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

Abstract

Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved.

The beetle amnion and serosa functionally interact as apposed epithelia

“…5B, Figure 5-figure supplement 1). Rotation occurs irrespective of whether the embryo's long axis is orthogonal (this study) or parallel (Strobl and Stelzer, 2014) to gravity (Strobl and Stelzer, 2014), suggesting an inherent anisotropy of the early egg. However, there is no counterpart in later development (n= 118), including for 15 embryos filmed continuously and which exhibited typical frequencies of early rotation.…”

Light Sheet Microscopy to Measure Protein Dynamics

“…Due to the low phototoxicity and rapid imaging speed, LSM has proven to be a new standard for long‐term 3D/4D imaging of developmental procedures at cellular resolution in a wide range of species, including growing plant roots, the invertebrates Drosophila melanogaster , Tribolium castaneum , and Caenorhabditis elegans , as well as the vertebrates zebrafish, Medaka, and remarkably the developing mouse embryo (various examples are depicted in Fig. ; Huisken et al, ; Keller and Stelzer, ; Maizel et al, ; Wu et al, ; Ichikawa et al, ; Strobl and Stelzer, ). Live organ function, such as the zebrafish heartbeat, can be visualized in real‐time precision (Mickoleit et al, ).…”

Light Sheet Fluorescence Microscopy (LSFM)

“…Hence, multi-layer mounting approaches have been developed to ensure stable conditions, such as the multi-layer approach applied by Kaufmann et al (2012) for imaging developing zebrafish embryos. LSFM has also been used to image the development of Caenorhabditis elegans embryos (Chardes et al, 2014), amoeboid movements (Takao et al, 2012), bacterial symbioses in developing zebrafish larvae (Taormina et al, 2012) and development of Tribolium castaneum embryos (Strobl and Stelzer, 2014). Despite the overwhelming focus on embryonic development, LSFM has also been used to image large cellular specimens such as three-dimensional spheroid cultures Pampaloni et al, 2013) and monitor cell division dynamics within large spheroid tumor models (Lorenzo et al, 2011).…”