3D visualization and analysis of cell-matrix ... · 3D visualization and analysis of cell-matrix transformations in whole-mount and live embryos using confocal and multi-photon microscopy

3D visualization and analysis of cell-matrix transformations in whole-mount and live embryos using confocal and multi-photon microscopy

Martins, G.G.1,2, Rifes, P. 1,2, Amândio1,2, R., Campinho1§∗, P., Palmeirim, I3., and Thorsteinsdóttir, S.1,2 1Departamento de Biologia Animal/Centro de Biologia Ambiental. Faculdade de Ciências, Universidade

de Lisboa, 1749-016 Lisboa, Portugal 2Instituto Gulbenkian de Ciência. R. da Quinta Grande, 6, 2780-156 Oeiras, Portugal 3 Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho, 4710-057 Braga, Portugal §Present address: Max Planck Institute of Molecular Cell Biology and Genetics Dresden, Germany

We study the cellular transformations that occur during embryonic development of the tissue that forms skeletal muscles and axial bones in vertebrates. Traditionally whole embryos (both normal and mutants) are studied either through low-magnification images, or, when more detailed images of inner tissues are required, by histological sectioning. However, sectioning distorts tissues, leads to a loss of the 3D context and seriously compromises the possibility of live-cell imaging. To address these problems and facilitate the interpretation of the complex 3D tissue architecture inside whole embryos, we use confocal and multi-photon microscopy and digital image reconstruction techniques to obtain highly detailed images of fixed and stained whole-mount embryos, as well as 3D time-lapse (4D) images of live embryos. Here we describe how we visualize and analyse the complex 3D movements and transformations of cells and their extracellular matrix during embryonic tissue reorganizations. This type of analysis provides rigorous information for studying embryonic malformations due to altered cell behavior induced by genetic alterations or chemical treatments.

Keywords 3D visualization; confocal, multi-photon; embryos

1. Introduction

During early embryonic development, the tissue that later originates the body skeletal musculature and axial bones in vertebrates undergoes dramatic reorganizations; the presomitic mesoderm (PSM; Fig 1) becomes segmented into repetitive structures – the somites (Fig 1), which underlie the formation of vertebrae, body skeletal muscles and impose the segmented organisation of the peripheral nervous system [1,2,3]. Somitogenesis involves a complex morphological transition in that a group of undifferentiated, mesenchymal PSM cells aggregate and ultimately form a sphere of polarised epithelial cells with a few mesenchymal cells in the lumen. Though much has been learned about the genetic/cell-signalling control of segmentation [1,2,3], we still know little about the actual cell and extracellular matrix transformations that shape the somites. Somite segmentation has traditionally been studied at low magnification to recognize the anterior-posterior progression of events (e.g. patterns of expression of cyclic genes, polarization of somites in the antero-posterior direction, determination of the time of formation of inter-somitic clefts [4]. More detailed images of inner tissues are obtained by histological sectioning, but the three-dimensional (3D) nature of the tissues is lost and, even if sections can in some circumstances be kept alive in culture [5], only cell and tissue rearragements that normally occur within

∗ Email of author for correspondence: [email protected]

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the plane of the section can be studied. Normally early embryonic tissues rapidly develop a complex 3D architecture with both structural and functional asymmetries along all the embryonic axii. Confocal and multi-photon microscopy [6,7] facilitate the observation of these phenomenae in deep embryonic tissues by producing series of optical sections, which can then be digitally reconstructed to allow examination of tissue and cellular organization in 3D. Using these methods, we have obtained unprecedented high resolution 3D images of chick embryo PSM cells undergoing mesenchyme to epithelium transition. This allows e.g. the study of alterations in cell shape and behaviour, as well as in the levels of expression and the spatial distribution of gene products (mRNAs and proteins) that are thought to play important roles in segmentation. This methodology has important applications for the study of the effects of chemicals or genetic abnormalities during embryonic development, and produces images that more precisely illustrate the complex phenomenology of both normal and abnormal development.

