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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 ventralectoderm, 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.
Fig1. Expression patterns of HoxA1 and HoxD1. Expression was examined by whole mount in situ hybridization as described under Materials and Methods. In all cases anterior is left. (A) HoxA1 expression: mid-neurula (Stage 15) lateral view. Black arrows, anterior and posterior boundaries of HoxA1 expression. (B) HoxA1 expression: mid-neurula (stage 17) dorsal view. sc, spinal cord; arrows, anterior and posterior boundaries of expression. (C) HoxA1 expression: tailbud (stage 24), lateral view. sc, spinal cord; arrow, anterior boundary of spinal cord expression. (D) HoxA1 expression: late tailbud (stage 30), lateral view. el, endolymphatic duct; hb, hindbrain; mb, midbrain. (Inset) HoxA1 expression relative to en-2. Closed arrows, HoxA1 expression; open arrow, en-2 expression. (E) HoxD1 expression: mid gastrula (stage 11) dorsal view. bp, blastopore; arrow dorsal lip. (F) HoxD1 expression: mid gastrula (stage 11), ventral view. (G) HoxD1 expression: early neurula (stage 13), dorsal view. (H) HOXD1 early neurula (stage 13), lateral view.(I) HoxD1 expression: mid neurula (stage 16), dorsal view. (J) HoxD1 expression: mid neural (stage 16), lateral view. Arrow, anterior boundary of expression.
Fig3. Expression patterns of HoxA1 and HoxD1 are altered by retinoic acid. The expression of HoxA1 and HoxD1 was analayzed by whole mount in situ hydridization as described under Materials and Methods. Embryos were treated continuously with 1um all trans retinoic acid from mid-bastula onward unless otherwise indicated. (A) HoxA1 expression: mid gastrula (stage 11) bp= blastopore. (B) HoxA1 expression neurula (stage 14), lateral view. Arrow= anterior boundary. (C) HoxA1 expression: tailbudembryo (stage 22) treated with 1uM RA for 3hr prior to collection. (D) HoxA1 expression: (stage 29) treated with 1uM RA for 3hr prior to collection. (E) HoxD1 expression: midgastrula (stage 11) bp. blastopore. (F) HoxD1 expression: early neurula (stage 13) dorsolateral view. Arrow, gap in expression. O = Otic vesicle. (H) HoxD1 expression: late tailbudembryo (stage 29-30) treated with RA for 3hr prior to collection. Arrows, intersomitic fissures; n= notochord; t=tailbud; s=anterior spinal cord.
Fig8. HoxD1 expression is ablated by the misexpression of dominant negative receptors. Data is detailed in Table 1. Albino embryos were micro-injected with a mixture of B-galactosidase mRNA (as a tracer) and dominant negative mRNA in one blastomere of a 2-cell embryo as described under Materials and Methods (thyroid hormone receptor: c-erbA (Barattino et al. 1993); dominant negative activin receptor: XAR delta (Hemmati-Brivanlo et al 1992); dominant negative fibroblast growth factor receptor: XFD (Amaya et al 1991). Embryos were allowed to develop to late gastrula-early neurula stage (Stage 12.5-13) and expression of HoxD1 was examined by in situ hybridization as described above. The expression of the mesodermal marker Xbra was also examined as a control. Xbra is expressed around the blastopre and in the notochord. At the stage examined (stage 12.5-13) the notochord has just begun to extend, so that notochord staining is visible to varying degrees (Nieuwkoop and Faber 1967). lacZ staining (light blue) localizes the injected mRNA. this staining is punctate due to a nuclear localization signal linked to the lacZ protein. Failure to see overlap of lacZ stain and HoxD1 mRNA in the normal domain of HoxD1 expression indictates ablation of expression by the dominant negative receptor. (A) HoxD1 expression; CAT RNA injected, dorsal view. (B) HoxD1 expression: c-erbA RNA- injected, dorsolateral view. (C) Xbra expression: c-erbA RNA-injected, dorsolateral-view. (D) HoxD1 expression: XARdelta RNA-injected, dorsal view. (E) Xbra expression, XARdelta RNA injected. (F) HoxD1 expression: XFD RNA-injected, dorsal view. (G) Xbra expression: XFD RNA-injected. Anterior is to the right. D= dorsal midline; bp= blastopore.
