Tribolium Extraembryonic Development

=Background= Embryonic development of most pterygote (winged) insects includes the formation and morphogenesis of two distinct extraembryonic epithelial sheets: the serosa and the amnion (reviewed in Panfilio 2008 and Schmidt-Ott et al. 2010). In Drosophila these tissues are secondarily reduced and have evolved into what is called the amnioserosa. Therefore, despite extensive literature on the Drosophila amnioserosa (e.g. Lynch et al 2013), other insect models such as the red flour beetle Tribolium castaneum (Fig. 1) need to be explored in order to further our understanding of the development and evolution of insect extraembryonic membranes.

Tribolium castaneum develops two extraembryonic membranes. The outer membrane, the serosa, envelopes the embryo-proper, amnion and yolk. The amnion remains attached to the embryo and covers it ventrally [Figure]. It follows that the amnion needs to open in order for the embryo to close dorsally. Indeed, at around 50-55 hrs after egg laying, the serosa and amnion rupture at what appears to be a predetermined position (at the ventral anterior of the egg). Shorty thereafter both membranes retract and form the so-called dorsal organ: a dense accumulation of apoptotic cells, dorsally on top of the yolk, in between the lateral flanks of the embryo that now gradually approach each other (initiating dorsal closure) [Ref].

In order to study these morphogenetic process in Tribolium, our lab recently characterised several GEKU enhancer trap lines (Koelzer et al. 2014). One of these lines specifically expresses EGFP in the serosa (G12424 or "serosa" line). A second line marks, among other tissues, precursor cells of the heart (G04609 or "heart" line). Enhancer trap lines are of particular interest for studies of the extramembryonic membranes, because it is very difficult to stain late embryonic stages with, for example, antibodies, due to the formation of cuticle. Also, these lines allow live imaging of the development of particular tissues in vivo.

Live imaging of Tribolium using conventional microscopy generally involves fixing a specimen in place and observing it from a fixed point of view. This static arrangement is not ideal for studying processes that are dynamic over a large area of the surface of an egg, such as extraembryonic morphogenesis, or major rearrangements of the embryo proper. Also, with conventional mounting methods, it is difficult to observe the surface of the poles of the longitudinally elongated eggs of Tribolium. Recent advances in selective plane illumination microscopy (SPIM) have the potential of solving these problems, as the optical arrangement of detection and illumination objectives in these microscopes has pushed the development of mounting methods that allow rotation of the specimen and hence multiview imaging.

=4 experiments=

The following four datasets were collected during the 2014 EMBO practical course on light sheet microscopy. All experiments were performed using the F1 progeny of a cross between female G12424 and male G04609 beetles. Before sample preparation, beetles were kept at 32 according to standard methods. Capillary embedding. 1% LM Agarose. Bead size 500nm.

Imaging:
Huisken SPIM3 3 views (-30, 0 and + 30 degrees). -Detection objective 16x NA..., illumination objectives ... Temperature: 28 degrees Celcius

Data analysis:
Data analysis: maximum intensity projection

=Discussion=

=Literature=

Koelzer S, Kölsch Y, Panfilio KA. (2014) Visualizing Late Insect Embryogenesis: Extraembryonic and Mesodermal Enhancer Trap Expression in the Beetle Tribolium castaneum. PLoS ONE.

Lynch, Holley E., et al. (2013) "Cellular mechanics of germ band retraction in Drosophila." Developmental biology 384.2: (205-213)

Panfilio, KA. (2008) Extraembryonic development in insects and the acrobatics of blastokinesis. Dev Biol 313, 471–491

Schmidt-Ott, U., Rafiqi, A. M., & Lemke, S. (2010). Hox3/zen and the Evolution of Extraembryonic Epithelia in Insects. In Hox Genes (pp. 133-144). Springer New York