Next Article in Journal
Coronary Vasculature in Cardiac Development and Regeneration
Next Article in Special Issue
Vortex Dynamics in Trabeculated Embryonic Ventricles
Previous Article in Journal
Epigenetic Regulation of Organ Regeneration in Zebrafish
Previous Article in Special Issue
The Memory of the Heart
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JCDD
Volume 5
Issue 4
10.3390/jcdd5040058
jcdd-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM)

by
Wolfgang J. Weninger
1,*,
Barbara Maurer-Gesek
1,
Lukas F. Reissig
1,
Fabrice Prin
2,
Robert Wilson
2,
Antonella Galli
3,
David J. Adams
3,
Jacqueline K. White
3,
Timothy J. Mohun
2 and
Stefan H. Geyer
1
1
Division of Anatomy & MIC, Medical University of Vienna, 1090 Vienna, Austria
2
The Francis Crick Institute, London NW1 1AT, UK
3
Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK
*
Author to whom correspondence should be addressed.
J. Cardiovasc. Dev. Dis. 2018, 5(4), 58; https://doi.org/10.3390/jcdd5040058
Submission received: 23 November 2018 / Revised: 9 December 2018 / Accepted: 11 December 2018 / Published: 15 December 2018
(This article belongs to the Special Issue Functional Morphology of the Developing and Mature Cardiovascular System)
Browse Figures
Versions Notes

Abstract

:
The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.

1. Introduction

During gastrulation, embryos form a simple tubular heart. It pumps blood into a similarly simple and symmetrically arranged arterial system. During the following embryo periods and, to smaller extent even peri- and postnatally, both the heart and vascular system undergo extensive remodelling, which transforms them into complexly shaped and asymmetrically arranged organs.
The remodelling processes are driven and orchestrated by genetic and biomechanical factors, which interact in a strict temporal and spatial corset. Errors result in moderate to severe cardiovascular disorders but can potentially trigger abnormal formation and growth of all organ systems throughout the body.
Due to the central role the cardiovascular system plays in the embryonic, foetal and postnatal life, the genetic and biomechanical factors regulating normal cardiovascular development and the mechanisms underlying its pathologies are the focus of biomedical research [1,2,3,4,5,6,7]. For such research, a large number of tools for labelling active genes and gene products and for modifying or physically challenging their interplay during critical steps of development and growth were created and successfully established. Yet, all these tools produce effects that need to be properly visualised. Hence the existence of decent visualisation methods is at the core of cardiovascular research.
In particular visualisation and descriptive analysis of in situ distribution of gene expression information and of subtle morphologic defects caused by gene deletions, hemodynamic experiments or by alteration of the physical or chemical environment in embryos of model organisms remains a major challenge. The traditional imaging approach, which is careful analysis of series of two-dimensional (2D) histologic sections, is highly inefficient. This chiefly for two reasons: Firstly, embryos and their cardiovascular systems are of a complex three-dimensional (3D) arrangement, making it virtually impossible to comprehend the exact topology on the basis of two-dimensional (2D) information. Secondly, the production and examination of histologic sections is slow and time-consuming.
For coping with the drawbacks of conventional histological sections and section-based imaging methods [8,9,10], the last few decades have seen the development of large numbers of innovative 3D imaging technologies covering the meso- and microscopic range. The most promising are micro computed tomography (µCT)—even termed as virtual histology [11,12,13,14,15,16,17,18,19], micro-magnetic resonance imaging (µMRI) [20,21,22,23,24,25,26,27,28,29,30], optical projection tomography (OPT) [31,32,33,34,35,36,37], optical coherence tomography (OCT) [38,39,40,41], photoacoustic tomography (PAT) [40,42], serial block face microscopy [43,44], ultrasound microscopy [45,46,47,48,49], Episcopic 3D microscopy (Epi3D) [50], Episcopic Fluorescence Image Capturing (EFIC) [51,52] and High Resolution Episcopic Microscopy (HREM) [53,54,55,56]. A similar, episcopic approach is used for serial block-face electron microscopy (SBFSEM), which offers ultrastructural 3D information [57,58,59].
This paper aims to focus on one of these techniques, HREM, and its potential for visualising the cardiovascular system of normal and genetically altered embryos of biomedical models. In addition, it will briefly introduce the ability of using HREM for visualising the vascular system in biopsy material stemming from human tissue.

2. What Is High-Resolution Episcopic Microscopy (HREM)

High resolution episcopic microscopy is a technique for visualising the morphology of materials embedded in eosin stained methacrylate resin JB4. While cutting through the sample, series of inherently aligned digital images are created from subsequently exposed block surfaces. These images are virtually stacked and converted to volume data of high resolution and quality.
HREM volume data fit excellently for in situ visualisation, volumetry, planimetry and mathematical analysis of the components of the cardiovascular system of genetically normal, genetically engineered and physically and chemically challenged embryos of all popular model organisms. In selected cases HREM also permits 3D visualisation of gene expression and gene product patterns and the characterisation of cell distributions in their natural context. Hence, it was already successfully employed in several projects studying normal embryogenesis [60,61,62,63,64,65] and the role genes play in selected human diseases [66,67,68,69,70]. But, most importantly, HREM was introduced for the deciphering the mechanisms of the developmental disorders program (DMDD, www.dmdd.org.uk) as a method for screening the morphological phenotype of mouse embryos harvested at embryonic day (E) 14.5. In DMDD nearly 700 mouse embryos, stemming from 87 knock out lines, which produce embryonically lethal offspring were screened and their annotated data are provided on a freely accessible webpage [71,72,73,74].
The workflow of HREM data creation is simple and will be briefly outlined in the following chapters.

2.1. Sample Preparation

Sample preparation involves fixation and dehydration. A broad spectrum of fixation solutions and dehydration in series of increasing ethanols or methanols is suitable. Yet, for gaining excellent data quality, sample processing has to be slightly adapted to meet the specific requirements of tissues, organisms and developmental stages [53,75]. Also, if possible, perfusion with physiologic solutions for washing out the blood of cardiac chambers and/or blood vessels should be performed prior to embedding.

2.1.1. Fixation

Most commonly, fresh material and loose neonatal and adult tissues are fixed in 4% PFA/PBS or Bouin’s solution for 24 h to several days, depending on sample size. Fixation time lasts for at least 1 day and does not exceed several weeks. Adult tissues, especially biopsy material harvested from the human skin and heart, are fixed in 2% PFA/PBS containing 4% carbolic acid and the samples are kept in this solution for at least one week and a maximum of twelve months [76]. If whole mount staining with dye reactions, e.g., the NBT/BCIP detection system was conducted prior to dehydration and resin embedding, the samples must be immersed in fixation solution before and after the staining procedure.

