Cell Division Control During Leech Development

从一个受精卵发育成一个复杂的多细胞生物体需要无数轮的细胞分裂。 虽然所有的有丝分裂细胞都受到内部检查点的控制，但多细胞生物体中的有丝分裂细胞也受到外部影响的调节。 例如，在发育中的胚胎中，细胞分裂必须与其他发育过程协调，如细胞命运特化，细胞迁移和细胞分化。 本研究的目的是研究胚胎发育过程中细胞分裂的时间和空间控制。 这些研究将在Helobdella水蛭胚胎中进行，这些胚胎经历异步和不平等的细胞分裂的不变模式，以产生可单独识别的细胞，其细胞谱系，细胞周期特性和发育命运是已知的。 本研究项目的第一部分将调查细胞分裂的时间是如何在不同类别的水蛭识别细胞内调节的。 特别是，将检查的高度保守的细胞周期控制蛋白（Cdc25）的表达，活性调节和功能的模式。 例如，初步数据显示Cdc25的过表达诱导节段性创始细胞中的过早细胞分裂。有证据表明，Cdc25的绝对水平和细胞的年龄都不能决定过早分裂的发生，而是当这些细胞到达胚胎中的特定位置时触发过早分裂。 实验的目的是确定这种外部信号的来源。 本研究的第二部分将探讨如何在一个特定的早期卵裂球子集的卵裂方向进行调节。 以前的工作已经表明，在合子转录的情况下，这些细胞的有丝分裂纺锤体不承担不平等分裂的正确方向，因此，它们总是平等地分裂。 为了鉴定参与纺锤体定位的合子表达基因，使用消减杂交和差异筛选技术产生了差异表达的cDNA库。 一系列的筛选程序将被用来确定推定的“纺锤体定位”序列之间的cDNA的集合。在分离全长cDNA后，将根据相应RNA恢复转录抑制胚胎的这些细胞中的不等切割的能力来鉴定参与纺锤体定位的cDNA。 这些研究将有助于对复杂生物体中细胞用于调节细胞分裂的时间和对称性的不同机制的认识。

The development of a complex multicellular organism from a single fertilized egg requires numerous rounds of cell division. While all mitotic cells are subject to internal checkpoint controls, those in a multicellular organism are also regulated by external influences. In a developing embryo, for example, cell division must be coordinated with other developmental processes, such as cell fate specification, cell migration, and cell differentiation. The objectives of this research are to examine the temporal and spatial controls of cell division during embryonic development. These studies will be conducted in Helobdella leech embryos, which undergo invariant patterns of asynchronous and unequal cell division to produce individually identifiable cells whose cell lineages, cell cycle properties, and developmental fates are known. The first part of this research project will investigate how the timing of cell division is regulated within the different classes of leech identified cells. In particular, the patterns of expression, regulation of activity, and function of a highly conserved cell cycle control protein (Cdc25) will be examined. For example, preliminary data revealed that overexpression of Cdc25 induces premature cell divisions in the segmental founder cells. Evidence suggests that neither the absolute level of Cdc25 nor the age of the cell determines the onset of premature division, but rather premature division is triggered when these cells reach a particular location in the embryo. Experiments are designed to identify the source of this external signal. The second part of this research will investigate how the orientation of cleavage is regulated in a specific subset of early blastomeres. Previous work has shown that, in the absence of zygotic transcription, the mitotic spindles of these cells do not assume the proper orientation for unequal cleavage and, as a consequence, they always cleave equally. In order to identify the zygotically expressed genes involved in spindle positioning, a pool of differentially expressed cDNAs has been generated using subtractive hybridization and differential screening techniques. A series of screening procedures will be used to identify putative "spindle positioning" sequences among this collection of cDNAs. Upon isolation of full-length cDNAs, those involved in spindle positioning will be identified on the basis of the ability of the corresponding RNA to restore unequal cleavages in these cells of transcriptionally inhibited embryos. These studies will contribute to the emerging appreciation of the diverse mechanisms used by cells in complex organisms to regulate the timing and symmetry of cell division.