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The 20th century brought about the realization that genetics, through the analysis of mutations, was key to identifying factors required for embryonic development. Indeed Genesis is the Journal of Genetics and Development. However, as we enter the 21st century we increasingly recognize that a detailed understanding of the complex molecular and cellular events that drive embryonic development requires integration of multi-disciplinary approaches. Thus, a major challenge to better understanding organismal development is to integrate multiscale information from single cells, to tissue, and intrinsic physical forces, within a unified framework. Unfortunately, technology development and biological insights often occur simultaneously, but are reported in non-overlapping journal articles and conferences, making it tremendously difficult to appreciate the exciting existing and future synergies.

Microscopy is an extremely valuable tool in developmental biology and provides a platform to collect multiscale information. Over the past two to three decades, unprecedented progress has been made in three distinct but convergent areas, each key for live imaging. First, development of genetically-encoded reporters, stemming from the isolation of GFP has demonstrated its use in a heterologous system. Second, data acquisition has tremendously benefitted from improvements in confocal imaging, including selective plane illumination, as well as multi-photon excitation. Third, in silico methodologies propelled by digital media have advanced image analysis, mathematical modeling and synthetic biology to better extract and utilize information from imaging data. In this way, microscopy is a tool that can bridge biological information across multiple spatio-temporal scales.

In this special issue, we bring imaging experts together with biologists to discuss advances in the visualization and interrogation of complex cell dynamics in the embryo. We have learned through this process that there are a wealth of new fluorescent reporter models and cell labeling strategies, advances in technology development and high throughput-like automated imaging and computing to visualize in vivo cell behaviors in 4D. We highlight examples of these achievements and hope you will enjoy the articles and share our excitement of this rapidly moving field of bioimaging!

A Historical Perspective

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

To start off this issue, we thought it was appropriate and fun to look back a bit on the early days of imaging embryos, both to understand how pioneering time-lapse experiments were carried out and highlight how far the field has come in a short time. Rachel Fink (Fink, pp. 484–487) entertains us with a light-hearted view of her cell migration studies at the Woods Hole Marine Biological Laboratory during the mid-1980's, during the birth of video microscopy. She describes the simple elegance of just being able to capture what your eyes could see down the microscope so that a post-doc and her advisor could accurately discuss their observations. This seems a far cry, almost other worldly, from today's techology where someone can receive the latest multi-dimensional movies on their cell phone in seconds! We hope you enjoy this personal and amusing account of how video microscopy “opened windows never thought possible”.

Explorations at the Frontier of Light Microscopy

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

Next, Keller and colleague (Khiary and Keller, pp. 488–513) review the state-of-the art technology for live imaging. By describing a variety of biological processes, they focus on telling us about fluorescence light microscopy techniques that range from multiphoton to light sheet microscopy for systems-level investigation (often referred to as in toto imaging) of animal and plant development. Their discussions range from fluorescence based imaging methods for quantitative mapping of gene expression patterns to the use of genetically-encoded fluorescent protein reporters for cell tracking and dynamic lineage analysis. To handle large image data sets, they discuss computational approaches to image segmentation and provide a comprehensive overview of a variety of recent methods. The authors link this to emerging methods for automated data annotation and biophysical modeling. By drawing on their own studies in zebrafish, they describe how information on cell position (by virtue of nuclear labeling) and cell shape (plasma membrane labeling) may lead to whole embryo digital reconstruction, with single-cell resolution. By carefully leading us through individual processes, the authors successfully paint the big picture of a generally applicable pipeline for microscopy analysis and hypothesis testing.

In an example of imaging development and application, a technology report contributed by Liebling and colleagues (Ohn et al. pp. 514–521) describes automated methods to register high-speed, multi-color images of repeating dynamic processes. Here, the authors extend their previously defined approaches to register 4-D heart contraction datasets to multicolor acquisition of rapid, repetitive dynamics without the need for simultaneous image collection from multiple aligned detectors. This strategy uses the sequential acquisition of high frame-rate fluorescent data and the methods are adopted from those used in medical imaging with X-ray tomography, MRI and PET scans. Their data concludes that these new methods can be used to register 512x512 images taken at 30 frames per second using two different fluorochromes as well as brightfield images using a single detector.