2.1.2. Dehydration

After fixation, the samples are dehydrated in a series of methanols or ethanols with increasing concentrations (50%, 70%, 80%, 90%, 96%). Additional dehydration steps might be added for minimizing shrinkage artefacts. Depending on the volume of the specimens, they are kept from approximately 20 min (early zebrafish embryos) to up to 3 h (dense skin biopsy material) in each alcohol. Tissue contrast in large tissue samples profits from adding eosin to the higher alcohols [75,76].

2.1.3. Infiltration

After dehydration, the samples are infiltrated with and embedded in JB4-resin (Polysciences) containing eosin (0.4 g/100 mL) or alternatively eosin and acridine orange (0.275 g eosin/100 mL and 0.055 g acridine orange/100 mL). Except for stirring eosin/arcridine orange into the solutions, the infiltration and embedding solutions are prepared following the instructions of the Polysciences embedding kit. Infiltration times depend on specimen size and range between 3–4 h (small zebrafish embryos) and 48 h (4 mm skin biopsy material).

2.1.4. Embedding

Following infiltration, the specimens are placed in embedding moulds, filled with liquid embedding solution, composed of JB4-resin (Polysciences), which contains eosin (0.4 g/100 mL) or alternatively eosin and acridine orange (0.275 g eosin/100 mL and 0.055 g acridine orange/100 mL). The typical size of commercial embedding moulds ranges between 6 × 8 × 5 mm3 and 8 × 10 × 16 mm3.
After setting the block holders on top of the moulds, the moulds with block holders are sealed airproof with paraplast or by covering the surface with mineral oil to support hardening of the resin. Placing the airproof covered moulds in the fridge for approximately two days reduces the likeliness of the occurrence of bubbles or cavities inside the resin.
After hardening is completed, the polymerised blocks are either baked at 70–90 °C for 1 to 2 days for accelerated hardening and darkening of the resin, or simply stored for at least a week at room temperature. Blocks are then transferred to the room in which they are sectioned and stored for a few days before they are mounted on the microtome of the HREM apparatus. Storage for months is possible, although blocks older than one year tend to be brittle and break more easily during sectioning.

2.2. HREM Data Generation

The resin blocks are mounted in an HREM apparatus and sectioned, while series of digital images showing freshly cut block surfaces are captured. Sectioning is performed in an air-conditioned room at 18° to 20° Celsius, as high temperatures and humidity soften the block. The specifications of the components comprising a HREM-setup and the exact workflow during sectioning have been described in detail previously [56,77].

2.2.1. HREM Apparatus

An HREM apparatus essentially comprises a microtome or device for sectioning resin blocks in 1 to 5 µm steps. The sectioning device is equipped with a photo-position, at which the block containing the sample reproducibly stops after each cut. A fluorescence optics is aligned with the photo-position. Its optical axis comprises a GFP or YFP filter cube. If specimens are specifically stained with bluish stains, GFP or YFP and Texas Red filter cubes are subsequently placed in the optical axis. On the phototube of the optics sits a digital camera, which is connected to a PC operating a data generation software that orchestrates cutting, image capturing and image storage. A device for focusing and for adjusting the field of view completes the set up (Figure 1).
Various prototypes of HREM apparatuses are in use [53,78]. But recently a ready to use setup has also been made commercially available by Indigo Scientific Ltd. (Baldock, UK) (OHREM, http://www.indigo-scientific.co.uk/).
In routine projects, HREM apparatuses produce volume data comprised of 3000–4000 inherently aligned section images in about 8 h. The voxel dimensions of the created volume data range between 2 × 2 × 2 µm3 and 5 × 5 × 5 µm3, depending on the desired field of view. Larger and smaller dimensions are possible but require customised set ups and skilled operators.

2.2.2. Data Processing

HREM data are either contrast enhanced by routines and archived, or directly loaded into 3D visualisation software, such as Amira® (versions 5–6, ThermoFisher Scientific, Merignac, France), Osirix® (www.osirix-viewer.com, versions 6–8, Pixmeo SARL, Bernex, Switzerland) or Imaris® (version 9, Bitplane AG, Zürich, Switzerland). If section thickness does not correspond with the pixel size of the digital images, scaling of the single images to match the section thickness by using Photoshop® (version 11, Adobe Systems, San Jose, CA, USA) or IrfanView (irfanview.com, version 4, Irfan Škiljan, Wiener Neustadt, Austria) routines is of advantage for later metric analysis.

2.2.3. Data Visualisation

HREM data are ideal for immediate volume rendering and thus 3D models with exceptional resolution can be generated within seconds. The high contrasts of HREM data enable fully automated segmentation of body surfaces and subsequent surface rendering. 3D surface modelling of embryo organs, such as the central nervous system, lungs, liver pancreas, endocrine glands and others requires semi-automatic segmentation by, e.g., using region growing algorithms and interactive demarcation of the organ borders from adjacent structures. 3D surface modelling of structures such as blood vessels, nerves and ganglia requires manual tracing in most cases.
Rapid visualization of specifically labelled structures (e.g., LacZ or NBT/BCIP stained gene products) is possible by using multi-channel imaging. Two images are captured from each block surface. One with a GFP or YFP and one with a Texas Red filter set. Overall embryo morphology is visualised by using volume rendering in the GFP/YFP image set. Specifically stained structures are visualised as surface models after automated threshold segmentation in the image set captured with the Texas Red filter (Leica-Microsystems, Wetzlar, Germany).

3. Examples

Since its first publication in 2006, HREM has been used to analyse a variety of embryonic and adult samples. The following section provides examples of the successful use of HREM for analysing cardiovascular components.
Use of all animals was in accordance with UK Home Office regulations, the UK Animals (Scientific Procedures) Act of 1986 and approved by the Wellcome Trust Sanger Institute’s Animal Welfare and Ethical Review Body.

3.1. Skin Vasculature

The dermal layer of the skin harbours a highly complex system of blood vessels. They guarantee smooth supply of nutrients, oxygen and endocrine signals to all cells in all skin layers and, at the same time aid thermoregulation of the entire body.
In a few anatomical studies, HREM proved to be highly useful for analysing the topology and architecture of the dermal arteries in various regions of the human skin [76,79,80]. Yet it was also successful in visualising neovascularisation in wound healing models [81,82,83] (Figure 2). In this context we like to emphasise that the dense tissues of adult humans and animals prevent automatic detection of blood vessels, where blood vessel segmentation has to be performed by manual tracing.

