Three-dimensional reconstruction of gene expression patterns during cardiac development

Soufan, Alexandre T.; Ruijter, Jan M.; Hoff, Maurice J.B. van den; Boer, Piet A. J. de; Hagoort, Jaco; Moorman, Antoon F.M.

doi:10.1152/physiolgenomics.00182.2002

Cited by 75 publications

(83 citation statements)

References 25 publications

(29 reference statements)

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“…Fluorescent in situ hybridization (FISH) was performed using the Tyramide Signal Amplification (TSA) system (Perkin-Elmer) (51). A 3D reconstruction of gene expression was done with the Amira 3.0 software as previously described (52). Quantitative data were analyzed using Student's t test.…”

Section: Methodsmentioning

confidence: 99%

A right-sided pathway involving FGF8 / Snai1 controls asymmetric development of the proepicardium in the chick embryo

Schlueter

Brand

2009

Proc. Natl. Acad. Sci. U.S.A.

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The proepicardium (PE) is a transient structure that forms at the venous pole of the embryonic vertebrate heart. This cardiac progenitor cell population gives rise to the epicardium, coronary vasculature, and fibroblasts. In the chicken embryo, the PE displays left-right (L-R) asymmetry and develops only on the right side, while on the left only a vestigial PE is formed, which subsequently gets lost by apoptosis. In this study, we analyzed how the L-R asymmetry pathway affects PE formation. Experimental manipulation of left-side determinants such as Shh, Nodal, and Cfc as well as forced expression of Pitx2 had no effect on the sidedness of PE development. In contrast, inhibition of early-acting regulators of L-R axis formation such as H ؉ /K ؉ -ATPase or primitive streak apoptosis affected the sidedness of PE development. Experimental interference with the right-side determinants Fgf8 or Snai1 prevented PE formation, whereas ectopic left-sided expression of Fgf8 or Snai1 resulted in bilateral PE development. These data provide novel insight into the molecular control of asymmetric morphogenesis suggesting that also the right side harbors an instructive signaling pathway that is involved in the control of PE development. This pathway might be of general relevance for setting up L-R asymmetries at the venous pole of the heart. left-right asymmetry ͉ Tbx18 ͉ Pitx2 ͉ venous pole morphogenesis T he left-right (L-R) axis controls asymmetric organogenesis in vertebrates (1-3). In lower chordates, Xenopus, and chick, breaking of the initial bilateral body symmetry involves the asymmetric distribution of ion channels and transporters, resulting in the development of membrane potential differences between the left and right body side, which may drive an asymmetric transport of small molecules through gap junctions (4, 5). In mammals, Xenopus, and zebrafish, ciliated cells in the organizer generate a fluid flow to the left that transports L-R determinants (6). In mammals, this nodal flow is believed to be the sole mechanism by which L-R asymmetry is initiated (7). In the chicken embryo, asymmetric gene expression in Hensen's node of gastrula stage embryos is an important intermediate step of establishing L-R asymmetry (2). Shh, for example, is asymmetrically expressed on the left side of Hensen's node and induces Nodal expression in a small left-sided domain adjacent to Hensen's node (8). Nodal establishes a large expression domain in the left lateral plate mesoderm (LPM) and induces the expression of Pitx2, a homeobox gene of the bicoid class (7). Pitx2 determines morphological L-R asymmetries in most organ systems (9-12) via modulation of cell-cell adhesion, cell morphology, extracellular matrix composition, spindle orientation, and cell proliferation (13-15).A right-sided regulatory cascade of gene expression is also initiated in Hensen's node including Bmp4 that induces asymmetric expression of Fgf18 and Fgf8 on the right side (16-18). It is believed that the right side does not initiate its own sidespecific morphogene...

