Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos

Research on insect model organisms is an indispensable element in developmental biology (Bodmer and Venkatesh, 1998; Kumar, 2001; Bellen et al., 2010). Although around one million species from the insect order have been described (Patel, 2000), the fruit fly Drosophila melanogaster has an almost unchallenged status in the insect developmental biology community. The red flour beetle Tribolium castaneum is considered an emerging model organism and has become the second most important insect species after Drosophila (Klingler, 2004; Schröder et al., 2008; Brown et al., 2009). In contrast to Drosophila, Tribolium exhibits short-germ development, in which segments are added sequentially to the posterior growth zone (Liu and Kaufman, 2005; Roth and Hartenstein, 2008; Schröder et al., 2008), and embryonic leg development (Grossmann and Prpic, 2012). Other aspects of Tribolium development are also considered representative for the insect order, such as extensive extra-embryonic membrane formation (Handel et al., 2005; Panfilio, 2008; Benton et al., 2013; Panfilio et al., 2013) and non-involuted head development (Peel, 2008; Schinko et al., 2008; Posnien and Bucher, 2010). On the methodology side, some molecular biological tools have a higher potency in Tribolium than in Drosophila, and since the Tribolium genome was recently sequenced (Richards et al., 2008), RNA interference (Bucher et al., 2002; Tomoyasu and Denell, 2004; Tomoyasu et al., 2008) has become a very powerful technique for the analysis of gene functions.

A recent approach to study Tribolium development has been fluorescence live imaging. First reports have described blastoderm formation, gastrulation, the beginning of germband elongation and dorsal closure by imaging the transgenic EFA-nGFP line or transiently labeled embryos by confocal microscopy (El-Sherif et al., 2012; Sarrazin et al., 2012; Benton et al., 2013; Panfilio et al., 2013). However, no long-term fluorescence live imaging of Tribolium embryogenesis has been available until now.

Recently, light sheet-based fluorescence microscopy (LSFM) has emerged as the alternative to confocal fluorescence microscopy and thus constitutes one of the most valuable novel tools in developmental biology (Hell et al., 1994; Stelzer and Lindek, 1994; Huisken et al., 2004; Keller et al., 2008). The complete morphogenesis of Drosophila has already been imaged with great success (Keller et al., 2010). In contrast to an epifluorescence arrangement, LSFM uses at least two independently operated lenses. The lenses used in the excitation of the fluorophores are arranged at an angle of 90° relative to those used for the detection of the three-dimensional fluorophore density distribution. Special optical arrangements illuminate only a thin planar section centered on the focal planes of the detection lenses. Hence, true optical sectioning as well as no phototoxic damage and no photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The energy required to excite the fluorophores while recording a three-dimensional stack of images is reduced by two to four orders of magnitude relative to conventional, confocal and two-photon fluorescence microscopy. Since modern cameras are used to record millions of pixels in parallel, tens to hundreds of images with subcellular resolution can be recorded within a few seconds (Keller and Stelzer, 2010).

Here, we describe a novel mounting method for non-invasive long-term fluorescence live imaging of Tribolium embryos in LSFM. We show all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement continuously in the same embryo and in four orientations and provide a comprehensive chronological description of Tribolium embryogenesis. As an example of the quality and applicability of our data, we provide evidence that serosa window closure and serosa opening, two processes that occur more than 1 day apart, are functionally linked via a previously undescribed structure, the serosa scar. All of our long-term imaging datasets are available as a resource for the community.

The development of Tribolium embryos stops after a few hours when they are embedded in agarose columns, a mounting method that, by contrast, works well with Drosophila embryos (Keller et al., 2011). We assume that this is caused by mechanical constraints, reduced gas exchange or a combination of both. Therefore, we developed a novel mounting method in which only a minor fraction of the egg surface is attached to the top of an agarose hemisphere, while the major fraction of the egg surface is exposed to the buffer (supplementary material Fig. S1A). This method allowed us to image Tribolium for more than 2 days by LSFM (supplementary material Fig. S1B) until the onset of regular muscular movement (supplementary material Fig. S1C).

With the novel mounting method, we obtained three non-invasive long-term fluorescence live imaging datasets of three embryos from the EFA-nGFP transgenic line (Sarrazin et al., 2012). All three embryos were able to hatch into larvae (supplementary material Fig. S1D), developed into adults that showed no abnormalities, produced healthy and fertile progeny and were still alive at the end of the study. Therefore, we infer that our data were obtained in an as close-to-natural fashion as possible, establishing quality standards that have to be met by future studies.

