Molecular and biophysical investigation of epithelial cell sheet invagination

早期的形态发生运动将新胚胎从一个无定形的细胞球转变为一个复杂的可比较的结构，其中主要器官被布置在其中。这种运动的例子有原肠形成和神经形成，前者形成原始的肠道，后者形成脊椎弦。内陷是一种形态发生运动，在此过程中细胞片弯曲(在神经形成的情况下沿着一条线，在原肠形成的情况下沿着一个圆圈)。内陷异常会导致脊柱裂等疾病，大约每1000名活产儿中就有一人受到影响。细胞片在分支器官(肺、肾、血管)的形成过程中也会发生内陷，或者在癌症期间，当肿瘤导致新血管生长以提供营养时，细胞膜也会内陷。在内陷过程中，一组细胞必须表达与周围细胞不同的基因。内陷在胚胎中得到了广泛的研究，并提出了几种理论来解释细胞膜内陷的机制。然而，由于胚胎中的所有细胞都经历了一系列明确的基因表达，因此很难知道内陷的原因是什么，什么只是副作用。所有提出的内陷理论都要求细胞群体的一个子集表达一组与周围细胞不同的基因。现代分子生物学技术使我们能够迫使细胞表达所选择的基因。微印刷技术和微流控技术使我们能够以微米级的精度选择性地处理细胞。通过将这两套技术结合起来，我将设计一个实验系统，允许我迫使单层内的细胞子集表达我选择的基因。此外，这些细胞将被培养在可以变形的柔软基质上。这个系统将使我能够直接测试为内陷提出的每一种不同的理论。对于每个提出的理论，我将通过迫使单层内的一条细胞经历该理论假设是内陷原因的运动类型来复制所提出的运动。这将通过强制基因表达或通过化学处理来实现。然后，我将简单地观察24小时的细胞片，看看它是否内陷。一旦我找到了引起内陷的治疗方法，我就会检查起作用的机械力。这个项目将使我们能够更好地了解内陷的机制，并识别在癌症进展过程中可以靶向抑制内陷的蛋白质。

Early morphogenetic movements transform the new embryo from an amorphous ball of cells into a complex compartimentalised structure in which the main organs are laid out. Examples of such movements are gastrulation which creates the primitive gut and neurulation which creates the spinal chord. Invagination is a morphogenetic movement during which a cell sheet buckles (along a line in the case of neurulation and along a circle in the case of gastrulation). Abnormalities in invagination lead to conditions such as spina bifida that affect approximately 1 in 1000 live births. Cell sheets also undergo invagination during the creation of branched organs (lungs, kidneys, blood vessels) or during cancer when the tumour causes new blood vessels to grow to provide it with nutrients. During invagination, one group of cells has to express different genes than the surrounding cells. Invagination has been extensively studied in embryos and several theories have been proposed to explain the mechanism through which a cell sheet invaginates. However, since all cells in embryos undergo a defined sequence of gene expression, it is difficult to know what is responsible for invagination and what is just a side effect. All of the proposed theories for invagination require that one subset of the cell population express a different set of genes than the surrounding cells. Modern molecular biology techniques enable us to force cells to express a gene of choice. Microprinting techniques and microfluidic techniques enable us to selectively treat cells with micrometer precision. By combining both sets of techniques, I will devise an experimental system that will allow me to force a subset of cells within a monolayer to express a gene of my choice. In addition, the cells will be cultured on a soft substrate that they can deform. This system will enable me to test each of the different theories proposed for invagination directly. For each proposed theory, I will replicate the proposed movement by forcing a stripe of cells within the monolayer to undergo the type of movement that the theory hypothesizes is the cause of invagination. This will be done either through forced gene expression or through chemical treatment. Then, I will simply observe the cell sheet over a period of 24 hours and see if it invaginates. Once I have found which treatments give rise to invaginations, I will examine the mechanical forces at play. This project will enable us to better understand the mechanisms of invagination and identify proteins that can be targeted to inhibit it during cancer progression.