Into the Fourth Dimension using Integrated Approaches and Automation

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

Multi-disciplinary approaches may include wet lab experimentation, image analysis and mathematical modeling. Focusing on dorsal closure, a well-studied morphogenetic process during Drosophila embryogenesis, Gorfinkel and colleagues (Gorfinkel et al., pp. 522–533) illustrate how an integrative approach that combines genetics with image acquisition and analysis provides new and unexpected insights into how individual cells organize and produce macroscopic tissue movements. The authors discuss how the physical forces driving dorsal closure have been uncovered, in part through the use of laser ablation studies. They discuss details of how correlative analyses between fluorescence intensities and cell shapes have revealed the importance of the actomyosin network. They argue by example that the availability of quantitative information on the kinematic properties of cells and tissues provide a critical basis for mathematical models of dorsal closure. In turn, mathematical model predictions provide a rapid feedback mechanism to test at the bench. Thus, multidisciplinary approaches have the potential to yield novel multi-scale information on dorsal closure and so provide a blueprint for better understanding of other complex, related morphogenetic processes, such as neural tube closure and wound healing.

One of the focused reviews in this special issue discusses imaging advances in neural development. Sagasti and colleagues present technical considerations of time-lapse imaging in the zebrafish embryo (Rieger et al., pp. 534–545). They begin their article by presenting fluorescent cell labeling techniques and the recent strides in using transposon-based methods to increase the frequency at which exogeneous DNA integrates into chromosomes (Kikuta and Kawakami,2009). As an example of the power of time-lapse imaging, they discuss a problem in central nervous system development. Recent studies have shed light on two important questions in neural development during retina and hindbrain formation. First, whether nuclear position correlates to cell cycle stage during interkinetic nuclear migration and the patterns of cell division near the apical (luminal) side of the neural tube (NT). Sagasti and colleagues present findings that highlight how using time-lapse imaging was crucial in determining the details of asymmetric cell division. During asymmetric cell division, they describe details that reveal the progenitor cell closer to the apical (luminal) side of the NT becomes the neuron (evaluated by HuC expression). In contrast, the basal daughter cell becomes the progenitor (Alexandre et al.,2010). Secondly, that cell behaviors in the NT are complex and nuclear position does not correlate with cell cycle stage; contrary to a long-held model.

In another example of how the zebrafish model is making great strides as a tool for the study of neural development, Bruses and colleague (Zelenchuk and Bruses, pp. 546–554) discuss motor neuron labeling. One of the challenges to examining the molecular mechanisms that regulate axonal pathfinding has been the ability to visualize the dendritic tree and axonal arborization of single motor neurons in fixed and live animals. The authors characterize a 125-bp mnx1 enhancer that directs transgene expression in zebrafish spinal cord motor neurons. Notably, this cis-regulatory element was active beyond embryonic development and could be used to image cells in adult fish. They describe how a promoter containing three copies of the 125-bp mnx1 enhancer was generated in a Tol2 vector and used to drive EGFP expression either directly or in combination with the Gal4/UAS transcriptional activation system. In beautiful still images from confocal time-lapse imaging of fluorescently labeled primary motor neurons, an entire cell including the axonal arborization and dendritic extensions are vividly clear. The authors point out that the mnx1 enhancer can be cloned upstream of a protein coding sequence or combined with other methods for example using site specific recombinases (e.g. Cre/loxP) or steroid receptors to provide further temporal control of transgene activation. In summary, use of this type of promoter element, may reveal novel insights of motor neuron development, and has the potential to shed light on gene products involved in neurodegenerative disorders.

Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high throughput strategies. In a comprehensive review, Supatto and colleague (Truong and Supatto, pp. 555–569) identify the key challenges for applying such strategies in developmental biology. Using examples from Drosophila and zebrafish models, the authors discuss the progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation and data mining. All of these components are seamlessly stitched together in an experimental workflow proposed by the authors for high content/high-throughput imaging and analysis of embryonic morphogenesis. Lastly, the article provides an extremely useful set of references presented in table format to guide readers through the recent progress in these lead areas.

