Novel approaches to define tissue fusion mechanisms in embryonic development

在人类发育过程中，组织融合方式的中断是导致出生缺陷的常见原因，在英国，大约每500人中就有一人受到影响。这项研究之所以被推动，是因为尽管我们尽了最大的努力，但大多数天生患有腭裂、脊柱裂和心脏缺陷等问题的患者仍然没有确定他们疾病的遗传原因。这往往会影响遗传咨询和预防工作。我们也不确定母亲的环境对这些情况(如疾病、维生素缺乏或药物滥用)有什么影响。我的目标是通过进行变革性研究来解决这些问题，以揭示正常组织融合所需的关键基因、细胞行为和分子系统，并提供我们对这些如何被干扰的知识的阶段性改变。为了加强我们对融合的理解，我们需要实验上通用和适当的模型系统。我将集中讨论眼部缺损的原因，这是导致妊娠头7周眼部视裂闭合(OFC)融合缺陷引起的主要遗传性失明原因。这种缺陷在视网膜和视神经上留下了一个无法治愈或修复的永久性缺口。我已经建立了鸡眼作为一种强大和易驯服的新融合模型，因为它具有独特的特征组合：(I)它与人类的OFC非常相似；(Ii)可以在鸡蛋内进行实验；(Iii)鸡眼足够大，可以准确地解剖融合组织，以便进一步实验。在罗斯林研究所，我们还开发了独特的带有荧光细胞的转基因鸡，我可以在整个OFC中观察它们的行为，并从这些细胞中选择性地分离特定的细胞，以揭示它们独特的基因表达谱。这项工作将把这些新技术与DNA测序、活细胞成像、基因网络分析和细胞行为建模方面的其他尖端技术相结合，以生成迄今为止最健壮和准确的OFC和组织融合信息框架。该项目的第一个目标是为直接参与OFC融合过程的细胞揭示基因表达水平和分子特征。我还将使用数学和计算机模拟来确定这些细胞的形态、运动和组织的变化，并将其与我的分子数据相结合。接下来，通过使用基因编辑技术选择性地去除OFC特异性基因，我将确定它们丢失或失调的后果，以了解更多关于融合过程中特定细胞行为的遗传驱动因素和修饰物。了解Netrin-1基因的功能是我们工作的关键部分，因为我最近表明，这是不同物种正常OFC的关键因素，也是其他发育融合环境(眼睛、耳朵和上颚)所必需的。然后，我将继续揭示Netrin-1和其他融合特异性基因是如何受到全基因组网络的调控的，使我们能够更好地了解在人类患者中发现的突变的生物学后果，并准确预测新的致病候选基因和非遗传因素对融合中基因表达调控的影响。这是转变融合生物学的强大工具组合。它将提供有价值的信息，说明胚胎发育过程中的特定挑战如何扰乱遗传和细胞程序，直接或间接导致发育融合缺陷。我透露的信息将帮助临床医生发现新的致病突变，并为支持受影响家庭的遗传咨询提供证据。从长远来看，我的系统将揭示母体环境如何影响融合缺陷的原因，并帮助确定减少其发生率的策略。最后，我的数据将揭示基因功能如何与细胞行为联系起来，为健康和疾病的广泛生物学背景提供洞察力。

Disruptions to how tissues fuse together during human development is a common cause of birth defects, affecting approximately 1 in 500 people in the UK. This research has been prompted because despite our best efforts, most patients born with problems such as cleft palate, spina bifida, and heart defects still don't have the genetic cause of their disorder identified. This often impacts genetic counselling and efforts towards prevention. We also remain unsure what impacts maternal environment have on these conditions (e.g. illness, vitamin deficiency, or substance abuse). I aim to address these by performing transformative research to reveal the key genes, cell behaviours, and molecular systems required for normal tissue fusion, and provide a step-change in our knowledge of how these can be perturbed.To enhance our understanding of fusion, we require experimentally versatile and appropriate model systems. I will focus on the causes of ocular coloboma, the leading inherited cause of blindness arising from a fusion defect of optic fissure closure (OFC) in the eye in the first 7 weeks of pregnancy. This defect leaves a persistent gap in the retina and optic nerve which cannot be cured or repaired. I have established the chick eye as a powerful and tractable new model for fusion because of a unique combination of features: (i) it closely resembles OFC in humans; (ii) experiments can be performed inside the egg; and (iii) chick eyes are sufficiently large to accurately dissect fusing tissues for further experimentation. At Roslin Institute we have also developed unique transgenic chickens with fluorescent cells whose behaviours I can observe throughout OFC, and from which I can selectively isolate specific cells to reveal their unique gene expression profiles. This work will combine these new technologies with other cutting-edge techniques in DNA sequencing, live-cell imaging, gene-network analysis and cell behaviour modelling to generate the most robust and accurate framework of OFC and tissue fusion information available to date.The first aim of this project is to reveal gene expression levels and molecular signatures for the cells directly taking part in the OFC fusion process. I will also determine the changes in morphology, movement and organisation of these cells using mathematical and computer simulations, and integrate this with my molecular data. Next, by selectively ablating OFC-specific genes using gene-editing techniques, I will determine the consequence of their loss or dysregulation to learn more about the genetic drivers and modifiers for specific cell behaviours during fusion. Understanding the function of the Netrin-1 gene is a key part of our work, as I have recently shown that this is an essential factor for normal OFC in diverse species, and that it is required for other developmental fusion contexts (eye, ear and palate). I will then go on to reveal how Netrin-1 and other fusion-specific genes are regulated by genome-wide networks, allowing us to understand better the biological consequences of mutations identified in human patients, and to accurately predict new disease-causing candidate genes and the effect of non-genetic factors on the regulation of gene expression in fusion.This is a powerful combination of tools to transform fusion biology. It will provide valuable information for how specific challenges during embryogenesis can disrupt genetic and cellular programmes, directly or indirectly leading to developmental fusion defects. The information I reveal will help clinicians to uncover new causative mutations and provide evidence to support genetic counselling in affected families. In the longer term, my system will reveal how the maternal environment can influence the causes of fusion defects, and help define strategies to reduce their incidence. Lastly, my data will reveal how gene function links to cell behaviour, providing insight for a broad range of biological contexts in health and disease.