Our data allow us to summarize nearly the complete course of Tribolium embryogenesis from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in a dynamic fashion, including four embryogenesis events that are considered fundamental throughout the insect class – gastrulation, germband elongation, germband retraction and dorsal closure (Fig. 1; supplementary material Movie 1). Furthermore, we created a chronological description of Tribolium embryogenesis that links our observations with the current literature (Table 1).

After blastoderm formation, gastrulation begins with the rearrangement of the uniform blastoderm, which differentiates into the serosa and the embryonic rudiment. The serosa migrates over the posterior pole and grows over the embryonic rudiment, initiating the invagination process. The embryonic rudiment condenses and sinks into the yolk (Fig. 1A). Once the serosa window closes, the embryonic rudiment is released into the interior of the serosa, differentiating into the germband, thereby initiating germband elongation. Segments are now added sequentially to the posterior growth zone. The germband tips curl around both poles, migrating dorsally towards each other. Segmentation becomes more prominent in the thoracic region and leg development begins (Fig. 1B). Germband retraction is accompanied by extensive broadening and the germband flanks bend laterally towards the dorsal side. Legs reach their maximum length while pleuropodia develop at the first abdominal segment and the head becomes clearly separated from the thorax (Fig. 1C). When the germband has retracted completely, dorsal closure begins. After opening, the serosa first migrates over the anterior, then over the posterior pole and turns inside-out on the dorsal side, forming the dorsal organ. Finally, the dorsal organ is incorporated into the dorsal center when both germband flanks close (Fig. 1D). Shortly after dorsal closure, regular muscular movement sets in.

In contrast to Drosophila, Tribolium develops appendices during embryogenesis. We investigated how far these structures can be visualized with LSFM. During dorsal closure, the head performs a ∼45° turn from an anterior to an anterior-ventral position (Fig. 2A). On the single planes, we were able to resolve and follow structures such as the labial palps and the mandibles over time (Fig. 2B; supplementary material Movie 2). Even deep within the embryo, we could still resolve the trachea as well as the esophagus with its butterfly-shaped inner lumen above the subesophageal ganglion (Fig. 2C; supplementary material Movie 2).

An important issue with long-term fluorescence imaging is photobleaching. If datasets were recorded with an imaging interval of 00:06 h, moderate photobleaching occurred, whereas with an interval of 00:30 h photobleaching was barely visible (supplementary material Fig. S2), maintaining a signal-to-noise ratio that was sufficient to perform volume renderings of the complete dataset (supplementary material Movie 3). Volume rendering visualized certain structures more efficiently; for example, median groove folding during gastrulation or embryonic leg development (Fig. 2D). Furthermore, our LSFM setup was sufficiently sensitive to observe cell proliferation in the EFA-nGFP line. During gastrulation, dividing nuclei could be detected as light gray clouds over a dark gray background (Fig. 2E), and, even beyond gastrulation, sites of massive proliferation could be determined; for example, at the outer rims of the head lobes during germband elongation (Fig. 2F).

As an example of the practical application of our data, we characterized a previously undescribed transient structure that emerges and vanishes again in between serosa differentiation and internalization. After serosa window closure, in many, but not all, instances, the germband rotated slightly around the anterior-posterior axis. This rotation revealed an anterior-ventral cell cluster on the serosa, which we term the serosa scar (Fig. 3A; supplementary material Movie 4). For approximately one-third of embryogenesis, the serosa scar remained quiescent in the same location. Shortly before dorsal closure, the serosa scar was pulled apart in a circular fashion. The pulling redirected dorsally, which led to elimination of the space between the serosa and the germband. This was followed by the migration of the serosa over the anterior pole (Fig. 3B; supplementary material Movie 5).

The cell movement pattern suggests that the serosa scar is established at the end of gastrulation as a predetermined point for the formation of an anterior-ventral serosa opening during dorsal closure once pulling at the dorsal side begins. This view is supported by evidence that the space between the serosa and the germband is eliminated, which indicates a subsequent liquid outflow through the newly formed serosa opening.

Spatiotemporally separated and functionally distinct morphogenetic processes, such as serosa window closure (Handel et al., 2000; Benton et al., 2013) and serosa opening (van der Zee et al., 2005; Panfilio et al., 2013), have been described previously, but were not related to preceding and subsequent processes. Therefore, those investigations had not been able to demonstrate a higher order dynamics of embryogenesis, such as the functional link between serosa window closure and serosa opening that we describe here (Fig. 3C). This example illustrates the importance of non-invasive long-term fluorescence live imaging.