3.2. Biomedical Model Organisms

3.2.1. Zebrafish and Frog Embryos

HREM has been successfully used to image the heart of early zebrafish and frog embryos [53]. Due to the small sample size, also whole mount in situ hybridisation of mlc2a products could be used and allowed automated detection of heart cells. The insensitivity of HREM for artefacts caused by yolk and the high-resolution of the produced volume data allowed 3D imaging of the heart tube of 24–28 hpf zebrafish embryos in the context of the entire embryo sitting on the yolk sac. These data are excellently fitted for metric analysis of the cardiac axis of normal and mutated embryos in respect to the axis of the neural tube and other embryonic structures (Figure 3).

3.2.2. Chick Embryos

HREM was successfully applied for visualising the heart and great blood vessels of normally developed and biomechanically challenged early to late chick embryos, from developmental stage 5 to 35 according to the staging system suggested by Hamburger and Hamilton [84] (Figure 3). The data enabled precise descriptions of pharyngeal arch artery remodelling and, in early embryos 3D visualisation of the distribution of specifically labelled Tbx5 and nkx2.5 products in the myocardium. In addition, HREM was successfully applied for visualising cardiovascular morphology of experimentally produced mutants [85,86] and for metric descriptions of the dimensions and angles of the great arteries [60,86].
In early embryos HREM permits visualisation of the cardiovascular system in the context of the whole embryo. Embryos older than HH28 must be parted for HREM imaging and only the thoraces or isolated hearts can be processed.

3.2.3. Mouse Embryos

HREM proved to work excellently with normal and genetically engineered early to late mouse embryos. It was successfully used visualising the single steps during cardiovascular remodelling and for metric analysis of the dimensions of the great intrathoracic arteries and the lengths of vessel segments [60,62,63,87,88,89]. HREM also was applied for visualising structural abnormalities in various knock out lines serving as models for human diseases [68,90,91,92].
An important application of HREM was its use in the scope of the Deciphering the Mechanisms of Developmental Disorders (DMDD) program (https://dmdd.org.uk). HREM data of nearly 700 embryos of 87 knock out lines producing embryonically lethal offspring have been produced and thoroughly analysed in this program. Due to the unmatched spatial resolution and image quality of HREM and the availability of newly created reference data precisely defining normal cardiovascular morphology in the substages of E14.5 [93] vascular variations, large and small heart defects, but also entirely new and potentially life-threatening cardiovascular abnormalities were diagnosed and annotated [72] (Figure 4).

Author Contributions

W.J.W. and S.H.G. created data, assembled figures and wrote the article. B.M.-G. and L.F.R. created and processed data and contributed to preparing figures. F.P. and R.W. contributed to HREM data processing, data mining and creation of figures. A.G., D.J.A. and J.K.W. identified lethal mouse lines and provided and processed their embryos. T.J.M. produced heart models and contributed to finalization of the manuscript.

Funding

Deciphering the mechanims of developmental disorders (DMDD) was supported by the Wellcome Trust [100160], [FC001157], [FC001117]; Cancer Research UK [FC001157], [FC001117]; and the UK Medical Research Council [FC001157], [FC001117].

