinoids in the regulation of HoxD1 expression. Dominant interfering activin and FGF receptors prevented expression of HoxD1 in vivo, implicating these factors in the normal induction of HoxD1. Our data indicate that induction of labial-like homeodomain genes is complex and may require many factors.