2. Materials and Methods

Fertilized chicken eggs are incubated until the developmental stages of interest, and are then either i) excised, fixed and processed for in toto detection of specific proteins using immunocytochemistry [Rifes et al., submitted]. .These processed embryos are cleared to improve imaging in depth and image acquisition is done using either a Leica Spectra TCS SP2 or a Zeiss META confocal systems. Note that the same imaging techniques can be used on embryos processed for mRNA in situ hybridsation with fluorescently labelled probes. ii) In other experiments, the embryos are kept alive and stained with vital dyes in ovo, or undergo microelectroporation to produce GFP expressing cells [8]). Stained live embryos are excised from the egg and placed in a custom made culture chamber, where they are kept alive under physiological conditions while acquiring time-lapse 3D stacks using a either a BioRad 1024ES or Zeiss LSM 510 Meta multi-photon systems. In some experiments embryo halves are excised and then cultured separately to test the effects of chemicals on PSM segmentation (for example, drugs that affect extracellular matrix synthesis and/or organization). Confocal stacks of separate explants are then reassembled in 3D and placed side-by-side for direct comparison of effects. The Z-series obtained are first inspected and pre-processed using the ImageJ software (freely available from http://rsb.info.nih.gov/ij/ [9]) and then imported into TGS Amira software (Mercury Computer Systems, Inc.) for further analysis. Images from different contrasting techniques (or different fluorescence wavelengths) are assigned separate colours and merged into a single multi-coloured image (multi-modal). These images allow us to study the spatial relationships between different cell/tissue components, and perform colocalization analysis. Densitometry measurements in situ (profiling of protein content) are performed using linear or point “probes” placed through embryonic tissues in 3D, which measure the average grey levels in the vicinity of the probe (given a defined radius). This reveals the local variations of protein/mRNA content along the tissues. Cell/tissue shapes are studied after manual segmentation followed by iso-surface reconstruction. Some of the images presented are in the form of 3D stereo-pairs, which should be viewed using a stereoscope, or by using the parallel-eye method [10].

3. Results and Discussion

3.1 Multi-modal Z-series

We typically label the tissues with 2-3 different markers to obtain images of several cellular and extracellular matrix (the tissue material that surrounds cells) components. We use for example antibodies to N-cadherin (Ncad) which is a protein involved in cell-cell adhesion and plays an important role in somitogenesis [11]. Ncad is diffusely distributed in all cells of the PSM, but within somites it typically becomes localized in the apical part of the cell (the side towards the inside of the somite, Fig 1). Fibronectin (Fn) is an important component of the extracellular matrix [12]. This protein is also present in and around the PSM, and accumulates in the spaces between somites (i.e., in the inter-somitic clefts).

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D

A

C

E

B

Fig 1. Example of single-plane confocal images (from a stack of 90 µm optical slices spanning the full thickness of the PSM) in the anterior portion of a chick embryo PSM+two somites (rostral is to the right, and caudal to the left of the panels). A) Whole embryo at 33h, corresponding to the HH11 developmental stage (full length ~3 mm), showing the portion imaged by confocal microscopy and represented as slices in B-E). Three different images are presented showing N-cadherin (B), fibronectin (C) and Nomarski’s “differential interference contrast” (D). In E) all three images are combined into a multi-coloured image: N-cad (red), fibronectin (green) and DIC (grey). White bar = 100 µm.

Fig 2. Stereo-paired image of orthogonal slices reconstructed from the original confocal images show in Fig 1, with three planes crossing the interior of the tissues (coronal, sagittal and transversal planes). Red represents N-cadherin, green is fibronectin and grey is Nomarski’s DIC.

3.2 Mosaics of 3D images and tissue explants

Detailed 3D images of large tissues (which expand beyond the field of view of high magnification objectives) can be visualized and analyzed after reassembly into 3D mosaics of multiple high-resolution 3D stacks. 3D visualization software packages, such as Amira/ResolveRT (Mercury, Inc) allow interactive reassembly of the parts, or even automatic reassembly when the 3D stacks contain overlapping portions. This visualization procedure allows direct side-by-side comparison of two embryonic halves cultured and imaged differently to test (on one half) the effect of a chemical treatment. The natural variability among different embryos does not allow direct comparisons between different individuals, so treatments must be done using symmetrical tissues of the same individual, for comparison purposes. We have used this procedure to study the effects of manipulating extracellular matrix synthesis on somite formation. To better understand the effects seen we further characterize the distribution of important proteins (both cellular and extracellular) along the PSM (as shown in Fig 4), as well as important changes in cell and tissue shape as seen in surface reconstructions (see Fig 5).


