DESCRIPTION (provided by applicant): Biomechanical aspects of embryonic tissues are poorly understood, especially nuclear mechanics. Very early embryos are well-known to be very soft and have very low levels of the nuclear structure protein lamin-A, which we have shown by single cell manipulations means that the nucleus is softer than in almost any adult cell [Swift Science 2013]. Initial differentiation to tissue turns on lamin-A transcription in heart, which seems important because knockout mice exhibit 'developmental defects of the heart' and die shortly after birth [Kubben Nucleus 2011], but lamin-A protein characterization is lacking in intac embryonic tissues as studied here. Lamin-A mutations cause a range of diseases with various ages of onset, including dilated cardiomyopathy (DCM) and accelerated aging (Progeria) affecting heart. Lamin-A is also known to affect differentiation and cell survival - all of which motivates studies to see & perturb the lamina in beating hearts. With adult tissue and primary cells, we have found that lamin-A levels are nearly proportional to tissue stiffness E [Swift Science 2013]. Relatively stiff connective tissues bear high mechanical stress, such as bone and even heart, and they have high lamin-A, suggesting stiff nuclei resist the stress. In contrast, very soft tissues such as brain and marrow that bear little stress express low lamin-A. B-type lamins are comparatively constant in the solid tissues, so that lamin-A:B stoichiometry seems a mechanosensor of stiffness and stress in adult tissues. We have worked through the mathematics of a simple mechanobiological gene circuit that fits findings for adult cells and tissues. Our hypothesis here is that Lamins in normal embryos adjust developmentally in response to mechanical stresses. Our goal is to determine and perturb mechano- regulation of lamin gene circuits in developing embryos, with a focus on what develops into a stiff heart relative to fluid blood. We focus on the facile chick embryo system per our recent studies that demonstrate acute sensitivity of beating heart to matrix elasticity [Majkut Curr Biol 2013]. Chick has advantages including the fact that chick erythrocytes have lamins, but we will at the end compare to developing mouse tissues. First we will quantify lamin protein levels throughout development by Mass Spec, and we will assess their stress and stiffness sensitivity with novel measurements and perturbations. We will relate embryonic lamina measurements to nuclear rheology and perturb the levels to validate relationships and molecular mechanisms. Preliminary data shows that beating chick hearts are easily transfected, so that Lamin Promoter- Reporter constructs can be tested as in situ mechanosensors of stress and stiffness. The lamina also enhances maturation and differentiation, and initial data with adult cells indicates feedback to cytoskeletal gene expression and the retinoid pathway of therapeutic relevance. Our studies should ultimately reveal the nuclear lamina as a multi-factorial, embryonic stress sensor that feeds back into broader structural regulation. 16

PUBLIC HEALTH RELEVANCE: The heart is the first organ to form and is a mechanically stressed pump that is susceptible to diseases of structural proteins in the matrix, cytoskeleton, and even the nucleus. Lamin-A is a nuclear structure protein (related to keratins in nails and hair) that seems to turn on at the beginning of tissue differentiation, but we know little of its protein regulation nor its contributions to structure even though mutations cause Dilated Cardiomyopathies and Accelerated Aging (Progeria) that affects Heart. Blood develops soon after the Heart begins pumping, and we will compare Lamins in Heart and Blood cells because the latter also exhibit major changes in nuclear lamina proteins that already seem to contrast with those in Heart and could be equally revealing. Importantly, quantifying lamins in development could lead to a real-time means to monitor the stress and stiffness in tissues through expression of lamin Promoter-Reporter constructs. Regulatory roles for lamins in maturation- differentiation and cell survival suggest that regenerative cardiology with various stem cells should also benefit from a clarification of cell-level sensitivities to tissue stiffness especially since rigid post- infarct scars limit pumping by adult heart. Compounds that affect in situ lamina levels might thus be more easily identified by our fundamental studies and ultimately find use in disease and differentiation.