Imaging Modalities for the Study of Gastrulation Phase in Caenorhabditis elegans

By Sanaya Bhathena

ABSTRACT

Being structurally small, simple, transparent, and multicellular, Caenorhabditis elegans is an ideal model for understanding the fundamentals of various biological processes. The three-day life cycle of Caenorhabditis elegans involves four major developmental stages: embryonic, postembryonic, larval, and adult. Gastrulation begins at the 26-cell stage with ingression of the two endodermal precursors into the blastocoel, which is followed by the ingression of mesodermal precursors and germline precursors. Combining current genetic techniques, high resolution live cell imaging, and manipulations of the ingressing cells is key to understanding of organ formation within the nematode. Differential interference contrast microscopy provides excellent resolution and phase gradient to image C. elegans, thereby providing an overview of the sequential gastrulation with the identification of granular components within the cell. Combinations of fluorescence microscopy and confocal time-lapse microscopy are used with DIC imaging to obtain cellular details about gastrulation. Brightfield microscopy has also been used for larger picture analysis of the nematode’s anatomy but is not helpful in understanding the details of gastrulation. The electron micron microscope, with its current technological advancements, can be used to understand the gastrulation through cross-sections. Being transparent, cell migration at gastrulation can also be captured using green fluorescent protein. Using a holistic approach towards imaging the gastrulation in C. elegans may provide pertinent information that can be applied to larger eukaryotic organisms.

INTRODUCTION

Caenorhabditis elegans is a structurally small and simple nematode worm; research of its anatomical development allows the tracing of its complete genome sequence [1]. This organism is used as a genetic model for understanding of developmental biology and neurobiology. The transparency of its body, the constancy of cell number and cell position, a fast-paced life cycle of three days, an adult lifespan of three weeks, a small size of approximately 1.5 mm in length, and an ability to grow easily make it ideal for maintenance in a laboratory setting, multiple imaging techniques and genetic studies [2] [3]. The nematode’s optical transparency and in vivo growth capability make it a favorable multicellular model organism to represent the physiological systems of larger eukaryotic animals.

The life cycle of C. elegans, typically spanning over three days, begins with the embryonic stage, followed by progression through reproductive development via four larval stages (L1-L4) to adulthood. In the case of starvation, an alternate pathway leads to the Dauer stage in between the L2 and L4 [4] [5]. Worm development begins during fertilization as asymmetric cell divisions establish the anterior-posterior axis, followed by the establishment of the dorso-ventral axis between the two-cell and the four-cell stage. It finally concludes the embryonic stage with the establishment of the left-right axis [6].

Postembryonic development comprises of four larval stages (L1–L4) each separated by molts [7]. The larval stages involve two alternate pathways: a straightforward passage through the four larval stages or an entry into a Dauer stage that signifies growth-arrest [7]. Once the larval stages are complete, C. elegans enters adulthood. An ideal specimen for observation, its body is easily imageable with differential interference contrast microscopy and with more cell precision through laser microsurgery [2]. Live imaging, optical mapping, laser cell ablation, and fluorescent protein tagging microscopy can be performed throughout the intact, live adult animal [8]. The nematode’s motion during relatively high spatial resolution imaging may cause discrepancies while tracking cellular movement; therefore, immobilization may be required to avoid issues in cell identification caused by animal movement [8].

Gastrulation is the process by which an embryo is restructured due to cell and tissue movements. During this process, the three germ layers, i.e., the endoderm, ectoderm, and mesoderm, are arranged and the organ starts reconstructing with an objective to internalize surface layer cells [9]. In C. elegans, this process begins at the 26-cell stage. The ingression of the two endodermal precursor cells from the surface to the interior of the embryo is soon followed by the ingression of mesodermal precursors and germline precursors [10]. Combination current genetic techniques, high resolution live cell imaging, and manipulations of the ingressing cells aid in our understanding of these cell and organ formations within the nematode.

IMAGING GASTRULATION IN THE LIFE CYCLE OF CAENORHABBDITIS ELEGANS

Differential interference contrast (DIC) microscopy (also referred to as Nomarski microscopy) is an imaging tool used on live biological cells to gather mostly qualitative observations. The nonlinearity present between an object’s characteristics and the needed image intensity makes quantitative analysis challenging [11]. Nomarski images are formed by combining the phase gradient and amplitude of the specimen. C. elegans is a weak absorbing specimen; thus, the image produced consists of phase information that provides structural information about the transparent nematode based on the changes noticed in refractive index on encountering various cell components [12]. With DIC providing excellent resolution and phase gradient to image the thin transparent worm, it can provide an overview of the processes taking place underneath the structure of the specimen.

DIC microscopy, unlike phase contrast and Brightfield microscopy, allows high contrast images of the worm [13]. The light passes through a plane polarizer in the DIC microscope, and then splits into two components using a prism [14]. It interacts with the sample, combines into the objective with the use of a second prism and passes through an analyzer leading to the formation of highly contrasted images visible to the observer [14]. The type of images produced can be used to achieve videos showing various cellular processes over a time scale via time lapse DIC microscopy [15]. With such high contrast images produced through the Nomarski microscope, the resolution can also examine the larger cellular structures and the granular components within the cells. This makes it a great instrument to analyze details within C. elegans cells.