Acknowledgments

The authors thank Luciano da F. Costa (Sao Carlos Institute of Physics, University of Sao Paulo) for mathematic analysis of zebrafish embryos.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Olson, E.N. A decade of discoveries in cardiac biology. Nat. Med. 2004, 10, 467–474. [Google Scholar] [CrossRef]
  2. Srivastava, D. Making or breaking the heart: from lineage determination to morphogenesis. Cell 2006, 126, 1037–1048. [Google Scholar] [CrossRef] [PubMed]
  3. Pierpont, M.E.; Basson, C.T.; Benson, D.W., Jr.; Gelb, B.D.; Giglia, T.M.; Goldmuntz, E.; McGee, G.; Sable, C.A.; Srivastava, D.; Webb, C.L. Genetic basis for congenital heart defects: Current knowledge: A scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: Endorsed by the American Academy of Pediatrics. Circulation 2007, 115, 3015–3038. [Google Scholar] [CrossRef] [PubMed]
  4. Andelfinger, G. Genetic factors in congenital heart malformation. Clin. Genet. 2008, 73, 516–527. [Google Scholar] [CrossRef] [PubMed]
  5. Dees, E.; Baldwin, H.S. Making a heart: Advances in understanding the mechanisms of cardiac development. Curr. Opin. Pediatr. 2016, 28, 584–589. [Google Scholar] [CrossRef] [PubMed]
  6. Ivanovitch, K.; Esteban, I.; Torres, M. Growth and Morphogenesis during Early Heart Development in Amniotes. J. Cardiovasc. Dev. Dis. 2017, 4. [Google Scholar] [CrossRef] [PubMed]
  7. Zaidi, S.; Brueckner, M. Genetics and Genomics of Congenital Heart Disease. Circ. Res. 2017, 120, 923–940. [Google Scholar] [CrossRef] [Green Version]
  8. Weninger, W.J.; Streicher, J.; Müller, G.B. 3-dimensional reconstruction of histological serial sections using a computer. Wien. Klin. Wochenschr. 1996, 108, 515–520. [Google Scholar]
  9. Streicher, J.; Weninger, W.J.; Müller, G.B. External marker-based automatic congruencing: A new method of 3D reconstruction from serial sections. Anat. Rec. 1997, 248, 583–602. [Google Scholar] [CrossRef] [Green Version]
  10. Streicher, J.; Donat, M.A.; Strauss, B.; Spörle, R.; Schughart, K.; Müller, G.B. Computer-based three-dimensional visualization of developmental gene expression. Nat. Genet. 2000, 25, 147–152. [Google Scholar] [CrossRef]
  11. Metscher, B. MicroCT for comparative morphology: Simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol. 2009, 9, 11–24. [Google Scholar] [CrossRef] [PubMed]
  12. Metscher, B.D. MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions. Dev. Dyn. 2009, 238, 632–640. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Metscher, B.D.; Müller, G.B. MicroCT for molecular imaging: Quantitative visualization of complete three-dimensional distributions of gene products in embryonic limbs. Dev. Dyn. 2011, 240, 2301–2308. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Gignac, P.M.; Kley, N.J.; Clarke, J.A.; Colbert, M.W.; Morhardt, A.C.; Cerio, D.; Cost, I.N.; Cox, P.G.; Daza, J.D.; Early, C.M.; et al. Diffusible iodine-based contrast-enhanced computed tomography (diceCT): An emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues. J. Anat. 2016, 228, 889–909. [Google Scholar] [CrossRef] [PubMed]
  15. Degenhardt, K.; Wright, A.C.; Horng, D.; Padmanabhan, A.; Epstein, J.A. Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ. Cardiovasc. Imaging 2010, 3, 314–322. [Google Scholar] [CrossRef] [PubMed]
  16. Wong, M.D.; Dorr, A.E.; Walls, J.R.; Lerch, J.P.; Henkelman, R.M. A novel 3D mouse embryo atlas based on micro-CT. Development 2012, 139, 3248–3256. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Johnson, J.T.; Hansen, M.S.; Wu, I.; Healy, L.J.; Johnson, C.R.; Jones, G.M.; Capecchi, M.R.; Keller, C. Virtual histology of transgenic mouse embryos for high-throughput phenotyping. PLoS Genet. 2006, 2, e61. [Google Scholar] [CrossRef]
  18. Hsu, C.-W.; Wong, L.; Rasmussen, T.L.; Kalaga, S.; McElwee, M.L.; Keith, L.C.; Bohat, R.; Seavitt, J.R.; Beaudet, A.L.; Dickinson, M.E. Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages. Dev. Biol. 2016, 419, 229–236. [Google Scholar] [CrossRef]
  19. Núñez, J.A.; Goring, A.; Hesse, E.; Thurner, P.J.; Schneider, P.; Clarkin, C.E. Simultaneous visualisation of calcified bone microstructure and intracortical vasculature using synchrotron X-ray phase contrast-enhanced tomography. Sci. Rep. 2017, 7, 13289. [Google Scholar] [CrossRef] [Green Version]
  20. Cleary, J.O.; Modat, M.; Norris, F.C.; Price, A.N.; Jayakody, S.A.; Martinez-Barbera, J.P.; Greene, N.D.E.; Hawkes, D.J.; Ordidge, R.J.; Scambler, P.J.; et al. Magnetic resonance virtual histology for embryos: 3D atlases for automated high-throughput phenotyping. Neuroimage 2011, 54, 769–778. [Google Scholar] [CrossRef]
  21. Zamyadi, M.; Baghdadi, L.; Lerch, J.P.; Bhattacharya, S.; Schneider, J.E.; Henkelman, R.M.; Sled, J.G. Mouse embryonic phenotyping by morphometric analysis of MR images. Physiol. Genet. 2010, 42A, 89–95. [Google Scholar] [CrossRef] [PubMed]
  22. Cleary, J.O.; Price, A.N.; Thomas, D.L.; Scambler, P.J.; Kyriakopoulou, V.; McCue, K.; Schneider, J.E.; Ordidge, R.J.; Lythgoe, M.F. Cardiac phenotyping in ex vivo murine embryos using microMRI. NMR Biomed. 2009, 22, 857–866. [Google Scholar] [CrossRef] [PubMed]
  23. Berrios-Otero, C.A.; Wadghiri, Y.Z.; Nieman, B.J.; Joyner, A.L.; Turnbull, D.H. Three-dimensional micro-MRI analysis of cerebral artery development in mouse embryos. Magn. Reson. Med. 2009, 62, 1431–1439. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Petiet, A.E.; Kaufman, M.H.; Goddeeris, M.M.; Brandenburg, J.; Elmore, S.A.; Johnson, G.A. High-resolution magnetic resonance histology of the embryonic and neonatal mouse: A 4D atlas and morphologic database. Proc. Natl. Acad. Sci. USA 2008, 105, 12331–12336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Schneider, J.E.; Bose, J.; Bamforth, S.D.; Gruber, A.D.; Broadbent, C.; Clarke, K.; Neubauer, S.; Lengeling, A.; Bhattacharya, S. Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique. BMC Dev. Biol. 2004, 4, 16–27. [Google Scholar] [CrossRef]
  26. Schneider, J.E.; Bhattacharya, S. Making the mouse embryo transparent: Identifying developmental malformations using magnetic resonance imaging. Birth Defects Res. 2004, 72, 241–249. [Google Scholar] [CrossRef] [PubMed]
  27. Schneider, J.E.; Bamforth, S.D.; Farthing, C.R.; Clarke, K.; Neubauer, S.; Bhattacharya, S. Rapid identification and 3D reconstruction of complex cardiac malformations in transgenic mouse embryos using fast gradient echo sequence magnetic resonance imaging. J. Mol. Cell. Cardiol. 2003, 35, 217–222. [Google Scholar] [CrossRef]
  28. Smith, B.R. Magnetic resonance microscopy in cardiac development. Microsc. Res. Tech. 2001, 52, 323–330. [Google Scholar] [CrossRef] [Green Version]
  29. Dhenain, M.; Ruffins, S.W.; Jacobs, R.E. Three-dimensional digital mouse atlas using high-resolution MRI. Dev. Biol. 2001, 232, 458–470. [Google Scholar] [CrossRef]
  30. Jacobs, R.E.; Ahrens, E.T.; Dickinson, M.E.; Laidlaw, D. Towards a microMRI atlas of mouse development. Comput. Med. Imaging Graph. 1999, 23, 15–24. [Google Scholar] [CrossRef] [Green Version]
  31. Sharpe, J.; Ahlgren, U.; Perry, P.; Hill, B.; Ross, A.; Hecksher-Sorensen, J.; Baldock, R.; Davidson, D. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 2002, 296, 541–545. [Google Scholar] [CrossRef] [PubMed]
