Karavanova I D, Dove L F, Resau J H, Perantoni A O
Laboratory of Comparative Carcinogenesis, National Cancer Institute--Frederick Cancer Research and Development Center, MD 21702, USA.
Development. 1996 Dec;122(12):4159-67. doi: 10.1242/dev.122.12.4159.
Differentiation of metanephric mesenchyme is triggered by an inductive signal(s) from the epithelial ureteric bud. As a result of this induction, most of the metanephric mesenchyme converts into epithelium of a nephron. We have developed and characterized an explant culture system, in which metanephric mesenchyme can grow and completely differentiate in vitro in the absence of an inductive tissue. When separated 13 dpc rat metanephric mesenchymes were cultured in serum-free conditioned medium from a rat ureteric bud cell line (RUB1) in the presence of bFGF and TGFalpha, they were induced to differentiate into nephron epithelia and glomeruli-like structures. The nephric type of differentiation was confirmed by both morphological and molecular criteria and paralleled the developmental changes of nephron differentiation in vivo. Expression patterns of brush-border antigen as well as molecular markers of kidney differentiation Wt1, Lim1, Hgf and c-met, c-ret, Shh, Wnt4, Wnt7b, and Wnt11 were analyzed in explants by whole mount and tissue section in situ hybridization following 1-9 days in culture. The expression of secreted patterning molecules Bmp7 and Wnt7b, but not Shh or Wnt11, were demonstrated by RT-PCR and northern blot hybridization with RNA from the RUB1 cells. Our culture system lends itself to examining the relevance of these and other signaling molecules required for nephron differentiation.
后肾间充质的分化是由来自输尿管芽上皮的诱导信号触发的。这种诱导的结果是，大部分后肾间充质转化为肾单位上皮。我们开发并表征了一种外植体培养系统，在后肾间充质中，在没有诱导组织的情况下，它可以在体外生长并完全分化。当将13天龄大鼠的后肾间充质分离出来，在存在碱性成纤维细胞生长因子（bFGF）和转化生长因子α（TGFα）的情况下，在来自大鼠输尿管芽细胞系（RUB1）的无血清条件培养基中培养时，它们被诱导分化为肾单位上皮和肾小球样结构。通过形态学和分子标准证实了肾型分化，并且与体内肾单位分化的发育变化平行。在培养1-9天后，通过整装和组织切片原位杂交分析外植体中刷状缘抗原的表达模式以及肾分化的分子标志物Wt1、Lim1、Hgf和c-met、c-ret、Shh、Wnt4、Wnt7b和Wnt11的表达模式。通过RT-PCR以及与来自RUB1细胞的RNA进行Northern印迹杂交，证实了分泌型模式分子Bmp7和Wnt7b的表达，但未证实Shh或Wnt11的表达。我们的培养系统有助于研究这些以及其他肾单位分化所需信号分子的相关性。