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A A

B B

Fig 3. Stereo-paired images of reassembled portions of embryos. In A) two halves that had been separated and cultured individually, were imaged separately using the confocal system, and their 3D reconstructed volumes were then reassembled to compare side-by-side the effects of the chemical treatment applied to the left PSM (which made less somites than expected): notice the coloured surface reconstructions of somites and PSM shown inside the fibrous extraellular matrix (volume rendered in green). In B) the anterior portion of the PSM and somites I-III where imaged with high resolution in two separate 3D volumes, which were precisely reassembled in 3D to get a full high-resolution volume spanning both portions of the mesoderm. Using 3D image visualization software it is possible to reassemble 3D parts either interactively or even automatically.

3.3 Densitometry measurements in situ

To characterize quantitatively the distribution of important proteins (e.g, cytoskeletal or extracellular), we apply a digital spline-probe tool (available in the Amira/ResolveRT [Mercury, Inc.], drawn within the tissue in 3D to measure profiles of detected fluorescence intensity. These profiles give accurate quantifiable measurements of local variations of proteins of interest along the PSM and in somites. The same procedure can be done for embryos processed for in-situ hybridisation for detection of mRNA transcripts (thus revealing sites of specific gene expression). For example, in Fig 4B, it can be seen that F-actin (a cytoskeletal protein involved in cell shape changes and stabilization of cell-cell and cell-matrix adhesions) peaks at discrete points along the PSM (Fig 4B, asterisks), revealing that cells at these sites are actively contracting and making strong adhesions with neighbouring cells and the surrounding extracellular matrix in the forming somites, located at the anterior end of the PSM. It is also possible to obtain measurements of total protein, or mRNA load in a specific cell or tissue. After digitally segmenting and 3D reconstructing individual cells or tissues (for example, as represented in Fig 3A, where somites are represented with different coloured surface objects), the protein load inside each somite is calculated by adding the total gray level intenisities inside these 3D „objects“.


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Fig 4. Profiles of protein content measured along the chick PSM tissue. In A) arrows point at three different transverse confocal slices embedded within the 3D reconstructed PSM. Red colour represents the cytoskeletal protein F-actin, and green represents the extracellular-matrix protein fibronectin. B) Densitometrix profile of F-actin concentration along the PSM on the right embryonic side, as measured by the white „tubular“ probe inserted within the PSM (arrowheads). Peaks (*) represent the interior of recently formed somites.

3.4 3D iso-surface models and morphometrics

Surface models of complex embryonic tissues or organs can be extremely helpful to study their 3D shape and spatial relationship with surrounding tissues or cells. Surface models are produced after segmentation (an image processing technique that allows the isolation of specific portions of an image), either manually, by contouring regions of interest (ROI) in serial sections, or automatically, when a particular cell or tissue can be digitally isolated based on characteristic fluorescence intensity levels (or gray levels, as recorded in optical slices), a process often known as thresholding and/or flood filling segmentation. After segmentation, iso-surfacing produces simplified hollow objects (or 3D surfaces), which are accurate simplified representations of the “original” shape of the tissues/cells segmented. This is an advantage to the the “artistic” diagramatic drawings commonly used for publication and presentation purposes, not only because they are more realistic (and less prone to viewer interpretation), but also because they can be used to obtain accurate morphological measurements (e.g, volume, longest diameter, surface area, protein load inside surface, sphericity, etc.). The 3D visualization and analysis software (e.g., Mercury’s Amira; or Bitplane’s Imaris) also allows the user to draw lines between points in these objects to obtain measurements manually and interatively, similar toi what is done in mechanical engineering and modeling of machinery parts. One further advantage of generating such 3D models of biological specimens is that they can easily be made available online to other researchers, for example as vrml/X3D files (see example in Fig 5), which can be inspected in a browser using freely available software (e.g, Cosmo Player or Cortona viewer). Because these models are simplified surface objects, they occupy considerably less digital space, and are more easily distributed online. These models are also


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interesting didatic materials to developmental biology classes, where students often have difficulty understadning the complex spatial organization of embryonic organs and tissues.

Fig 5. Iso-surface 3D surface models of individual somites of a chick embryo, produced by iso-surfacing after manual contouring of somite boundaries in a confocal stack. The image shows an a vrml model as viewed using a freely available viewer plugin for a www browser. These images can be interactively inspected on-line, with the possibility of adding or removing surface objects from the view, or simultaneously study transversal, sagitall and coronal sections. The model can be rotated in 3D, zoomed/panned using the mouse.