  32. Sharpe, J. Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 2003, 202, 175–181. [Google Scholar] [CrossRef] [Green Version]
  33. Lickert, H.; Takeuchi, J.K.; Von Both, I.; Walls, J.R.; McAuliffe, F.; Adamson, S.L.; Henkelman, R.M.; Wrana, J.L.; Rossant, J.; Bruneau, B.G. Baf60c is essential for function of BAF chromatin remodelling complexes in heart development. Nature 2004, 432, 107–112. [Google Scholar] [CrossRef]
  34. Colas, J.F.; Sharpe, J. Live optical projection tomography. Organogenesis 2009, 5, 211–216. [Google Scholar] [CrossRef] [Green Version]
  35. Breckenridge, R.; Kotecha, S.; Towers, N.; Bennett, M.; Mohun, T. Pan-myocardial expression of Cre recombinase throughout mouse development. Genesis 2007, 45, 135–144. [Google Scholar] [CrossRef] [PubMed]
  36. Jeansson, M.; Gawlik, A.; Anderson, G.; Li, C.; Kerjaschki, D.; Henkelman, M.; Quaggin, S.E. Angiopoietin-1 is essential in mouse vasculature during development and in response to injury. J. Clin. Investig. 2011, 121, 2278–2289. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Anderson, G.A.; Wong, M.D.; Yang, J.; Henkelman, R.M. 3D imaging, registration, and analysis of the early mouse embryonic vasculature. Dev. Dyn. 2013, 242, 527–538. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Wang, S.; Garcia, M.D.; Lopez, A.L.; Overbeek, P.A.; Larin, K.V.; Larina, I.V. Dynamic imaging and quantitative analysis of cranial neural tube closure in the mouse embryo using optical coherence tomography. Biomed. Opt. Express 2017, 8, 407–419. [Google Scholar] [CrossRef]
  39. Singh, M.; Raghunathan, R.; Piazza, V.; Davis-Loiacono, A.M.; Cable, A.; Vedakkan, T.J.; Janecek, T.; Frazier, M.V.; Nair, A.; Wu, C.; et al. Applicability, usability, and limitations of murine embryonic imaging with optical coherence tomography and optical projection tomography. Biomed. Opt. Express 2016, 7, 2295–2310. [Google Scholar] [CrossRef]
  40. Liu, M.; Maurer, B.; Hermann, B.; Zabihian, B.; Sandrian, M.G.; Unterhuber, A.; Baumann, B.; Zhang, E.Z.; Beard, P.C.; Weninger, W.J.; et al. Dual modality optical coherence and whole-body photoacoustic tomography imaging of chick embryos in multiple development stages. Biomed. Opt. Express 2014, 5, 3150–3159. [Google Scholar] [CrossRef]
  41. Raghunathan, R.; Singh, M.; Dickinson, M.E.; Larin, K.V. Optical coherence tomography for embryonic imaging: a review. J. Biomed. Opt. 2016, 21, 50902. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Chen, Q.; Jin, T.; Qi, W.; Mo, X.; Xi, L. Label-free photoacoustic imaging of the cardio-cerebrovascular development in the embryonic zebrafish. Biomed. Opt. Express 2017, 8, 2359–2367. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Ewald, A.J.; McBride, H.; Reddington, M.; Fraser, S.E.; Kerschmann, R. Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution. Dev. Dyn. 2002, 225, 369–375. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Sivaguru, M.; Fried, G.A.; Miller, C.A.; Fouke, B.W. Multimodal optical microscopy methods reveal polyp tissue morphology and structure in Caribbean reef building corals. J. Vis. Exp. 2014, e51824. [Google Scholar] [CrossRef] [PubMed]
  45. Srinivasan, S.; Baldwin, H.S.; Aristizabal, O.; Kwee, L.; Labow, M.; Artman, M.; Turnbull, D.H. Noninvasive, in utero imaging of mouse embryonic heart development with 40-MHz echocardiography. Circulation 1998, 98, 912–918. [Google Scholar] [CrossRef]
  46. Phoon, C.K.; Ji, R.P.; Aristizabal, O.; Worrad, D.M.; Zhou, B.; Baldwin, H.S.; Turnbull, D.H. Embryonic heart failure in NFATc1-/- mice: Novel mechanistic insights from in utero ultrasound biomicroscopy. Circ. Res. 2004, 95, 92–99. [Google Scholar] [CrossRef] [PubMed]
  47. Kulandavelu, S.; Qu, D.; Sunn, N.; Mu, J.; Rennie, M.Y.; Whiteley, K.J.; Walls, J.R.; Bock, N.A.; Sun, J.C.; Covelli, A.; et al. Embryonic and neonatal phenotyping of genetically engineered mice. Ilar. J. 2006, 47, 103–117. [Google Scholar] [CrossRef]
  48. Phoon, C.K. Imaging tools for the developmental biologist: Ultrasound biomicroscopy of mouse embryonic development. Pediatr. Res. 2006, 60, 14–21. [Google Scholar] [CrossRef]
  49. Golden, H.B.; Sunder, S.; Liu, Y.; Peng, X.; Dostal, D.E. In utero assessment of cardiovascular function in the embryonic mouse heart using high-resolution ultrasound biomicroscopy. In Cardiovascular Development; Humana Press: Totowa, NJ, USA, 2012; pp. 245–263. [Google Scholar]
  50. Weninger, W.J.; Meng, S.; Streicher, J.; Müller, G.B. A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology. Anat. Embryol. 1998, 197, 341–348. [Google Scholar] [CrossRef]
  51. Weninger, W.J.; Mohun, T. Phenotyping transgenic embryos: A rapid 3-D screening method based on episcopic fluorescence image capturing. Nat. Genet. 2002, 30, 59–65. [Google Scholar] [CrossRef]
  52. Weninger, W.J.; Floro, K.L.; Bennett, M.B.; Withington, S.L.; Preis, J.I.; Barbera, J.P.; Mohun, T.J.; Dunwoodie, S.L. Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development. Development 2005, 132, 1337–1348. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Weninger, W.J.; Geyer, S.H.; Mohun, T.J.; Rasskin-Gutman, D.; Matsui, T.; Ribeiro, I.; Costa Lda, F.; Izpisua-Belmonte, J.C.; Müller, G.B. High-resolution episcopic microscopy: A rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology. Anat. Embryol. 2006, 211, 213–221. [Google Scholar] [CrossRef] [PubMed]
  54. Geyer, S.H.; Mohun, T.J.; Weninger, W.J. Visualizing vertebrate embryos with episcopic 3D imaging techniques. Sci. World J. 2009, 9, 1423–1437. [Google Scholar] [CrossRef] [PubMed]
  55. Mohun, T.J.; Weninger, W.J. Imaging heart development using high-resolution episcopic microscopy. Curr. Opin. Genet. Dev. 2011, 21, 573–578. [Google Scholar] [CrossRef] [PubMed]
  56. Geyer, S.H.; Maurer-Gesek, B.; Reissig, L.F.; Weninger, W.J. High-resolution Episcopic Microscopy (HREM)—Simple and Robust Protocols for Processing and Visualizing Organic Materials. J. Vis. Exp. 2017. [Google Scholar] [CrossRef] [PubMed]
  57. Denk, W.; Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2004, 2, e329. [Google Scholar] [CrossRef] [PubMed]
  58. Kalson, N.S.; Holmes, D.F.; Herchenhan, A.; Lu, Y.; Starborg, T.; Kadler, K.E. Slow stretching that mimics embryonic growth rate stimulates structural and mechanical development of tendon-like tissue in vitro. Dev. Dyn. 2011, 240, 2520–2528. [Google Scholar] [CrossRef]
  59. Smith, D.; Starborg, T. Serial block face scanning electron microscopy in cell biology: Applications and technology. Tissue cell 2018. [Google Scholar] [CrossRef]
  60. Weninger, W.J.; Maurer, B.; Zendron, B.; Dorfmeister, K.; Geyer, S.H. Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos. J. Microsc. 2009, 234, 173–190. [Google Scholar] [CrossRef]
  61. Geyer, S.H.; Maurer, B.; Pötz, L.; Singh, J.; Weninger, W.J. High-Resolution Episcopic Microscopy Data-Based Measurements of the Arteries of Mouse Embryos: Evaluation of Significance and Reproducibility under Routine Conditions. Cells Tissues Organs 2012, 195, 524–534. [Google Scholar] [CrossRef]
  62. Geyer, S.H.; Weninger, W.J. Some Mice Feature 5th Pharyngeal Arch Arteries and Double-Lumen Aortic Arch Malformations. Cells Tissues Organs 2012, 196, 90–98. [Google Scholar] [CrossRef] [PubMed]
