Department of Medicine, University of California, San Diego, CA 92093-0693, USA.
Dev Biol. 2010 Mar 15;339(2):354-65. doi: 10.1016/j.ydbio.2009.12.033. Epub 2010 Jan 6.
Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFbeta superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st(-)(/)(-) kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st(-/-) UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.
乙酰肝素硫酸蛋白聚糖（HSPGs）是发育过程中的重要调节剂，可能通过与生长因子（如 GDNF、FGF 和 TGFbeta 超家族成员、EGF 受体配体和 HGF）相互作用发挥作用。生物合成酶，乙酰肝素硫酸 2-O-磺基转移酶（Hs2st）的缺失会导致肾脏发育不全。通过使用体内和体外的新组合方法，我们重新分析了 Hs2st(-)(/)(-)肾脏形态发生缺陷。利用分别模拟肾脏分支形态发生不同阶段的测定法，我们发现 Hs2st(-/-)UB 能够在与对照 MM 重新组合时进行分支，并诱导间充质到上皮的转化，而分离的 Hs2st 缺失 UB 能够在嵌入细胞外基质时在存在外源性可溶性促分支生长因子的情况下进行分支形态发生，表明 UB 具有内在的能力。这与普遍的观点相反，即肾脏发育不全表型的缺陷是由于 2-O 磺化 HS 在 UB 分支中起主要作用。出乎意料的是，突变 MM 在与野生型组织的重组实验中也完全能够被诱导。因此，突变的 UB 和 MM 组织本身都有能力，但突变组织的组合在体内以及我们显示的器官培养中失败。我们假设在相互诱导过程中存在 2OS 依赖性缺陷，该缺陷可能在 UB 或 MM 侧，因为这两种祖细胞组织都表达 Hs2st。鉴于这些观察结果，我们专门检查了 HS 2-O 磺化修饰对 UB 和 MM 各自形态发生能力的作用。我们证明，早期 UB 分支形态发生不是主要由依赖 HS 2-O 硫酸盐修饰的因素调节的；然而，对 MM 诱导有贡献的因素对 2-O 磺化修饰非常敏感。这些数据表明，Hs2st 缺失肾脏的主要缺陷是 MM 无法通过相互诱导失败或 MM 形态发生的原发性失败来诱导。这导致正常的 UB 形成，但影响 T 形 UB 的形成或 T 形 UB 的迭代分支（可能是收集系统发育中依赖 HS 的两个独立阶段）。我们讨论了 HS 与 Wnts（例如 Wnt 9b）之间相互作用中断可能是观察到的表型的一个重要方面。这似乎是第一个 MM 缺陷阻止早期 UB 分支超过第一次分叉阶段的例子，这是早期肾脏发育的限制步骤之一。
