BMP morphogen gradient spatiotemporal modulation by metalloprotease activity

描述(申请人提供)：在脊椎动物和无脊椎动物中，骨形态发生蛋白(BMP)以浓度依赖的方式在形态梯度中作用于背腹(DV)轴。BMP形态梯度的形状是至关重要的：离散DV位置的BMP信号的精确量决定了不同的细胞命运。在……里面 斑马鱼胚胎，高BMP信号决定腹侧细胞命运，中间水平决定侧向命运，没有BMP信号导致背侧命运。随着DV组织从前向后逐渐形成图案，从囊胚到原肠胚期，BMP信号梯度形状不断地被调节。由于BMP还可以构筑神经管和发育中的肢体，并在癌症、骨质疏松症、骨关节炎、进行性骨发育不良等疾病的进展中起作用，了解DV构型中塑造BMP梯度的时空机制将为BMP在这些其他背景下的功能及其调控提供范例。这项建议的目标是确定BMP信号梯度在原肠形成过程中是如何形成的。我们可以通过核磷酸化Smad1/5的定量免疫荧光分析来确定BMP信号梯度的形状，Smad1/5是活性BMP信号的直接读数。最初的BMP信号梯度是在胚泡中期由Chordin(CHD)产生的，Chordin是一种关键的细胞外拮抗剂，可以结合和抑制BMP配体的信号传递。重要的是，我们观察到原肠形成开始时的梯度形状与原肠形成后期的形状不同。由于CHD的活动在整个原肠发育过程中都是必需的，因此确定CHD在时间和空间上是如何调节的是非常重要的。CHD的活性受两类关键蛋白质的调节：(I)同源金属蛋白酶Bmp1a和Tolloid(TLD)，它们裂解和钝化CHD；(Ii)金属蛋白酶抑制剂sizzed(SZL)。这两种金属蛋白酶功能的丧失如何在空间和时间上影响BMP梯度将被研究。此外，还将研究SZL函数的损失如何影响梯度。假设Bmp1a/TLD在原肠形成早期不受抑制地抑制CHD，从而产生BMP信号梯度，而SZL在原肠形成后期抑制Bmp1a/TLD，从而引导BMP信号梯度。在这个方案中，Bmp1a/TLD和SZL的时间需求，每个蛋白质如何影响BMP信号梯度的形状，以及TLD和SZL蛋白活性的有效范围将被确定。总之，这些实验将阐明金属蛋白酶活性的时空调节如何正确地塑造BMP信号梯度，从而形成斑马鱼发育中的DV轴。

DESCRIPTION (provided by applicant): In vertebrates and invertebrates, Bone Morphogenetic Proteins (BMPs) act in a morphogen gradient to pattern the dorsoventral (DV) axis in a concentration dependent manner. The shape of the BMP morphogen gradient is critical: precise amounts of BMP signaling at discrete DV positions specify distinct cell fates. In the zebrafish embryo, high BMP signaling specifies ventral cell fates, intermediate levels specify lateral fates, and no BMP signaling results in dorsal fates. The BMP signaling gradient shape is continually regulated from blastula through gastrula stages, as DV tissues are progressively patterned from anterior to posterior. Since BMPs also pattern the neural tube and developing limbs, and act in the progression of cancer, osteoporosis, osteoarthritis, fibrodysplasia ossificans progressiva, understanding the spatiotemporal mechanisms that shape the BMP gradient in DV patterning will provide paradigms for BMP function and its modulation in these other contexts. The goal of this proposal is to determine how the BMP signaling gradient is shaped during gastrulation. We can determine the shape of the BMP signaling gradient through a quantitative immunofluorescence assay of nuclear phosphorylated Smad1/5, a direct readout of active BMP signaling. The initial BMP signaling gradient is generated at mid-blastula stages by Chordin (Chd), a key extracellular antagonist that binds and inhibits BMP ligands from signaling. Importantly, we observe that the gradient shape at the start of gastrulation differs fro its shape during late gastrulation. Since Chd activity is required throughout gastrulation, it is vtal to determine how Chd is regulated in time and space. Chd activity is modulated by two key classes of proteins: (i) the homologous metalloproteases Bmp1a and Tolloid (Tld), which cleave and inactivate Chd, and (ii) the metalloprotease inhibitor Sizzled (Szl). How loss of function of both metalloproteases affects the BMP gradient spatially and temporally will be investigated. Additionally, how loss of Szl function affects the gradient will be examined. It is hypothesized that Bmp1a/Tld inhibit Chd without restraint early in gastrulation to generate the BMP signaling gradient, while Szl is critical to inhibit Bmp1a/Tld during the latter half of gastrulation to steeen the BMP signaling gradient. In this proposal, the temporal requirement for Bmp1a/Tld and Szl, how each protein affects the shape of the BMP signaling gradient, and the effective ranges of Tld and Szl protein activity will be determined. Together, these experiments will elucidate how the spatiotemporal regulation of metalloprotease activity correctly shapes the BMP signaling gradient to pattern the DV axis of the developing zebrafish embryo.