Chan D C, Wynshaw-Boris A, Leder P
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Development. 1995 Oct;121(10):3151-62. doi: 10.1242/dev.121.10.3151.
Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. Since the ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed formins), we wished to understand their role in the formation of these organs. Therefore, we first examined the embryonic expression patterns of the four major ld mRNA isoforms. Isoforms I, II and III (all containing a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney including the ureteric bud. Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney including the ureteric bud. Using a lacZ reporter assay in transgenic mice, we show that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, we analyzed the expression of a number of genes thought to play a role in limb development. Most significantly, we find that although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus we conclude that one or more formins are necessary to initiate and/or maintain fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, we further conclude that while fgf-4 is capable of supporting distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including sonic hedgehog (shh), a marker for the polarizing region and Hoxd-12, a marker for posterior mesoderm. We propose that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis.
隐性肢体畸形（ld）突变的纯合子小鼠表现出肢体和肾脏缺陷。肢体缺陷，即少指畸形和并指畸形，已追溯到顶外胚层嵴（AER）的分化异常和肢体前后轴的缩短。肾脏缺陷通常为肾发育不全，被认为是输尿管芽生长失败的结果。由于ld基因座产生多种编码几种不同蛋白质（称为formin）的RNA异构体，我们希望了解它们在这些器官形成中的作用。因此，我们首先检查了四种主要ld mRNA异构体的胚胎表达模式。异构体I、II和III（均含有碱性氨基末端）在背根神经节、颅神经节以及包括输尿管芽在内的发育中的肾脏中表达。异构体IV（含有酸性氨基末端）在脊索、体节、肢芽的顶外胚层嵴（AER）以及包括输尿管芽在内的发育中的肾脏中表达。利用转基因小鼠中的lacZ报告基因检测，我们表明异构体IV的这种差异表达源于其第一个外显子上游不同的调控序列。这些表达模式表明所有四种异构体可能都参与输尿管芽的生长，而异构体IV可能参与AER的分化。为了进一步确定ld肢体缺陷的发育后果，我们分析了一些被认为在肢体发育中起作用的基因的表达。最显著的是，我们发现虽然ld肢芽的AER表达几种AER标记，但它们不表达可检测水平的成纤维细胞生长因子4（fgf - 4），而fgf - 4被认为是向中胚层发出的AER信号。因此我们得出结论，一种或多种formin对于在远端肢体中启动和/或维持fgf - 4的产生是必需的。由于ld肢体形成诸如指（趾）等远端结构，我们进一步得出结论，虽然fgf - 4能够支持在经过操作的肢体中远端肢体的生长，但它对于正常肢体发育中的远端生长并非必不可少。此外，ld肢体中几种中胚层标记的表达严重下降，包括作为极化区域标记的音猬因子（shh）和作为后中胚层标记的Hoxd - 12。我们提出ld肢芽中AER的不完全分化导致极化活性降低和沿前后轴的缺陷。
