Simon H G, Tabin C J
Department of Genetics, Harvard Medical School, Boston, MA 02115.
Development. 1993 Apr;117(4):1397-407. doi: 10.1242/dev.117.4.1397.
Adult urodele amphibians can regenerate their limbs and tail. Based on their roles in other developing systems, Hox genes are strong candidates for genes that play a role in regulating pattern formation during regeneration. There are four homologous clusters of Hox genes in vertebrate genomes. We isolated cDNA clones of two newt homeobox genes from homologous positions within two Hox clusters; Hox-4.5 and Hox-3.6. We used RNase protection on nonamputated (normal) and regenerating newt appendages and tissue to compare their transcriptional patterns. Both genes show increased expression upon amputation with similar kinetics. Hox-4.5 and Hox-3.6 transcription is limited to the mesenchymal cells in the regenerates and is not found in the epithelial tissue. In addition to regenerating appendages, both genes are transcriptionally active in adult kidney of the newt. Striking differences were found in the regulation of Hox-4.5 and Hox-3.6 when they were compared in unamputated limbs and in regenerating forelimbs versus regenerating hindlimbs. Hox-4.5 is expressed in the blastema of regenerating fore- and hindlimbs, but Hox-4.5 transcripts are not detectable in normal limbs. In contrast, Hox-3.6 transcripts are found exclusively in posterior appendages, but are present in normal as well as regenerating hindlimbs and tails. Hox-4.5 is also expressed at a higher level in proximal (mid-humerus) regenerates than in distal ones (mid-radius). When we proximalized the positional memory of a distal blastema with retinoic acid, we find that the early expression level of Hox-4.5 is also proximalized. When the expression of these genes is compared to the expression of two previously reported newt Hox genes, a consistent pattern emerges, which can be interpreted in terms of differential roles for the different Hox clusters in determining regenerative limb morphology.
成年有尾两栖动物能够再生其四肢和尾巴。基于Hox基因在其他发育系统中的作用，它们很有可能是在再生过程中参与调节模式形成的基因。脊椎动物基因组中有四个Hox基因的同源簇。我们从两个Hox簇中的同源位置分离出了蝾螈同源异型框基因的cDNA克隆；Hox - 4.5和Hox - 3.6。我们对未截肢（正常）和再生的蝾螈附肢及组织进行了核糖核酸酶保护实验，以比较它们的转录模式。两个基因在截肢后均表现出表达增加，且动力学相似。Hox - 4.5和Hox - 3.6的转录仅限于再生组织中的间充质细胞，在上皮组织中未发现。除了再生附肢外，这两个基因在成年蝾螈肾脏中也具有转录活性。当在未截肢的肢体以及再生前肢与再生后肢中比较Hox - 4.5和Hox - 3.6时，发现了显著差异。Hox - 4.5在再生前肢和后肢的芽基中表达，但在正常肢体中检测不到Hox - 4.5转录本。相反，Hox - 3.6转录本仅在后肢附肢中发现，但在正常以及再生的后肢和尾巴中均存在。Hox - 4.5在近端（肱骨中部）再生组织中的表达水平也高于远端（桡骨中部）。当我们用视黄酸使远端芽基的位置记忆近端化时，我们发现Hox - 4.5的早期表达水平也近端化了。当将这些基因的表达与之前报道的两个蝾螈Hox基因的表达进行比较时，出现了一种一致的模式，这可以根据不同Hox簇在确定再生肢体形态中的不同作用来解释。
