Altizer A M, Moriarty L J, Bell S M, Schreiner C M, Scott W J, Borgens R B
Institute for Applied Neurology, Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907-1244, USA.
Dev Dyn. 2001 Aug;221(4):391-401. doi: 10.1002/dvdy.1158.
A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.
稳定的离子电流从正在发育和再生的两栖动物肢体中流出。在无尾目和有尾目动物的发育肢体中，局部向外的电流（0.5 - 2微安/平方厘米）预示着产生早期芽体的间充质积累的位置。在此，我们报告通过使用非侵入性的自参考电极测量细胞外电流，对小鼠和鸡胚胎肢体芽发育过程中类似的向外离子电流的测量结果。在小鼠和鸡胚胎中，胁腹电流通常是向内的——这是外胚层摄取钠离子的方向。与小鼠肢体芽相关的向外电流范围为0.04 - 10.8微安/平方厘米。小鼠肢体芽和胁腹电流与在两栖动物幼虫中测量到的相似，因为它们在应用30微摩尔氨氯吡脒（一种钠离子通道阻滞剂）后会可逆地消失和/或反转。与两栖动物胚胎不同，在前后轴上与小鼠肢体芽相邻的胁腹外胚层通常与向外的离子电流相关。这增加了在这个发育平面上间充质细胞所经历的细胞外电压梯度与在肢体芽其他区域观察到的不同或变化的可能性。在鸡胚胎中，在体节尾侧的胁腹，在肢体芽形成前三个发育阶段，向内的胁腹电流显著反转，变为非常大（约100微安/平方厘米）的向外电流。我们通过使用人工施加的“逆流”来反转这种向外的离子电流，该“逆流”通过插入胚胎尾侧外胚层下方的微电极拉动，以此来测试这种向外的离子电流与鸡胚胎肢体形成的相关性。这种操作在 hindlimb bud 形成前2.5 - 3个发育阶段进行约6小时。该方法导致一些胚胎在妊娠第10天时肢体形成异常，而所有对照胚胎发育正常。这些数据表明肢体发育存在早期生理控制。