Next Gen Fluorescent Mice

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

Traditional fluorescent marking of distinct cell populations in mouse embryos is technically challenging. Current methods encompass the injection or electroporation of reporters (including chromogenic and fluorescent dyes, and fluorescent proteins), as well as grafting of cells or tissues, and the generation of chimeras or mosaics. Fate mapping through binary genetic approaches employing recombinase systems such as the Cre/loxP, Flp/FRT, or Dre/Rox can be used in mice. Thus, mice that exhibit widespread, readily detectable expression of reporters that are otherwise indistinguishable from wild-type non-transgenic littermates, represent essential tools for non-invasive cell labeling of specific cell populations for live imaging experiments.

The ROSA26 (R26) locus has been demonstrated to exhibit widespread transcriptional activity during embryonic development (Friedrich and Soriano,1991; Soriano,1999). In this issue, Aizawa, Behringer and colleagues (Shoi et al., pp. 570–578) have excitingly developed R26-RG, a mouse reporter strain that simultaneously marks cell membranes with a red fluorescent protein and cell nuclei with a green fluorescent protein upon Cre excision of a floxed Pgk-Neo-pA cassette. In their method, a self-cleaving 2A sequence was used in a single construct to drive bicistronic expression of two reporters: a plasma membrane-bound myrostoylated-mCherry and a nuclear-localized H2B-EGFP fusion. In beautiful time-lapse movies, heterozygous and homozygous embryos for the Cre-deleted dual tag reporter allele reveal the power of this method to visualize individual cell positions and processes. Focusing on preimplantation and early postimplantation stages, as well as in mouse embryo fibroblasts (MEFs), the authors visualized stereotypical changes in the shape, as well as division of individual cells. This new line of reporter micerepresents an additional valuable tool to the available repertoire of reporters (e.g., Trichas et al.,2008; Nowotschin et al.,2009) for double labeling and tracking populations of cells in vivo in mice.

One limitation of widely expressed fluorescent reporters is that in complex tissue populations it is often advantageous to selectively label cells of interest. In mice, this can be circumvented several ways, including the use of (1) widely expressed but conditionally activated (e.g. Cre) reporters strains, (2) widely expressed photomodulatable reporters, and (3) tissue-specific reporters. Thus, by further validating and extending their analysis of the R26-RG strain, Aizawa, Behringer, and colleagues went on to label and image selected populations of cells upon Cre excision. Tissue-specific RG expression was examined at various sites after Cre-mediated floxed Pgk-Neo-pA cassette excision with a Sox9:Cre and subsequent RG reporter activation.

Extending their use of spectrally-distinct subcellularly-localized bicistronic dual tag reporter cassettes Aizawa and colleagues (Abe et al., pp. 579–590) go on to report the generation of an additional series of 12 (!) Cre reporter strains. The authors surveyed a variety of fluorescent proteins for live imaging applications. The reporter lines they generated mark the nucleus of a cell, its mitochondria, Golgi apparatus, plasma membrane, microtubule, actin filaments or focal adhesions. Distinct double labeling was also shown for nucleus and plasma membrane or Golgi apparatus. The construction of targeting vectors or insertion of a series of fluorescent transgenes into the R26 locus was made routine using the Gateway cloning system. This now becomes a powerful tool to analyze complex cell behaviors, including the tracking of cell division, cell death and cell movements throughout the early mouse embryo. Lastly, the authors suggest that this tool will have wide applicability to the developmental community, since when recombined by Cre, these reporters become cell specific tools for visualizing particular events of morphogenesis. In this way, characterized Cre driver strains can be used with any of Aizawa's cohort of Cre reporters.

Photogenicity of the Embryo

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

Photomodulatable fluorescent proteins are one of an emerging class of genetically-encoded fluorescent proteins. However, unlike other model systems, the application of photomodulatable fluorescent protein technology in mice is still not commonplace. Behringer and colleagues (Griswald et al., pp. 591–598) report the generation and validation of an iUBC-KikGR mouse line exhibiting widespread expression of the photoconvertible green-to-red fluorescent protein KikGR. Unlike existing mouse lines exhibiting widespread KikGR expression,generated by targeting the R26 locus (Kurotaki et al.,2007) or using the CAG promoter (Nowotschin et al.,2009), Behringer and colleagues have placed KikGR under the control of the human Ubiquitin C promoter. In this way, KikGR was expressed robustly and broadly in hemizygous transgenic embryos from the 2-cell stage onward, and in the adult. Wonderful examples of photoconversion that range from the entire embryo to subregions of an organ and to a few individual cells are shown. This new line of KikGR expressing mice represents yet another tool for optical highlighting and tracking defined sub-populations of cells in vivo in mice.

