Rationale: The formation and maintenance of a functional vasculature is essential for normal embryonic development, and genetic changes that affect the vasculature underlie pathogenesis in many human diseases. In vivo imaging in mouse models is required to understand the full complexity of mammalian vascular formation, which is a dynamic and 3-dimensional process. Optical microscopy of genetically expressed fluorescent reporter proteins offers high resolution but limited depth of penetration in vivo. Conversely, there are a plethora of molecular probes for alternative in vivo vascular imaging modalities, but few options for genetic control of contrast enhancement. Objective: To develop a reporter system for multimodal imaging of genetic processes involved in mammalian vascular biology. Methods and Results: To approach this problem, we developed an optimal tagging system based on Biotag-BirA technology. In the resulting Biotag reporter system, coexpression of 2 interacting proteins results in biotin labeling of cell membranes, thus enabling multimodal imaging with “avidinated” probes. To assess this approach for in vivo imaging, we generated transgenic mice that expressed the Biotag-BirA transgene from a minimal Tie2 promoter. A variety of imaging methods were used to show the utility of this approach for quantitative analysis in embryonic and adult models of vascular development, using intravascular injection of avidinated probes for near infrared, ultrasound, and magnetic resonance imaging. Conclusions: The present results demonstrate the versatility of the Biotag system for studies of vascular biology in genetically engineered mice, providing a robust approach for multimodal in vivo imaging of genetic processes in the vasculature.

The mouse is the preferred model organism for genetic studies of mammalian brain development. MRI has potential for in utero studies of mouse brain development, but has been limited previously by challenges of maximizing image resolution and contrast while minimizing artifacts due to physiological motion.

Vascular system development involves a complex, three-dimensional branching process that is critical for normal embryogenesis. In the brain, the arterial systems appear to develop in a stereotyped fashion, but no detailed quantitative analyses of the mouse embryonic cerebral arteries have been described. In this study, a gadolinium-based contrast perfusion method was developed to selectively enhance the cerebral arteries in fixed mouse embryos. Three-dimensional magnetic resonance micro-imaging (micro-MRI) data were acquired simultaneously from multiple embryos staged between 10 During mammalian embryogenesis, the vasculature forms as a highly complex three-dimensional (3D) system of interconnected blood vessels that provide the developing tissues with essential nutrients, growth factors, and oxygen. Studies using genetically engineered mice have provided critical data on the genetic and molecular factors controlling vascular development during embryogenesis and also during disease progression involving abnormal formation of blood vessels. In these studies, quantitative 3D analysis of vascular patterns is seldom performed since commonly used histologic methods are inherently two dimensional and prone to sectioning artifacts, making 3D reconstructions difficult. Volumetric imaging methods such as magnetic resonance micro-imaging (micro-MRI) could provide powerful new tools to study vascular patterning in both normal and mutant mouse embryos.Vascular development during embryogenesis can be divided into two key processes: vasculogenesis and angiogenesis. The earliest step, vasculogenesis, involves the recruitment and assembly of endothelial-cell precursors, called angioblasts, into a primary vascular plexus (1). These primitive blood vessels are then extended and elaborated by the process of angiogenesis, which involves cell proliferation, differentiation of the arterial and venous components, and the establishment of the final 3D patterns of the vascular networks (2). While vasculogenesis is a process that occurs only during early embryonic development, angiogenesis is an ongoing process that can also occur after birth, depending on the needs of different tissues, which can be advantageous, as in angiogenesis after ischemia (3), or pathologic, as in tumor angiogenesis (4).The development of the mammalian vasculature results in apparently stereotypical patterns in a number of vascular subsystems, including the cerebral arteries. However 3D vascular patterning during brain development has not been investigated in detail, in large part due to the lack of analytical tools. We are particularly interested in analyzing the 3D patterns of the cerebral arterial inputs and how these patterns change during normal development, and in mouse mutants after genetic alterations that disrupt vascular patterning. The primary vascular inputs into the developing brain are the basilar artery that perfuses the more posterior parts of the brain and the carotid arteries that supply blood to the more anterior brain regions. In this report...

Both the availability of methods to manipulate genes and the completion of the mouse genome sequence have led to the generation of thousands of genetically modified mouse lines that provide a new platform for studying mammalian development and developmental diseases. Phenotyping of mouse embryos has traditionally been performed on fixed embryos by the use of ex vivo histological, optical and high-resolution MRI techniques. Although potentially powerful, longitudinal imaging of individual animals is difficult or impossible with conventional optical methods due to the inaccessibility of mouse embryos inside the maternal uterus. To address this problem we present a method of imaging the mouse embryo from stages as early as embryonic date (E) 10.5, close to the onset of organogenesis in most physiological systems. This method uses a self-gated MRI protocol combined with image registration to obtain whole-embryo high resolution (100 μm isotropic) three-dimensional images. Using this approach, we demonstrate high-contrast in the cerebral vasculature, limbs, spine and central nervous system without the use of contrast agents. These results indicate the potential of MRI for longitudinal imaging of developing mouse embryos, in utero, and for future applications in analyzing mutant mouse phenotypes.

The vasculature is the earliest developing organ in mammals and its proper formation is critical for embryonic survival. Magnetic resonance imaging (MRI) approaches have been used previously to analyze complex three-dimensional (3D) vascular patterns and defects in fixed mouse embryos. Extending vascular imaging to an in utero setting with potential for longitudinal studies would enable in vivo, dynamic analysis of the vasculature in normal and genetically engineered mouse embryos. In this study we utilized an in utero MRI approach that corrects for motion, using a combination of interleaved gated acquisition and serial co-registration of rapidly acquired 3D images. We tested the potential of this method by acquiring and analyzing images from wildtype and Gli2 mutant embryos, demonstrating a number of Gli2 phenotypes in the brain and cerebral vasculature. These results show that in utero MRI can be used for in vivo phenotype analysis of a variety of mutant mouse embryos.