Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.

Background-Serious congenital heart defects occur as a result of improper atrioventricular septum (AVS) development during embryogenesis. Despite extensive knowledge of the genetic control of AVS development, few genetic lesions have been identified that are responsible for AVS-associated congenital heart defects. Methods and Results-We sequenced 32 genes known to be important in AVS development in patients with AVS defects and identified 11 novel coding single-nucleotide polymorphisms that are predicted to impair protein function. We focused on variants identified in the bone morphogenetic protein receptor, ALK2, and subjected 2 identified variants to functional analysis. The coding single-nucleotide polymorphisms R307L and L343P are heterozygous missense substitutions and were each identified in single individuals. The L343P allele had impaired functional activity as measured by in vitro kinase and bone morphogenetic protein-specific transcriptional response assays and dominant-interfering activity in vivo. In vivo analysis of zebrafish embryos injected with ALK2 L343P RNA revealed improper atrioventricular canal formation. Conclusion-These data identify the dominant-negative allele ALK2 L343P in a patient with AVS defects. (Circulation.2009;119:3062-3069.)Key Words: ALK2 protein, human Ⅲ genes Ⅲ heart defects, congenital Ⅲ screening Ⅲ signal transduction T he primitive heart tube of vertebrates consists of an inner layer of endothelial cells, the endocardium, and an outer muscular layer of myocardial cells. After formation of the heart tube, endothelial cells delaminate from the endocardium and migrate into an extracellular matrix, called cardiac jelly, which resides between the endocardium and myocardium. These invading endothelial cells undergo an endothelium-tomesenchyme transition (for detailed review, see elsewhere 1 ) and give rise to swellings known as endocardial cushions (ECs). ECs contribute to the valves and septa of the heart, and disruptions in their formation result in valvular and septal defects. 2 A number of signaling pathways, including vascular endothelial growth factor signaling, Notch, Wnt/␤-catenin, bone morphogenetic protein (BMP)/transforming growth factor-␤ signaling have been implicated in atrioventricular septum (AVS) development either in vitro or in vivo. 1 Clinical Perspective on p 3069This extensive knowledge of the genetic control of AVS development has yet to be translated into a broader clinical knowledge of the genetic determinants of congenital heart defects (CHDs). This is due largely to the complex pathogenesis of CHD and the scarcity of large families with multiple affected individuals suitable for conventional genetic analyses. More recently, candidate screening approaches have been used to circumvent this limitation. Such approaches, when coupled with kindred linkage and/or detailed functional analyses, can identify novel causative mutations in genes previously suspected to function in AVS development. [3][4][5] In an effort to identify genetic lesions that may cause C...

The zebrafish (Danio rerio) has become a popular vertebrate model organism to study organ formation and function due to its optical clarity and rapid embryonic development. The use of genetically modified zebrafish has also allowed identification of new putative therapeutic drugs. So far, most studies have relied on broad overexpression of transgenes harboring patient-derived mutations or loss-of-function mutants, which incompletely model the human disease allele in terms of expression levels or cell-type specificity of the endogenous gene of interest. Most human genetically inherited conditions are caused by alleles carrying single nucleotide changes resulting in altered gene function. Introduction of such point mutations in the zebrafish genome would be a prerequisite to recapitulate human disease but remains challenging to this day. We present an effective approach to introduce small nucleotide changes in the zebrafish genome. We generated four different knock-in lines carrying distinct human cardiovascular-disorder-causing missense mutations in their zebrafish orthologous genes by combining CRISPR/Cas9 with a short template oligonucleotide. Three of these lines carry gain-of-function mutations in genes encoding the pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2, ABCC9) subunits of an ATP-sensitive potassium channel (KATP) linked to Cantú syndrome (CS). Our heterozygous zebrafish knock-in lines display significantly enlarged ventricles with enhanced cardiac output and contractile function, and distinct cerebral vasodilation, demonstrating the causality of the introduced mutations for CS. These results demonstrate that introducing patient alleles in their zebrafish orthologs promises a broad application for modeling human genetic diseases, paving the way for new therapeutic strategies using this model organism.