In vivo assessment of granulin dependent myeloid cell formation

在了解颗粒蛋白(GRN)如何调节髓系细胞分化方面存在着根本的差距。 这一缺口的持续存在是一个重要的问题，因为在它被填补之前，理解如何 颗粒素在急性髓系白血病发生中的作用尚不清楚，因此 操纵颗粒蛋白途径来治疗这些血液系统恶性肿瘤将仍然是遥不可及的。这个 长期目标是通过扩大他们的治疗范围来改善急性髓系白血病患者的预后 治疗选择。总体目标是在体内确定需要颗粒的髓系细胞群 以促进髓系细胞的正常发育，以及颗粒蛋白激活的分子途径。中环 假设颗粒蛋白对粒细胞、中性粒细胞和 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。其基本原理是 对于拟议的研究来说，了解造血的基本分子机制 颗粒蛋白的调节有可能转化为更好地理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，只有24%。在强劲的初步数据指引下，这 假说将通过追求两个具体目标来检验：1)在体内鉴定需要 用于正常发育的gRNA；以及2)确定哪些STAT家族成员通过颗粒激活 适当的髓系分化。在第一个目标下，一个已公布的gRNA突变体，已经由 髓系细胞数量减少的申请者，将被用于在体内鉴定髓系细胞群 它的发育受到胚胎和成人造血过程中缺乏gRNA的影响。 斑马鱼胚胎、组织学技术和RNA杂交探针中强大的活体显微镜工具 手头上已经有的东西将会被使用。在第二个目标下，定量聚合酶链式反应以精确定位STAT家族 由gRNA激活的成员，并为gRNA突变胚胎中的每个STAT候选注入mRNA将是 用来修复髓系缺陷的手术。利用斑马鱼的优势提出了一种创新的方法 基因组复制导致两个拷贝的祖先颗粒蛋白基因(gRNA和grnb)在 由于gRNA的特化，颗粒蛋白的造血功能史无前例 造血过程。由于gRNA是特定于造血过程的，这避免了对其他 纸巾。此外，斑马鱼在体内造血发育可视化方面的能力是 被剥削。这项拟议的研究意义重大，因为它有望从纵向上促进人们对 颗粒蛋白可能在急性髓系白血病中起作用，以及这种蛋白是如何被操纵来治疗的 这些障碍。

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.