3.5 4 D Imaging of live embryonic tissues

Because live tissues and embryos cannot be cleared and processed as is done with fixed biological samples, live-imaging in depth encompasses further problems and limitations. One alternative is the use of water-immersion objectives which are becoming less expensive and more widely available. However, we have found that for thick specimens such as the chick embryo, a “dry” 10 or 20x objective produces images of comparable quality in deep tissues (in fact, non-immersion objectives have some advantages over immersion objectives because they do not require physical contact between the sample and the front-lens, so there is less heat dissipation and less physical distortions introduced by moving of mechanical parts). Using two-photon microscopy we were able to image the full thicknes of the PSM and somites (~120µm), while minimizing the effects of prolonged laser exposure and physical constraints of the imaging chamber. We typically image chick embryos which have been previously electroporated to incorporate a plasmid coding for the GFP protein. This procedure is well suited for tracking the behaviour of single cells, because GFP is produced continuously by the cells, and because of the inefficiency of the electroporation procedure, it is only expressed by a few cells, which are then easily distinguished from neighbouring, non-GFP-expressing cells. From these images we can follow the movements and shape changes of single cells whithin the PSM and nascent somite. The observations of multiple cells undergoing mitosis throughout the 5-6h imaging period shows that the cells are viable. The possibility of imaging live embryos and obtain highly detailed images of single cell behaviours also allow us to directly test the effects of chemically induced malformations, by unilateral microinjection of drugs that affect cell signaling or disrupt cell movements and/or extracellular matrix organization. In this way, it is not necessary to bisect embryos and culture separated halfs, as described above (section 3.2).


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Fig 6. Stereo-paired images of the formation of an inter-somitic boundary in a chick embryo whose GFP-expressing cells were 4D imaged using two-photon microscopy. Ventral is towards viewer, rostral is up direction and medial is left directrion. The upper pair shows the cells in the anterior PSM at t=0:00, whereas the lower pair shows the same field after 3 h; a new somite has formed and a clear intersomitic boundary appeared. Drawn within the 4D images are the tracks of three individual cells (white lines) whose centroids are represented as small spheres, and the final boundary limits of the most recently nascent somite (shown as gray circular contous). These 3D contours and tracks allow us to follow and better understand the movements that cells make during the formation of a morphological somite. Because cells move in 3D inside the PSM, their movements can only be followed in 3D time-lapse sequences (4D). In neural tube epithelium, positioned in the the medial (left) side of the images, it can be seen that mitosis has produced more GFP expressing cells after the 3 h, indicating good tissue viability. The field width and depth are approx 150 and 80 µm, respectively.

4. Conclusions

3D image reconstruction and processing methodologies offer great advantages for studying complex developmental phenomena imaged by either confocal or multi-photon microscopy. Though it is possible to 3D reconstruct embryos and tissues from series of histological sections, obtaining intact whole series is cumbersome, often introduces mechanical distortions, and requires realignment of the series. One disadvantage of the confocal method is that satisfactory labelling is often difficult to obtain, and imaging in depth requires tissue-clearing, which sometimes affects the labelling itself (and is not possible in live tissues). However, as we demonstrate here, high quality confocal images can be obtained in whole chick embryos, providing valuable 3D information. The information obtained by the in situ “3D probes” would not be possible without sectioning or extracting the tissues from the embryo, which compromise its 3D nature, the spatial relationships between tissues, and its viability. The 3D images produced are exact representations of the actual shape and characteristics of the embryonic tissues, and not mere artistic 3D representations. Such 3D models can be converted to a format easily distributed via internet (e.g., vrml/X3D), are considerably easier to study and can also be inspected in 3D by the viewer, using simple and freely available software (i.e. Cosmo Player or Cortona Viewer). They can be used to aid teachers and researchers in studying complex embryonic developmental phenomena, or even be used for illustrations or mathematical modelling. The high-resolution original Z-series can also be used later for other types of analysis (since the whole 3D structure of the tissues is recorded). This is typically not possible with traditional 2D imaging methods, whose application is mostly limited to the original publication. This type of analysis is essential for the rigorous testing of effects of drug treatments or


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genetic mutations on cell behaviour, and their role in embryonic malformations. The observation/tracking of cells in 3D within live embryonic tissues is also essential for understanding the complex movements that occur during the developmental organization of embryonic tissues (here exemplified with vertebrate somitogenesis).

Acknowledgements: This research is supported by the Portuguese Foundation for Science and Technology (FCT; POCI/BIA-BCM/59201/2004 ) and by the Network of Excellence “Cells into Organs” (EU/FP6).

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3D visualization and analysis of cell-matrix ... · 3D visualization and analysis of cell-matrix transformations in whole-mount and live embryos using confocal and multi-photon microscopy