  63. Geyer, S.H.; Weninger, W.J. Metric characterization of the aortic arch of early mouse fetuses and of a fetus featuring a double lumen aortic arch malformation. Ann. Anat. 2013, 195, 175–182. [Google Scholar] [CrossRef] [PubMed]
  64. Bruneel, B.; Matha, M.; Paesen, R.; Ameloot, M.; Weninger, W.J.; Huysseune, A. Imaging the zebrafish dentition: from traditional approaches to emerging technologies. Zebrafish 2015, 12, 1–10. [Google Scholar] [CrossRef] [PubMed]
  65. Pokhrel, N.; Cohen, E.B.; Genin, O.; Ruzal, M.; Sela-Donenfeld, D.; Cinnamon, Y. Effects of storage conditions on hatchability, embryonic survival and cytoarchitectural properties in broiler from young and old flocks. Poult. Sci. 2018, 97, 1429–1440. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Perez-Garcia, V.; Fineberg, E.; Wilson, R.; Murray, A.; Mazzeo, C.I.; Tudor, C.; Sienerth, A.; White, J.K.; Tuck, E.; Ryder, E.J.; et al. Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 2018, 555, 463–468. [Google Scholar] [CrossRef] [PubMed]
  67. Aiello, V.D.; Spicer, D.E.; Anderson, R.H.; Brown, N.A.; Mohun, T.J. The independence of the infundibular building blocks in the setting of double-outlet right ventricle. Cardiol. Young 2017, 27, 825–836. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Lana-Elola, E.; Watson-Scales, S.; Slender, A.; Gibbins, D.; Martineau, A.; Douglas, C.; Mohun, T.; Fisher, E.M.; Tybulewicz, V. Genetic dissection of Down syndrome-associated congenital heart defects using a new mouse mapping panel. Elife 2016, 5. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Spicer, D.E.; Henderson, D.J.; Chaudhry, B.; Mohun, T.J.; Anderson, R.H. The anatomy and development of normal and abnormal coronary arteries. Cardiol. Young 2015, 25, 1493–1503. [Google Scholar] [CrossRef] [PubMed]
  70. Anderson, R.H.; Mohun, T.J.; Brown, N.A. Clarifying the morphology of the ostium primum defect. J. Anat. 2015, 226, 244–257. [Google Scholar] [CrossRef] [Green Version]
  71. Mohun, T.; Adams, D.J.; Baldock, R.; Bhattacharya, S.; Copp, A.J.; Hemberger, M.; Houart, C.; Hurles, M.E.; Robertson, E.; Smith, J.C.; et al. Deciphering the Mechanisms of Developmental Disorders (DMDD): A new programme for phenotyping embryonic lethal mice. Dis Model. Mech. 2013, 6, 562–566. [Google Scholar] [CrossRef]
  72. Weninger, W.J.; Geyer, S.H.; Martineau, A.; Galli, A.; Adams, D.J.; Wilson, R.; Mohun, T.J. Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy. Dis Model. Mech. 2014, 7, 1143–1152. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Wilson, R.; McGuire, C.; Mohun, T.; Adams, D.; Baldock, R.; Bhattacharya, S.; Collins, J.; Fineberg, E.; Firminger, L.; Galli, A.; et al. Deciphering the mechanisms of developmental disorders: Phenotype analysis of embryos from mutant mouse lines. Nucleic Acids Research 2016, 44, D855–D861. [Google Scholar] [CrossRef] [PubMed]
  74. Wilson, R.; Geyer, S.H.; Reissig, L.; Rose, J.; Szumska, D.; Hardman, E.; Prin, F.; McGuire, C.; Ramirez-Solis, R.; White, J.; et al. Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice. Wellcome. Open Res. 2016, 1, 1. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Mohun, T.J.; Weninger, W.J. Embedding Embryos for High-Resolution Episcopic Microscopy (HREM). Cold Spring Harb. Protoc. 2012, 2012, 678–680. [Google Scholar] [CrossRef] [PubMed]
  76. Geyer, S.H.; Nöhammer, M.M.; Mathä, M.; Reissig, L.; Tinhofer, I.E.; Weninger, W.J. High-Resolution Episcopic Microscopy (HREM): A Tool for Visualizing Skin Biopsies. Microsc. Microanal. 2014, 20, 1356–1364. [Google Scholar] [CrossRef] [PubMed]
  77. Geyer, S.H.; Mohun, T.J.; Kamolz, L.P.; Weninger, W.J. High resolution episcopic microscopy - current applications. Curr. Biotech. 2012, 1, 281–286. [Google Scholar] [CrossRef]
  78. Mohun, T.J.; Weninger, W.J. Generation of volume data by episcopic three-dimensional imaging of embryos. Cold Spring Harb. Protoc. 2012, 2012, 681–682. [Google Scholar] [CrossRef] [PubMed]
  79. Geyer, S.H.; Nohammer, M.M.; Tinhofer, I.E.; Weninger, W.J. The dermal arteries of the human thumb pad. J. Anat. 2013, 223, 603–609. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Tinhofer, I.E.; Zaussinger, M.; Geyer, S.H.; Meng, S.; Kamolz, L.P.; Tzou, C.H.; Weninger, W.J. The dermal arteries in the cutaneous angiosome of the descending genicular artery. J. Anat. 2018. [Google Scholar] [CrossRef]
  81. Geyer, S.H.; Tinhofer, I.E.; Lumenta, D.B.; Kamolz, L.P.; Branski, L.; Finnerty, C.C.; Herndon, D.N.; Weninger, W.J. High-resolution episcopic microscopy (HREM): A useful technique for research in wound care. Ann. Anat. 2015, 197, 3–10. [Google Scholar] [CrossRef]
  82. Ertl, J.; Pichlsberger, M.; Tuca, A.-C.; Wurzer, P.; Fuchs, J.; Geyer, S.H.; Maurer-Gesek, B.; Weninger, W.J.; Pfeiffer, D.; Bubalo, V.; et al. Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds. Placenta 2018, 65, 37–46. [Google Scholar] [CrossRef]
  83. Wiedner, M.; Tinhofer, I.E.; Kamolz, L.P.; Seyedian Moghaddam, A.; Justich, I.; Liegl-Atzwanger, B.; Bubalo, V.; Weninger, W.J.; Lumenta, D.B. Simultaneous dermal matrix and autologous split-thickness skin graft transplantation in a porcine wound model: A three-dimensional histological analysis of revascularization. Wound Repair Regen. 2014, 22, 749–754. [Google Scholar] [CrossRef] [PubMed]
  84. Hamburger, V.; Hamilton, H.L. A series of normal stages in the development of the chick embryo. J. Morph. 1951, 88, 49–92. [Google Scholar] [CrossRef] [PubMed]
  85. Maurer, B.; Geyer, S.H.; Weninger, W.J. A Chick Embryo with a yet Unclassified Type of Cephalothoracopagus Malformation and a Hypothesis for Explaining its Genesis. Anat. Histol. Embryol. 2013, 42, 191–200. [Google Scholar] [CrossRef] [PubMed]
  86. Maurer-Gesek, B. Malformations of the Great Intrathoracic Arteries caused by Hemodynamic Alterations in Chick Embryos. Ph.D. Thesis, Medical University of Vienna, Vienna, Austria, 2016. [Google Scholar]
  87. Bamforth, S.D.; Chaudhry, B.; Bennett, M.; Wilson, R.; Mohun, T.J.; Van Mierop, L.H.; Henderson, D.J.; Anderson, R.H. Clarification of the identity of the mammalian fifth pharyngeal arch artery. Clin. Anat. 2013, 26, 173–182. [Google Scholar] [CrossRef] [PubMed]
  88. Anderson, R.H.; Brown, N.A.; Mohun, T.J. Insights regarding the normal and abnormal formation of the atrial and ventricular septal structures. Clin. Anat. 2016, 29, 290–304. [Google Scholar] [CrossRef] [PubMed]
  89. Paun, B.; Bijnens, B.; Cook, A.C.; Mohun, T.J.; Butakoff, C. Quantification of the detailed cardiac left ventricular trabecular morphogenesis in the mouse embryo. Med. Image Anal. 2018, 49, 89–104. [Google Scholar] [CrossRef]
  90. Le Garrec, J.F.; Dominguez, J.N.; Desgrange, A.; Ivanovitch, K.D.; Raphael, E.; Bangham, J.A.; Torres, M.; Coen, E.; Mohun, T.J.; Meilhac, S.M. A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics. Elife 2017, 6. [Google Scholar] [CrossRef] [Green Version]
  91. Dunlevy, L.; Bennett, M.; Slender, A.; Lana-Elola, E.; Tybulewicz, V.L.; Fisher, E.M.; Mohun, T. Down’s syndrome-like cardiac developmental defects in embryos of the transchromosomic Tc1 mouse. Cardiovasc. Res. 2010, 88, 287–295. [Google Scholar] [CrossRef]
