BMP morphogen gradient spatiotemporal modulation by metalloprotease activity

金属蛋白酶活性对 BMP 形态发生素梯度时空调节

• 批准号: 9029181
• 负责人: Francesca Bartoloni Tuazon
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9029181
• 关键词: Adult; Affect; Anterior; Binding; Biological; Biological Assay; Bone Morphogenetic Proteins; Bone Regeneration; Cell Transplants; Cells; Cleaved cell; Colorectal; Defect; Degenerative polyarthritis; Development; Disease; Dorsal; Embryo; Endodontics; Family; Foundations; Gastrula; Goals; Health; Heat-Shock Response; Histocompatibility Testing; Homeostasis; Image; Immunofluorescence Immunologic; Invertebrates; Investigation; Kidney Diseases; Lateral; Ligands; Limb structure; Malignant Neoplasms; Medical; Metalloproteases; Neural tube; Nuclear; Organ; Orthopedics; Osteoporosis; Pancreas; Pattern; Phenotype; Positioning Attribute; Proteins; Pulmonary Hypertension; Regulation; Reporting; Role; Shapes; Signal Transduction; Signaling Molecule; Specific qualifier value; Staging; Time; Tissue Engineering; Tissues; Tracer; Transgenes; Transplantation; Vertebrates; Zebrafish; blastocyst; chordin; extracellular; gastrulation; in vivo; inhibitor/antagonist; knock-down; loss of function; morphogens; mutant; progressive myositis ossificans; promoter; protein function; research study; response; restraint; spatiotemporal; tumor progression

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.

描述（由申请人提供）：在脊椎动物和无脊椎动物中，骨形态发生蛋白（BMP）以浓度依赖性方式以形态发生梯度作用于背腹（DV）轴。BMP形态原梯度的形状是至关重要的：在离散DV位置的BMP信号传导的精确量指定不同的细胞命运。在 在斑马鱼胚胎中，高BMP信号传导指定腹侧细胞命运，中等水平指定侧向命运，并且没有BMP信号传导导致背侧命运。BMP信号梯度形状从囊胚到原肠胚阶段不断调节，因为DV组织从前部到后部逐渐形成图案。由于BMP还对神经管和发育中的肢体进行图案化，并在癌症、骨质疏松症、骨关节炎、进行性骨化性纤维发育不良的进展中起作用，因此理解在DV图案化中形成BMP梯度的时空机制将为BMP功能及其在这些其他背景下的调节提供范例。本提案的目标是确定如何BMP信号梯度在原肠胚形成过程中形成。我们可以通过核磷酸化Smad 1/5的定量免疫荧光测定来确定BMP信号梯度的形状，这是活性BMP信号的直接读数。最初的BMP信号梯度在囊胚中期由Chordin（Chd）产生，Chordin是一种关键的细胞外拮抗剂，其结合并抑制BMP配体的信号传导。重要的是，我们观察到原肠胚形成开始时的梯度形状与原肠胚形成晚期的形状不同。由于Chd活性在整个原肠胚形成过程中是必需的，因此确定Chd在时间和空间上是如何调节的是有价值的。Chd活性受两类关键蛋白质调节：（i）同源金属蛋白酶Bmp 1a和Tolloid（Tld），其切割和抑制Chd，以及（ii）金属蛋白酶抑制剂Sizzled（Szl）。将研究两种金属蛋白酶的功能丧失如何在空间和时间上影响BMP梯度。此外，还将检查Szl功能丧失如何影响梯度。推测Bmp 1a/Tld在原肠胚形成早期无限制地抑制Chd以产生BMP信号梯度，而Szl在原肠胚形成的后半期抑制Bmp 1a/Tld以使BMP信号梯度变陡是关键的。在该提议中，将确定Bmp 1a/Tld和Szl的时间要求、每种蛋白质如何影响BMP信号梯度的形状以及Tld和Szl蛋白质活性的有效范围。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，以形成发育中的斑马鱼胚胎的DV轴。