Dynamic smad-mediated BMP signaling revealed through transgenic zebrafish

Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Developmental Dynamics (Impact Factor: 2.38). 03/2011; 240(3):712-22. DOI: 10.1002/dvdy.22567
Source: PubMed


Bone morphogenic protein (BMP) signaling is fundamental to development, injury response, and homeostasis. We have developed transgenic zebrafish that report Smad-mediated BMP signaling in embryos and adults. These lines express either enhanced green fluorescent protein (eGFP), destabilized eGFP, or destabilized Kusabira Orange 2 (KO2) under the well-characterized BMP Response Element (BRE). These fluorescent proteins were found to be expressed dynamically in regions of known BMP signaling including the developing tail bud, hematopoietic lineage, dorsal eye, brain structures, heart, jaw, fins, and somites, as well as other tissues. Responsiveness to changes in BMP signaling was confirmed by observing fluorescence after activation in an hsp70:bmp2b transgenic background or by inhibition in an hsp70:nog3 background. We further demonstrated faithful reportage by the BRE transgenic lines following chemical repression of BMP signaling using an inhibitor of BMP receptor activity, dorsomorphin. Overall, these lines will serve as valuable tools to explore the mechanisms and regulation of BMP signal during embryogenesis, in tissue maintenance, and during disease.

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    • "galFF/UAS:h2a-gfp) as described above, Tg(myl7:galFF, UAS:Kaede) and Tg(myl7:nlsKikGR) hsc6 (Lazic and Scott, 2011), hereafter referred to as Tg(myl:nlsKikGR), Tg(hsp70:dkk1-gfp) (Stoick-Cooper et al., 2007), Tg(-5.1myl7:nDsRed2) f2 (Mably et al., 2003), referred to as [(Tg(myl7: dsred)], Tg(hsp70:wnt8a-gfp) (Weidinger et al., 2005), Tg(Bre:GFP) p77 (Alexander et al., 2011), Tg(bre:dmKO2) fish (Collery and Link, 2011) and Tg(hsp70I:Nog3) fr14 (Chocron et al., 2007). "
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    ABSTRACT: Glypicans are heparan sulphate proteoglycans (HSPGs) attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, and interact with various extracellular growth factors and receptors. The Drosophila division abnormal delayed (dally) was the first glypican loss-of-function mutant described that displays disrupted cell divisions in the eye and morphological defects in the wing. In human, as in most vertebrates, six glypican-encoding genes have been identified (GPC1-6), and mutations in several glypican genes cause multiple malformations including congenital heart defects. To understand better the role of glypicans during heart development, we studied the zebrafish knypek mutant, which is deficient for Gpc4. Our results demonstrate that knypek/gpc4 mutant embryos display severe cardiac defects, most apparent by a strong reduction in cardiomyocyte numbers. Cell-tracing experiments, using photoconvertable fluorescent proteins and genetic labeling, demonstrate that Gpc4 'Knypek' is required for specification of cardiac progenitor cells and their differentiation into cardiomyocytes. Mechanistically, we show that Bmp signaling is enhanced in the anterior lateral plate mesoderm of knypek/gpc4 mutants and that genetic inhibition of Bmp signaling rescues the cardiomyocyte differentiation defect observed in knypek/gpc4 embryos. In addition, canonical Wnt signaling is upregulated in knypek/gpc4 embryos, and inhibiting canonical Wnt signaling in knypek/gpc4 embryos by overexpression of the Wnt inhibitor Dkk1 restores normal cardiomyocyte numbers. Therefore, we conclude that Gpc4 is required to attenuate both canonical Wnt and Bmp signaling in the anterior lateral plate mesoderm to allow cardiac progenitor cells to specify and differentiate into cardiomyocytes. This provides a possible explanation for how congenital heart defects arise in glypican-deficient patients. © 2015. Published by The Company of Biologists Ltd.
    Development 05/2015; 142(10):1767-76. DOI:10.1242/dev.113894 · 6.46 Impact Factor
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    • "Therefore, as in the case of blood vessels (Kim et al., 2012; Larrivée et al., 2012; Moya et al., 2012; Wiley et al., 2011), it appears that BMP signaling may modulate development and/or maintenance of lymphatic vessels in a context-dependent manner. Considering the complex regulation of BMP signaling in other systems (Collery and Link, 2011; David et al., 2009; Farnsworth et al., 2011; Hartung et al., 2006; Miyazono et al., 2010), it is seemingly possible that distinct BMP ligands, of which distribution is spatiotemporally regulated, may exert pro- or anti-lymphangiogenic effects during development. Our analyses identify Smad5 as the most important downstream mediator of BMP signaling during lymphatic development. "
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    ABSTRACT: Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.
    Moleculer Cells 03/2014; 37(3). DOI:10.14348/molcells.2014.0005 · 2.09 Impact Factor
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    • "detecting cell signaling changes during early stages of differentiation (Collery and Link 2011; Wang et al. 2012) or testing small chemical effects in short time ranges (Molina et al. 2009). However, the use of destabilized proteins often suffers from many drawbacks, including the low amount of detectable accumulating protein during a biological event. "
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    ABSTRACT: In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.
    MGG Molecular & General Genetics 05/2013; 288(5-6). DOI:10.1007/s00438-013-0750-z · 2.73 Impact Factor
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