In vivo assessment of granulin dependent myeloid cell formation

在理解颗粒蛋白（GRN）如何调节髓样细胞分化方面存在根本性的差距。 这一差距的继续存在是一个重要的问题，因为在填补这一差距之前， 粒蛋白对急性髓细胞白血病的发展的贡献是未知的，因此， 操纵颗粒蛋白途径来治疗这些造血系统恶性肿瘤仍然是不可能的。的 长期目标是通过扩大急性髓细胞白血病患者的治疗范围， 治疗选择总体目标是在体内确定需要颗粒蛋白的髓样细胞群。 以及粒蛋白在骨髓细胞中激活的分子途径。中央 假设颗粒蛋白对于粒细胞、嗜中性粒细胞 巨噬细胞通过激活信号转导和转录激活因子（STAT）家族成员。 这一假设是根据申请人提供的初步数据提出的。的理由 因为这项研究的目的是了解造血的基本分子机制， 调节颗粒蛋白有可能转化为更好地了解急性胰腺炎的发病机制， 髓系白血病、血液恶性肿瘤治愈率低，仅为24%。在强大的初步数据指导下， 将通过追求两个具体目标来测试假设：1）在体内鉴定需要在体外培养的骨髓细胞群。 2）确定哪些STAT家族成员通过颗粒蛋白被激活， 适当的髓样分化在第一个目标下，已发表的grna突变体， 申请人的骨髓细胞数量减少，将用于体内鉴定骨髓细胞群 在胚胎和成体造血过程中，其发育受到缺乏Grna的影响。 斑马鱼胚胎、组织学技术和RNA杂交探针中强大的体内显微镜工具 现有的将被使用。在第二个目标下，定量PCR定位STAT家族 成员激活的Grna，并在grna突变胚胎中注射每个STAT候选人的mRNA， 用于挽救骨髓缺陷。提出了一种利用斑马鱼的创新方法 基因组复制导致两个拷贝的祖先颗粒蛋白基因（grna和grnb），以了解在 一个前所未有的方式的造血功能的颗粒，由于专业化的grna， 造血过程由于grna对造血过程是特异性的， 组织中此外，斑马鱼用于体内造血发育可视化的能力是 被剥削拟议的研究是重要的，因为它有望纵向推进了解如何 颗粒蛋白可能导致急性髓细胞白血病以及如何操纵这种蛋白质来治疗 这些紊乱。

There is a fundamental gap in understanding how Granulin (GRN) regulates myeloid cell differentiation. Continued existence of this gap represents an important problem because, until it is filled, understanding of how Granulin contributes to the development of acute myeloid leukemia would be unknown, and therefore the manipulation of the Granulin pathway to treat these hematopoietic malignances will remain unreachable. The long-term goal is to improve the prognosis of patients suffering from acute myeloid leukemia by expanding their therapeutic options. The overall objective is to define in vivo the myeloid cell populations that require Granulin for proper development as well as the molecular pathway activated by Granulin in myeloid cells. The central hypothesis is that Granulin is essential for proper myeloid lineage differentiation of granulocytes, neutrophils and macrophages through the activation of signal transducers and activators of transcription (STAT) family members. This hypothesis has been formulated on the basis of preliminary data produced by the applicant. The rationale for the proposed research is that understanding the fundamental molecular mechanisms of hematopoietic regulation by Granulin has the potential to translate into better understanding of the pathogenesis of acute myeloid leukemia, blood malignancies with a low cure rate of 24%. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify in vivo the myeloid cell populations that require Grna for proper development; and 2) Determine which STAT family members are activated through Granulin for proper myeloid differentiation. Under the first aim, a published grna mutant which has been described by the applicant to have decreased myeloid cell numbers, will be used to identify in vivo the myeloid cell populations whose development is affected by the absence of Grna, both during embryonic and adult hematopoiesis. Powerful in vivo microscopy tools in the zebrafish embryo, histological techniques, and RNA-hybridization probes that are already on hand will be used. Under the second aim, quantitative PCR to pinpoint the STAT family members activated by Grna, and injection of mRNA for each STAT candidate in grna mutant embryos will be performed to rescue myeloid defects. An innovative approach is proposed by taking advantage of the zebrafish genome duplication that resulted in two copies of the ancestral Granulin gene (grna and grnb) to understand in an unprecedented manner the hematopoietic function of Granulin due to the specialization of grna in hematopoietic processes. Since grna is specific to hematopoietic processes, this avoids disruption of other tissues. In addition, the power of the zebrafish for visualization of in vivo development of hematopoiesis is exploited. The proposed research is significant, since it is expected to vertically advance understanding of how Granulin could be contributing to acute myeloid leukemia and how this protein could be manipulated to treat these disorders.