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Section: Methodsmentioning

confidence: 99%

A right-sided pathway involving FGF8 / Snai1 controls asymmetric development of the proepicardium in the chick embryo

Schlueter

Brand

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Such topological information is entirely lost. This is one of the main problems of physical section based 3D reconstruction methods [35][36][37]57]. They have to use relatively thick sections in order to reduce the number of sections.…”

Section: Episcopic 3d Imaging Methods -Resolu-tion Of Volume Datamentioning

confidence: 99%

“…Thus the last decades saw the development of a vast number of methods for creating 3D information of cell, tissue, and organ morphology, 3D information of gene expression and gene product patterns, or both. Examples for techniques capable of analyzing small specimens, such as tissue samples or embryos are: in vivo microscopy [7][8][9][10], microcomputed tomography ( CT) [11][12][13], micro-magnetic resonance imaging ( MRI) [9,[14][15][16][17][18], ultrasound biomicroscopy (UBM) [19][20][21], optical projection tomography (OPT) [22,23], confocal microscopy [24][25][26][27][28], atomic force microscopy [29][30][31], 3D electron tomography [32,33], histological or macroscopic section based 3D reconstruction methods [34][35][36][37][38], and 3D episcopic imaging methods (see below). This paper does not compare all the different methods for volume data generation and gene expression analysis.…”

Section: In Situ Gene Expression Analysismentioning

confidence: 99%

Episcopic 3D Imaging Methods: Tools for Researching Gene Function

2013

Advances in Genome Science: Changing Views on Living Organisms

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This work aims at describing episcopic 3D imaging methods and at discussing how these methods can contribute to researching the genetic mechanisms driving embryogenesis and tissue remodelling, and the genesis of pathologies. Several episcopic 3D imaging methods exist. The most advanced are capable of generating high-resolution volume data (voxel sizes from 0.5x0.5x1 m upwards) of small to large embryos of model organisms and tissue samples. Beside anatomy and tissue architecture, gene expression and gene product patterns can be three dimensionally analyzed in their precise anatomical and histological context with the aid of whole mount in situ hybridization or whole mount immunohistochemical staining techniques. Episcopic 3D imaging techniques were and are employed for analyzing the precise morphological phenotype of experimentally malformed, randomly produced, or genetically engineered embryos of biomedical model organisms. It has been shown that episcopic 3D imaging also fits for describing the spatial distribution of genes and gene products during embryogenesis, and that it can be used for analyzing tissue samples of adult model animals and humans. The latter offers the possibility to use episcopic 3D imaging techniques for researching the causality and treatment of pathologies or for staging cancer. Such applications, however, are not yet routine and currently only preliminary results are available. We conclude that, although episcopic 3D imaging is in its very beginnings, it represents an upcoming methodology, which in short terms will become an indispensable tool for researching the genetic regulation of embryo development as well as the genesis of malformations and diseases.

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“…The study of cardiac morphogenesis requires an understanding of inherent structural details in three dimensions (3D) and single labeled cardiac progenitor cell lineage tracing during cardiac development will promote the understanding of cardiac morphogenesis. A number of high-resolution section based tomography methods have been developed in the past few decades to image organ structure 5,6 ; however, these methods require expensive, specialized instruments, extensive labor, and lack detailed structural organization at single cell resolution in the final volumetric reconstructed image 7,8 . 3D volumetric imaging at the single cell level provides a means to study progenitor cell differentiation and cellular behavior in vivo 7 .…”

Section: Introductionmentioning

confidence: 99%

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Qureshi

Shieh

et al. 2016

JoVE

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Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-Cre ERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects. Video LinkThe video component of this article can be found at

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Three-dimensional reconstruction of gene expression patterns during cardiac development

Cited by 75 publications

References 25 publications

A right-sided pathway involving FGF8 / Snai1 controls asymmetric development of the proepicardium in the chick embryo

A right-sided pathway involving FGF8 / Snai1 controls asymmetric development of the proepicardium in the chick embryo

Episcopic 3D Imaging Methods: Tools for Researching Gene Function

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

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