We anticipate that LSFM will become a powerful tool in the analysis of fluorescently labeled transgenic Tribolium lines as well as mutants or embryos in which genes have been knocked down, especially in terms of the development of the head, legs and extra-embryonic membranes, in which Tribolium differs significantly from Drosophila.

With this work, the complete embryogenesis of a second insect species is now documented, which is an important step for comparative studies. We intend to investigate which other morphogenetic concepts exist besides the Drosophila paradigm and how they are executed, identifying fundamental principles as well as specialized alterations and thereby providing insights in the fields of developmental, cell and evolutionary biology.

The Tribolium castaneum (Herbst) Vermillion White EFA-nGFP transgenic line was used (Sarrazin et al., 2012). Rearing and embryo collection were performed as described previously (Brown et al., 2009); parameters are provided in supplementary material Table S1.

Simultaneous dechorionation of 10-15 embryos was performed as described previously (Benton et al., 2013). An embryo that exhibited reasonable fluorescence emission under a SteREO Discovery.V8 microscope (Carl Zeiss) with filter set 38 (Carl Zeiss) was picked with a small paint brush. A stainless steel pipe with an external diameter of 3 mm and an internal diameter of 1 mm (#R00306, Sawade Edelstahlrohre, Gottmadingen, Germany) was held vertically and filled with agarose from the top until a hemisphere formed and polymerized at the bottom end of the pipe. The hemisphere was then dipped into liquid agarose in order to apply a thin film that was used to mount the dechorionated embryo with the anterior side to the top of the hemisphere. Finally, 2 μl agarose were added around the gap between the embryo and agarose hemisphere for additional stability (supplementary material Fig. S1). Parameters are provided in supplementary material Table S1.

We used an advanced variant of LSFM termed monolithic digital scanned laser light sheet-based fluorescence microscopy (mDSLM), which is based on DSLM (Keller and Stelzer, 2010). Parameters are provided in supplementary material Table S1. Imaging started immediately after the embryo was inserted into the sample chamber.

When regular muscular movement could be detected (supplementary material Fig. S1C) imaging was stopped. The sample chamber was slowly drained and the agarose hemisphere with the embryo still mounted on top was retrieved and transferred to an object holder. The embryo was dried for 5 min at 35°C, then transferred to a moistening chamber and further incubated at 35°C. After hatching, the larva was transferred to a small glass tube and cultivated as stated above. After the larva had developed into an adult, it was provided with a mating partner. After an appropriate time, progeny production was checked.

Image processing was performed with Mathematica (Wolfram). Single planes (SP) were used to calculate the maximum projections. With those projections, two processing steps were performed. (1) Background correction (BC). A brightness threshold was applied that distinguishes the specimen from the background. The background was used to calculate the mean image background and then set to zero. Then, the mean image background was subtracted from the whole image. This led to an overall intensity decrease. (2) Mean transform (MT). For the desired series of images, the brightest and the dimmest images were selected and the mean intensity was calculated. For the desired series, the image closest to that intensity was determined and designated the reference image. All images were normalized to match the intensity of the reference image. This included both increase and decrease of intensities (supplementary material Fig. S3). Images that underwent this procedure are marked as maximum projections with image processing (MP). If necessary, images underwent equal brightness adjustment. Further details of image processing can be provided upon request. Images were cropped to 600×1000 pixels (XD×YD, supplementary material Table S1), maintaining the original line and pixel pitches used during the imaging recording process. Cell tracking in Fig. 3B was performed manually. Amira (FEI Visualization Science Group, Düsseldorf, Germany) was used for volume rendering (AV) in Fig. 2D and supplementary material Movie 3. Meta information about the three long-term imaging datasets is provided in supplementary material Table S1 (the original datasets, associated figures and movies in full quality are available at www.physikalischebiologie.de/bugcube).

We thank Andrew Peel and Michalis Averof for the Tribolium EFA-nGFP line; the Goethe University workshop for custom hardware; Alexander Schmitz for help with the Mathematica-based image processing; Daniel von Wangenheim for help with the generation of the movies and Amira-based volume rendering; and Isabell Smyrek, Christian Pohl, Anna Gilles and Michalis Averof for comments on the manuscript.