Combinations of various microscopy techniques are used to increase optical visibility in imaging of targeted structures and processes. Fluorescence can be combined with DIC imaging to obtain cellular detail, by the removal of the polarized light analyzer, preventing signal reduction by 30% [16,17]. In studies focusing on imaging gastrulation in C. elegans via DIC, many techniques involve using combinational microscopies such as acquiring simultaneous DIC and Green Fluorescence Proteins (GFP) images or DIC with Confocal Time-Lapse Microscopy [18,19]. These studies have suggested that as gastrulation occurs, the process can be captured with great contrast to the background as the Ea and Ep “sink”, and further division and movement towards the cleft of the progeny cell structures is noticeable in the embryo [18]. This result proves that the DIC and the time-lapse tool are a useful integrated technique to visualize the components involved in gastrulation through its stages.

A study by Harrell and Goldstein used DIC to understand the creation of cell lineages and identified each cell part involved in the internalization movement during gastrulation [19]. Their approach was to trace this process by combining DIC images taken over thirty-three multiplane recordings and four spinning disk confocal recordings of embryos containing plasma membrane markers. By combining these DIC time-lapse images with spinning disc microscope images, they concluded that there were specific lineage patterns in each embryo. They further applied DIC to map out known identities of gastrulating cells and embryonic cells in C. elegans. This set the boundaries to define the internalizing precursor gastrulating cells from the embryonic cells where mitosis was occurring. They were also able to identify individual gastrulating cells and find the correlation between the position of cells during internalization and its effects on the anteroposterior positions in the body later in development [19]. A similar study conducted by Baird and Yen to examine gastrulating cell lineages chose DIC imaging for gastrulating cell differentiation due to its high resolution and the strong magnification power of about 400-1000X [20]. Cell differentiation and development of the embryo could be captured schematically from planar sections and combining of digital images to create a 4D DIC microscope images of C. elegans embryo over a fixed time interval [21].

DIC imaging of C. elegans must be relatively precise and standard for live cell imaging; therefore, the gastrulation needs to be imaged at the right stage and conditions to facilitate the worm’s growth. Another study by Sullivan-Brown et. al describes using 4D microscopy to gain a holistic view of gastrulation. The technique for DIC imaging usually involves the C. elegans 1-4 cell stage embryos being mounted on poly-L-lysine coated coverslips, which are then mounted on a 2.5% agarose growth pad with egg buffer [22]. For four-dimensional DIC video microscopy, they imaged the process using a microscope and an additional camera to acquire images with a certain objective at a particular time interval with constant optical sectioning [22]. Altogether, DIC provides great magnification, clear contrast, modifiable image dimensionality, and time interval capture, making it an ideal imaging tool to understand stationary and dynamic cell structures.

In a bright field microscope, light enters from a source that focuses on a lens and is placed at the condenser [23]. The light passes through the lens to the specimen, and then through a second magnifying lens to reach the eyepiece of the observer [23].

With C. elegans, researchers use Brightfield microscopy for larger picture analysis such as the association between fat accumulation and a darker intestine. Under a Brightfield microscope, worms and their intestinal granules appear pale and close to transparent [24.25] [26,27,28]. Using bright field imaging for tracing gastrulation in C. elegans is challenging because it offers low magnification and a dull contrast [28,29]. The Brightfield image of the nematode provides a structural layout for the organism that has little cellular detail. Brightfield images can also be used at different life stages of the C. elegans to provide insight into the internal composition of the worm anatomy throughout its aging process [30]. Thus, it is not recommended for use on the gastrulation process, which needs higher resolution and phase contrast for the proper identification of precursors involved in ingression movement.

Electron microscopy has been used on C. elegans to image the organism in thin sections [31]. Current technological enhancements in electron microscopy that better focus the electron beam on delivery are making this tool more powerful by bettering the resolution and image handling [31]. A beam of electrons travels in a vacuum tube and focuses through various lenses to create a gray high contrast film based on various cross sections to give a powerful resolution image [32] [33].

Gastrulation of C. elegans can be seen with an electron microscope in cross-sections. Research on gastrulation in C. elegans using modern electron microscopy has created contrast photos of excellent resolution that trace the process by combining various frames [34]. Though electron microscopy is a relatively new technique to visualize C. elegans, it provides substantial detail about the internal structures of organelles. However, it is a cross-sectional imaging technique in a particular time frame, making it undesirable despite having superb magnification power as a film or putting together of various images to trace the gastrulation process.

Fluorescence microscope uses a high-powered light source to stimulate specific features with fluorescent tags attached or immunostained in the sample [35]. This fluorescent tag attached to the sample radiates a wavelength light that is low in energy but relatively long, in turn producing a magnified image [35]. It is crucial to use the right fluorescent markers becuase some markers, such as DsRed, are toxic at high expression levels [36].

