Dealy C N, Kosher R A
Department of Anatomy, University of Connecticut Health Center, Farmington 06030.
Dev Dyn. 1995 Jan;202(1):67-79. doi: 10.1002/aja.1002020107.
The apical ectodermal ridge (AER) promotes the proliferation and directed outgrowth of the subridge mesodermal cells of the developing limb bud, while suppressing their differentiation. Insulin-like growth factor-I (IGF-I) and its receptor are expressed by the subridge mesodermal cells of the chick limb bud growing out in response to the AER, and specific insulin receptors are present in the limb bud during its outgrowth. To study the possible roles of IGF-I and insulin in limb outgrowth, we have examined their effects on the morphogenesis of posterior and anterior portions of the distal tip of stage 25 embryonic chick wing buds subjected to organ culture in serum-free medium in the presence or absence of the AER and limb ectoderm. The distal mesoderm of control posterior explants lacking an AER or all limb ectoderm ceases expressing IGF-I mRNA, exhibits little or no proliferation, fails to undergo outgrowth, and rapidly differentiates. Exogenous IGF-I and insulin promote the outgrowth and proliferation and suppress the differentiation of distal mesodermal cells in posterior explants lacking an AER or limb ectoderm, thus mimicking at least to some extent the outgrowth promoting and anti-differentiative effects normally elicited on the subridge mesoderm by the AER. Furthermore, IGF-I and insulin-treated posterior explants exhibit high IGF-I mRNA expression, indicating that IGF-I and insulin maintain the expression of endogenous IGF-I by the subridge mesoderm. We have also found IGF-I and insulin can affect the morphology and activity of the AER. When the posterior portion of the wing bud tip is cultured with the AER intact in control medium, on day 4-5 the AER flattens, ceases expressing high amounts of the AER-characteristic homeobox-containing gene Msx2, and concomitantly an elongated cartilaginous element differentiates in the subridge mesoderm. In contrast, in the presence of exogenous IGF-I or insulin the AER of such explants does not flatten, continues expressing high amounts of Msx2, and the subridge mesoderm remains undifferentiated and proliferative. Thus, exogenous IGF-I and insulin maintain the thickness of the AER and sustain its expression of Msx2, while sustaining the anti-differentiative effect normally elicited on the subridge mesoderm by a thickened functional AER. Notably, we have also found that exogenous IGF-I and insulin induce the formation of a thickened ridge-like structure that expresses high amounts of Msx2 from the normally thin distal anterior ectoderm of the limb bud, while promoting dramatic outgrowth and proliferation of the anterior mesoderm, which normally undergoes little outgrowth or proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)
顶端外胚层嵴（AER）促进发育中肢体芽基嵴下中胚层细胞的增殖和定向生长，同时抑制其分化。胰岛素样生长因子-I（IGF-I）及其受体由响应AER而生长的鸡肢体芽基嵴下中胚层细胞表达，并且在肢体芽基生长期间肢体芽基中存在特异性胰岛素受体。为了研究IGF-I和胰岛素在肢体生长中的可能作用，我们检测了它们对处于第25阶段的胚胎鸡翼芽远端尖端前后部形态发生的影响，这些翼芽在无血清培养基中进行器官培养，培养条件为存在或不存在AER和肢体外胚层。缺乏AER或所有肢体外胚层的对照后部外植体的远端中胚层停止表达IGF-I mRNA，几乎不增殖或不增殖，无法生长，并迅速分化。外源性IGF-I和胰岛素促进缺乏AER或肢体外胚层的后部外植体中远端中胚层细胞的生长和增殖，并抑制其分化，从而至少在一定程度上模拟了AER通常对嵴下中胚层产生的促进生长和抗分化作用。此外，经IGF-I和胰岛素处理的后部外植体表现出高IGF-I mRNA表达，表明IGF-I和胰岛素维持嵴下中胚层内源性IGF-I的表达。我们还发现IGF-I和胰岛素可以影响AER的形态和活性。当翼芽尖端后部在对照培养基中完整保留AER的情况下进行培养时，在第4 - 5天AER变平，停止大量表达AER特征性的含同源框基因Msx2，同时一个细长的软骨元件在嵴下中胚层中分化。相反，在外源性IGF-I或胰岛素存在的情况下，此类外植体的AER不会变平，继续大量表达Msx2，并且嵴下中胚层保持未分化和增殖状态。因此，外源性IGF-I和胰岛素维持AER的厚度并维持其Msx2的表达，同时维持增厚的功能性AER通常对嵴下中胚层产生的抗分化作用。值得注意的是，我们还发现外源性IGF-I和胰岛素诱导从肢体芽基通常较薄的远端前部外胚层形成一个增厚的嵴状结构，该结构表达大量Msx2，同时促进前部中胚层的显著生长和增殖，而前部中胚层通常很少生长或增殖。（摘要截断于400字）
