Department of Cell, Development, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham Medical School, Birmingham, Alabama, USA.
Dev Dyn. 2022 Sep;251(9):1524-1534. doi: 10.1002/dvdy.328. Epub 2021 Mar 23.
Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited.
We describe a transgenic mouse line expressing CreER from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreER labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreER to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreER is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreER in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts.
Overall, the Alx4-CreER line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.
研究基因功能和前肢芽细胞命运的遗传工具非常有限。
我们描述了一种表达 CreER 的转基因小鼠品系，该基因来自 Aristaless-like 4（Alx4）启动子，可在前肢诱导重组。在胚胎第 8.5 天诱导 Cre 表达表明，使用 mTmG Cre 报告基因标记的 Alx4-CreER 细胞有助于前肢的指 I 到 III 以及前肢的桡骨。Cre 活性在后腿中沿 AP 轴进一步扩展，比在前肢中更广泛，导致一些 Cre 报告基因细胞有助于指 IV。在稍后的时间点诱导会标记逐渐局限于更靠前的指和近端结构的细胞。将 Alx4 启动子转基因中的 Cre 表达与内源性 Alx4 表达进行比较表明，与内源性 Alx4 表达相比，Cre 表达略有向后扩展。使用 Alx4-CreER 诱导内鞭毛运输 88（Ift88）缺失，这是一个对纤毛发生、 hedgehog 信号传导和肢体模式形成所必需的基因，不会导致明显的骨骼畸形。然而，删除的效率、Ift88 蛋白周转所需的时间以及纤毛的退化速度可能会阻碍使用这种方法来分析肢体中的纤毛。Alx4-CreER 也在中肾和肾管中活跃，这有助于成年肾单位的收集管和导管。在 Ift88 条件性品系中胚胎激活 Alx4-CreER 会导致收集管/导管中形成囊肿。
总体而言，Alx4-CreER 品系将成为评估前肢、中肾和肾管中细胞命运和分析基因功能的新工具。