  92. Captur, G.; Wilson, R.; Bennett, M.F.; Luxan, G.; Nasis, A.; de la Pompa, J.L.; Moon, J.C.; Mohun, T.J. Morphogenesis of myocardial trabeculae in the mouse embryo. J. Anat. 2016, 229, 314–325. [Google Scholar] [CrossRef] [Green Version]
  93. Geyer, S.H.; Reissig, L.F.; Husemann, M.; Hofle, C.; Wilson, R.; Prin, F.; Szumska, D.; Galli, A.; Adams, D.J.; White, J.; et al. Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21–S23. J. Anat. 2017, 231, 600–614. [Google Scholar] [CrossRef] [PubMed]
Jcdd 05 00058 g001 550
Figure 1. HREM apparatus.
Figure 1. HREM apparatus.
Jcdd 05 00058 g001
Jcdd 05 00058 g002 550
Figure 2. Thick skin of human thumb pad. Virtual sections through HREM data (A) together with surface models of dermal arteries (B), dermal veins (C) and both (D). Scalebars: 500 µm.
Figure 2. Thick skin of human thumb pad. Virtual sections through HREM data (A) together with surface models of dermal arteries (B), dermal veins (C) and both (D). Scalebars: 500 µm.
Jcdd 05 00058 g002
Jcdd 05 00058 g003 550
Figure 3. HREM for analysing the cardiovascular system of biomedical models. (A) Sagittal section through the head of a zebrafish embryo, 48 h after conception. (B) Surface models of the cranial parts of the central nerve system (cns), eye (e) and heart (h) with mathematically calculated and displayed planes through their main axes. (C,D) Surface models of the heart and arteries (red) of a chick embryo of stage 18 according to Hamburger Hamilton (HH) together with virtual resections through HREM data (C) and in the context of a semitransparent volume model of the embryo surface (D) respectively. (E,F) HH 34 chick embryo with cephalothoracopagus malformation. Surface models show a single heart and pharyngeal arch artery system (red) feeding the descending aorta of each twin (E). Additional surface models of the notochords (nc, green) in an opaque volume model of the surface with virtually excluded cranial parts from anterior (F). a, atrium; da, dorsal aorta; e, eye; in, intestine; li, liver; ll, lower limb; me, mesencephalon; nc, notochord; o, outflow tract; ov, otic vesicle; paa, pharyngeal arch arteries; rh, rhombencephalon; so, somite; t, telencephalon; ul, upper limb; v, ventricle. Scalebars: 100 µm (A), 500 µm (C–F).
Figure 3. HREM for analysing the cardiovascular system of biomedical models. (A) Sagittal section through the head of a zebrafish embryo, 48 h after conception. (B) Surface models of the cranial parts of the central nerve system (cns), eye (e) and heart (h) with mathematically calculated and displayed planes through their main axes. (C,D) Surface models of the heart and arteries (red) of a chick embryo of stage 18 according to Hamburger Hamilton (HH) together with virtual resections through HREM data (C) and in the context of a semitransparent volume model of the embryo surface (D) respectively. (E,F) HH 34 chick embryo with cephalothoracopagus malformation. Surface models show a single heart and pharyngeal arch artery system (red) feeding the descending aorta of each twin (E). Additional surface models of the notochords (nc, green) in an opaque volume model of the surface with virtually excluded cranial parts from anterior (F). a, atrium; da, dorsal aorta; e, eye; in, intestine; li, liver; ll, lower limb; me, mesencephalon; nc, notochord; o, outflow tract; ov, otic vesicle; paa, pharyngeal arch arteries; rh, rhombencephalon; so, somite; t, telencephalon; ul, upper limb; v, ventricle. Scalebars: 100 µm (A), 500 µm (C–F).
Jcdd 05 00058 g003
Jcdd 05 00058 g004 550
Figure 4. Cardiovascular features in HREM data of mouse embryos harvested at E14.5. (A,B) Surface rendered models of the arteries (red) together with a semi-transparent volume model from frontal (A) and lateral (B). (C) Surface rendered models of the head arteries (red) together with a sagittally sectioned volume model of the upper body half of an embryo. (D) Surface rendered models of the great intrathoracal arteries in an axially sectioned volume model. For details regarding artery topology see [87]. (E–J) Axial HREM sections, showing examples of cardiovascular malformations diagnosed in mutants produced in the DMDD program (E–G) and comparable sections through controls (H–J). Provided examples show persistent truncus arteriosus (pta) in a Psph mutant (E), additional opening of the coronary sinus (cs) into the right atrium (arrowhead) in a Cog6 mutant (F), and a complex malformation including common atrium (ca), double inlet left ventricle (div) and right sided descending aorta (da) in a Cfap53 mutant (g). (K–M) Volume rendered models of isolated hearts displaying heart malformations of a Rpgrip mutant. Perimembranous ventricular septal defect (pVSD) (K), muscular ventricular septal defect (mVSD) (L), double outlet right ventricle (DORV) (M). aa, ascending aorta; db, ductus arteriosus; e, eye; es, espophagus; h, heart; hb, hyoid bone; la, left atrial appendix; ll, lower limb; li, liver; lv, left ventricle; lvc, left vena cava superior; me, mesencephalon; nc, nasal cavity; ob, olfactory bulb; p, pinna; ra, right atrial appendix; rh, rhombencephalon; rv, right ventricle; rvc, right vena cava superior; sc, spinal chord; sn, snout; s1, septum primum; s2, septum secundum; t, telencephalon; th, thymus; to, tongue; tr, trachea; ul, upper limb; vs, ventricular septum. Scalebars 1 mm (A–D), 500 µm (E–M).
Figure 4. Cardiovascular features in HREM data of mouse embryos harvested at E14.5. (A,B) Surface rendered models of the arteries (red) together with a semi-transparent volume model from frontal (A) and lateral (B). (C) Surface rendered models of the head arteries (red) together with a sagittally sectioned volume model of the upper body half of an embryo. (D) Surface rendered models of the great intrathoracal arteries in an axially sectioned volume model. For details regarding artery topology see [87]. (E–J) Axial HREM sections, showing examples of cardiovascular malformations diagnosed in mutants produced in the DMDD program (E–G) and comparable sections through controls (H–J). Provided examples show persistent truncus arteriosus (pta) in a Psph mutant (E), additional opening of the coronary sinus (cs) into the right atrium (arrowhead) in a Cog6 mutant (F), and a complex malformation including common atrium (ca), double inlet left ventricle (div) and right sided descending aorta (da) in a Cfap53 mutant (g). (K–M) Volume rendered models of isolated hearts displaying heart malformations of a Rpgrip mutant. Perimembranous ventricular septal defect (pVSD) (K), muscular ventricular septal defect (mVSD) (L), double outlet right ventricle (DORV) (M). aa, ascending aorta; db, ductus arteriosus; e, eye; es, espophagus; h, heart; hb, hyoid bone; la, left atrial appendix; ll, lower limb; li, liver; lv, left ventricle; lvc, left vena cava superior; me, mesencephalon; nc, nasal cavity; ob, olfactory bulb; p, pinna; ra, right atrial appendix; rh, rhombencephalon; rv, right ventricle; rvc, right vena cava superior; sc, spinal chord; sn, snout; s1, septum primum; s2, septum secundum; t, telencephalon; th, thymus; to, tongue; tr, trachea; ul, upper limb; vs, ventricular septum. Scalebars 1 mm (A–D), 500 µm (E–M).
Jcdd 05 00058 g004

