Zebrafish smarcc1a mutants reveal requirements for BAF chromatin remodeling complexes in distinguishing the atrioventricular canal from the cardiac chambers.

BACKGROUND: Essential patterning processes transform the heart tube into a compartmentalized organ with distinct chambers separated by an atrioventricular canal (AVC). This transition involves the refinement of expression of genes that are first found broadly throughout the heart tube and then become restricted to the AVC. Despite the importance of cardiac patterning, we do not fully understand the mechanisms that limit gene expression to the AVC. RESULTS: We show that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, is critical for cardiac patterning. In smarcc1a mutants, myocardial differentiation and heart tube assembly appear to proceed normally. Subsequently, the smarcc1a mutant heart fails to exhibit refinement of gene expression patterns to the AVC, and the persistence of broad gene expression is accompanied by failure of chamber expansion. In addition to their cardiac defects, smarcc1a mutants lack pectoral fins, indicating similarity to tbx5a mutants. However, comparison of smarcc1a and tbx5a mutants suggests that perturbation of tbx5a function is not sufficient to cause the smarcc1a mutant phenotype. CONCLUSIONS: Our data indicate an important role for Smarcc1a-containing chromatin remodeling complexes in regulating the changes in gene expression and morphology that distinguish the AVC from the cardiac chambers.

High-resolution MRI of early-stage mouse embryos.

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 the study of 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 because of 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 day (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 the longitudinal imaging of developing mouse embryos in utero and for future applications in analyzing mutant mouse phenotypes.

Novel genetic approach for in vivo vascular imaging in mice.

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.

In utero phenotyping of mouse embryonic vasculature with MRI.

The vasculature is the earliest developing organ in mammals and its proper formation is critical for embryonic survival. MRI approaches have been used previously to analyze complex three-dimensional vascular patterns and defects in fixed mouse embryos. Extending vascular imaging to an in utero setting with potential for longitudinal studies would enable dynamic analysis of the vasculature in normal and genetically engineered mouse embryos, in vivo. In this study, we employed an in utero MRI approach that corrects for motion, using a combination of interleaved gated acquisition and serial coregistration of rapidly acquired three-dimensional 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.

Three-dimensional micro-MRI analysis of cerebral artery development in mouse embryos.

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 and 17 days of gestation, and a variety of image analysis methods was used to extract and analyze the cerebral arterial patterns. The results show that the primary arterial branches in the mouse brain are very similar between individuals, with the patterns established early and growth occurring by extension of the segments, while maintaining the underlying vascular geometry. To investigate the utility of this method for mutant mouse phenotype analysis, contrast-enhanced micro-MRI data were acquired from Gli2(-/-) mutant embryos and their wild-type littermates, showing several previously unreported vascular phenotypes in Gli2(-/-) embryos, including the complete absence of the basilar artery. These results demonstrate that contrast-enhanced micro-MRI provides a powerful tool for analyzing vascular phenotypes in a variety of genetically engineered mice.

Mn enhancement and respiratory gating for in utero MRI of the embryonic mouse central nervous system.

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. Manganese (Mn)-enhanced MRI (MEMRI) studies have demonstrated central nervous system (CNS) contrast enhancement in mice from the earliest postnatal stages. The purpose of this study was to expand MEMRI to in utero studies of the embryonic CNS in combination with respiratory gating to decrease motion artifacts. We investigated MEMRI-facilitated CNS segmentation and three-dimensional (3D) analysis in wild-type mouse embryos from midgestation, and explored effects of Mn on embryonic survival and image contrast. Motivated by observations that MEMRI provided an effective method for visualization and volumetric analysis of embryonic CNS structures, especially in ventral regions, we used MEMRI to examine Nkx2.1 mutant mice that were previously reported to have ventral forebrain defects. Quantitative MEMRI analysis of Nkx2.1 knockout mice demonstrated volumetric changes in septum (SE) and basal ganglia (BG), as well as alterations in hypothalamic structures. This method may provide an effective means for in utero analysis of CNS phenotypes in a variety of mouse mutants.