Because C. elegans are transparent, they are ideal for the expression of Green Fluorescent Protein (GFP) to observe physiological changes occurring during cell migration in gastrulation. With the knowledge of the organism’s genes and mutants, fluorescent microscopy can be paired with prior cell data to analyze and compare regular migratory cell pathways and behavior with mutations [37]. Another study by Gosai et. al aimed to integrate image acquisition and data analysis using a GFP tag to examine different biological processes occurring within the C. elegans, such as growth and tissue development via gastrulation as well as autophagy [38].

Since the recent advent of GFPs, cell identification and lineage analysis has reached its peak because GFPs can be identified during mutations [39]. Recent studies have integrated the images of DIC with fluorescent microscopy using GFP to identify unknowns about the mechanism of gastrulation [40,41,42,43]. A study using GFP with 4D DIC imaging could indicate that the endodermal internalization and regulation of gene expression determine endodermal cell lineage, further regulating gastrulation [44]. With a relatively large magnification and contrast produced specific to a targeted cell structure, fluorescence microscopy facilitates determination of single cells in a worm, making it useful for gastrulation study.

LIMITATIONS OF IMAGING MODALITIES ON GASTRULATION IN C. ELEGANS

In the sections above, the characteristics and principles of each imaging modality in relation to the cell ingression in C. elegans were described. Some imaging techniques have advantages such as having high resolution, providing a systematic approach, and creating contrast for species structure identification; however, these microscopy techniques have drawbacks as well.

Brightfield microscopy identifies the overall structure and composition of the organism well, but due to its uneven illumination, the formed images of the multi-well plates can be distorted [45]. Distortions in high throughput chemical and genetic screens usually make obtaining foreground-background intensity thresholding difficult [45].

Fluorescence microscopy provides great cellular detail due to its ability to highlight using fluorescent-specific targeted features within an organism. However, fluorescence microscopy cannot image lipids and other structures potentially occurring simultaneously with gastrulation. Coherent Anti-Stokes Raman scattering (CARS) microscopy is often substituted in its place as it involves chemicals targeted at known structures and allows label-free imaging in its living organisms [46]. The fluorescent-dye diet method can also be problematic when imaging other physiological processes such as food uptake efficiency, metabolism, dye distribution, and the components of the surrounding medium [46]. In principle, the lipid molecules could also be specifically tagged by fluorescent marker molecules [47]. However, the GFP tags affect the lipid structural properties due to their substantial makeup and impact the hydrophilic and hydrophobic components [48]; this in turn may change cellular lipid trafficking and metabolism. Multi-channel fluorescence with various wells is limited by the lack of fast shutters, filter wheels, and newer LED light sources causing imaging of well positions that would lead to disrupting image acquisition by reducing the time-point intervals [49].

For these reasons it is desirable to develop a microscopy technique that utilizes inherent properties of the sample for image contrast. C. elegans presents a reliable, flexible, small-scale organism system for in vivo imaging [50] with advantages over other model systems due to the organism’s simplicity and low-cost structure. With the increase in quality provided by various microscopes with different levels of complexity, it can be more difficult to obtain result-specific images as there is space for a mutation or lack of to approve correlation.

CONCLUSION AND FUTURE DIRECTION

Various imaging techniques such as differential interference contrast microscopy, phase contrast microscopy, fluorescence microscopy, Brightfield microscopy and confocal time-lapse microscopy can be used to complement each other to image gastrulation in the short developmental life cycle of C. elegans. By discussing the pros and cons of each technique individually, where some offer high contrast and others offer great resolution, we can infer that a middle ground needs to be achieved. This diversity will enhance the visibility in imaging of targeted structures and processes, which will help further our understanding of higher level multicellular eukaryotic organisms.

In studies focusing on imaging gastrulation in C. elegans, a great number of techniques involve using combination techniques, such as acquiring simultaneous DIC and GFP images or DIC with Confocal Time-Lapse Microscopy. These results turned out to be the most informative in detecting the correlation between various physiological processes. Combining multiple DIC time-lapse images with the spinning disc microscope recordings helped set boundaries to define the internalizing precursor gastrulating cells from the embryonic cells for cell division. The DIC contributed to the internal view of the processes occurring whereas the spinning disc and time-lapse feature made it possible for multiple points within that process to be captured. Each imaging technique presented here came with its own set of advantages and limitations; it would be beneficial to not only combine techniques, but also use the strong suits of one microscope to explore in depth the processes occurring.

ACKNOWLEDGEMENT

I would like to thank Jeff Hardin for his invaluable feedback on the paper. I would also like to thank former and current members of the Hardin lab for their invaluable support to introduce me to the C. elegans and teach me about their growth and maintenance protocol. I would also like to thank Paul Campagnola for organizing guest lectures for his Physics 619 class for which this paper was primarily written.

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2018-05-06T22:42:38+00:00 May 6th, 2018|