Staying with the theme of dynamic cell lineage tracing, Southard-Smith and colleagues present a beautiful application in the developing mouse enteric nervous system (Southard-Smith et al., pp. 599–618). They discuss the development of a mouse BAC transgenic line that drives an H2B-Venus reporter from Sox10 regulatory regions. The endogenous Sox10 pattern is cell type specific to neural crest-derived progenitors within the enteric nervous system. By focusing on Sox10, the authors present their model as a novel tool to study neural crest cell lineage segregation. Multipotent neural crest cells migrate through the fetal gut and differentiate to assemble enteric nervous system structures. Southard-Smith and colleagues very neatly show how H2B-Venus expression allows them to more accurately follow cell positions with confocal time-lapse microscopy as well as efficiently FACs sort cells based on nuclear localized fluorescence signal. It is also interesting to read their evidence for observing cell fragmentation, which they attribute to isolated cell death, and differences in transgene expression which are consistent with enteric nervous system assembly and the progression of multipotent neural crest cells towards non-glial fates, respectively. Thus, by using a nuclear localized fluorescent protein driven from Sox10 regulatory regions, the authors provide a powerful tool to study questions of neural crest cell lineage.

Avian transgenics – Fashionably Late to the Multicolor Party

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

A second focused review in this issue takes a look at dynamic cell lineage analysis during early avian embryogenesis. Lansford and colleagues review the progression and complications of past fate mapping techniques (Bower, pp. 619–643). This comprehensive review provides a firm backdrop for their arguments that the field of cell fate mapping is on a search for more accurate and robust models. Excitingly, they describe the development of transgenic quail that express various fluorescent proteins in a targeted fashion (Sato et al.,2010 and Bower, pp. 619–643), without the need for an externally applied contrast agent. When fluorescently labeled quail embryos are visualized using 4D confocal or multiphoton microscopy, individual cell movements can be dynamically followed during developmental events such as gastrulation, left-right asymmetry, and vasculogenesis. In the remainder of their article, the authors discuss how using their transgenic quail and targeted fluorescent reporters may overcome typical challenges in developmental imaging. These include diversity of fluorescence expression levels in cells, and the ability to uniquely resolve cell division and cell contact events in dense tissue. To address these issues within the framework of their transgenic quail, they present examples where clever targeting of fluorescent reporters can overcome these barriers. By using electroporation delivery of DNA, they show they can combine their transgenic quail (e.g., Tg (Tie:H2BeYFP)) with different fluorescent reporters, such as GAP43 membrane-tethered mCherry fluorescent protein. They go on to show how these cell labeling strategies can more accurately analyze cell divisions and complex cell movement events that make up dynamic cell lineage tracing.

Conclusion

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED

Our goal with this special live imaging issue has been to highlight emerging technologies and how they are being applied and encourage biologists who are tackling tough questions to describe them and think about futuristic approaches in order to inspire new developments. We hope that the exciting articles comprising this issue will continue to inspire imaging innovations, provide a framework for biologists to generate novel insights into complex cell dynamics in the embryo and stimulate communication between imaging experts and biologists. With this special imaging issue, genesis: The Journal of Genetics and Development has very kindly provided a chance for us to publicize our field. We hope more people will get involved in promoting live imaging and encourage readers to submit their manuscripts to this journal, read the articles, letters, and technical reports and visit the journal website for news and views.

LITERATURE CITED

  1. Top of page
  2. A Historical Perspective
  3. Explorations at the Frontier of Light Microscopy
  4. Into the Fourth Dimension using Integrated Approaches and Automation
  5. Next Gen Fluorescent Mice
  6. Photogenicity of the Embryo
  7. Avian transgenics – Fashionably Late to the Multicolor Party
  8. Conclusion
  9. LITERATURE CITED