Decoding the electric face: development and channelopathy-induced birth defects

描述（由申请人提供）：在易于处理的模型中，如非洲爪蟾的颅面发育，已经成为研究构建复杂结构的细胞活动的流行和高度有益的模型。关于颅面图案背后的化学和遗传过程已经收集了很多信息。作为对比和补充，我们的实验室发现了一层生物物理信号：不同细胞膜静息电位的调节分布，指导细胞行为和大规模形态发生。我们已经证明，眼睛发育、肢体再生和肿瘤发生都是由离子通道介导的电压梯度调节的。我们已经适应了电压敏感的荧光染料来观察这些梯度，并使用分子工具来修改它们，从而调节单个细胞的行为和重新编程整个器官原基。这些技术不同于经典的电场应用方法，不仅揭示了梯度的分子遗传来源，而且揭示了生物物理特性调节形态的表观遗传和转录下游步骤。最近，我们发现了早期颅面发育的一个显著特征：“电脸”——胚胎蛙面部的超极化和去极化的动态模式，它预示、预测和控制面部结构的形状和位置。扰乱这些模式，会导致关键基因表达的可预测变化和解剖结构的变化。这一发现为迄今为止神秘的观察提供了一种假设，即几种离子通道病变（由离子通道基因如KCNJ2突变引起的疾病）不仅会导致神经功能障碍和心律失常，还会导致颅面缺陷。这些通道参与胚胎面部正常生物电区域化的建立，这一事实可以解释为什么通道对颅面模式至关重要。我们的项目旨在：1)详细描述早期面部相对于基因表达域的生物电特性（融合转录和生物物理数据的物理组学图谱）；2)通过建立电偶联细胞内自组织的预测数学模型，探索生物电面模式的出现；3)揭示电压梯度如何调节特定下游面部图案基因表达域的分子细节；4)发展光遗传学技术，在活组织中读取/写入所需的电模式，以覆盖不正确的膜电压模式，从而防止缺陷。由此产生的数据将：作为未来试图合并生物物理和转录调控层的重要基础，以解释复杂的发育；建立定量模型，为电压依赖性图形的微创矫正操作提供处方；并探索利用生物电模式的引导自组装的新方法，以解决再生医学、病因学和出生缺陷治疗方面的问题，并通过合成生物工程产生新的杂交结构。

DESCRIPTION (provided by applicant): Craniofacial development in tractable models such as the frog Xenopus, has been a popular and highly rewarding model for studies of the cell activities that build complex structures. Much information has been gathered about the chemical and genetic processes that underlie craniofacial patterning. In contrast to and complementing that work, our lab has discovered a layer of biophysical signaling: a regulated distribution of different cell membrane resting potentials that instruct cell behavior and large-scale morphogenesis. We have showed that eye development, limb regeneration, and tumorigenesis are all regulated by ion channel-mediated voltage gradients. We have adapted voltage-sensitive fluorescent dyes to observe these gradients and used molecular tools to modify them thereby regulating individual cell behaviors and reprogramming whole organ primordia. These techniques are distinct from classical methods of electric field application, and reveal not only the molecular-genetic sources of the gradients but also the epigenetic and transcriptional downstream steps through which biophysical properties regulate morphology. Recently, we uncovered a remarkable feature of early craniofacial development: the "electric face" - dynamic patterns of hyper- and depolarization in the embryonic frog face that precede, predict, and control the shape and location of facial structures. Perturbing these patterns, results in predictable changes in expression of key genes and changes in anatomy. This finding suggests a hypothesis for the heretofore-mysterious observation that several channelopathies (diseases caused by mutations in ion channel genes such as KCNJ2) cause not only neurological deficits and cardiac arrhythmias but also craniofacial defects. The fact that these channels participate in the establishment of the normal bioelectric regionalization of the embryonic face may explain why channels are essential for craniofacial patterning. Our project aims to: 1) characterize in detail the bioelectric properties of the early face relative to gene expression domains (a physiomics atlas merging transcriptional and biophysical data); 2) explore the emergence of the bioelectric face patterns by formulating a predictive mathematical model of self-organization within electrically-coupled cells; 3) reveal molecular details of how voltage gradients regulate specific downstream face-patterning gene expression domains; and 4) develop optogenetic techniques to read/write desired electrical patterns in living tissue to override incorrect membrane voltage patterns thus preventing defects. The resulting data will: serve as an essential foundation for future attempts to merge biophysical and transcriptional regulatory layers in order to explain complex development; establish a quantitative model to prescribe minimally invasive corrective manipulations of voltage-dependent patterning; and to chart a new approach toward exploiting guided self-assembly of bioelectrical patterns to address issues in regenerative medicine, etiology and treatment of birth defects, as well as produce new hybrid constructs via synthetic bioengineering.