Share and Cite

MDPI and ACS Style

Weninger, W.J.; Maurer-Gesek, B.; Reissig, L.F.; Prin, F.; Wilson, R.; Galli, A.; Adams, D.J.; White, J.K.; Mohun, T.J.; Geyer, S.H. Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM). J. Cardiovasc. Dev. Dis. 2018, 5, 58. https://doi.org/10.3390/jcdd5040058

AMA Style

Weninger WJ, Maurer-Gesek B, Reissig LF, Prin F, Wilson R, Galli A, Adams DJ, White JK, Mohun TJ, Geyer SH. Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM). Journal of Cardiovascular Development and Disease. 2018; 5(4):58. https://doi.org/10.3390/jcdd5040058

Chicago/Turabian Style

Weninger, Wolfgang J., Barbara Maurer-Gesek, Lukas F. Reissig, Fabrice Prin, Robert Wilson, Antonella Galli, David J. Adams, Jacqueline K. White, Timothy J. Mohun, and Stefan H. Geyer. 2018. "Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM)" Journal of Cardiovascular Development and Disease 5, no. 4: 58. https://doi.org/10.3390/jcdd5040058

APA Style

Weninger, W. J., Maurer-Gesek, B., Reissig, L. F., Prin, F., Wilson, R., Galli, A., Adams, D. J., White, J. K., Mohun, T. J., & Geyer, S. H. (2018). Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM). Journal of Cardiovascular Development and Disease, 5(4), 58. https://doi.org/10.3390/jcdd5040058

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop