Contribution of cell proliferation to axial elongation in the red flour beetle Tribolium castaneum

Abstract

Most arthropods generate their posterior bodies by adding segments periodically, as the embryo grows, from a posteriorly located region called the segment addition zone. This mode of segmentation is shared with vertebrates and relies on oscillatory mechanisms, where the temporal periodicity of a clock is translated into repetitive spatial patterns. This ordered anterior-to-posterior pattern is achieved at the same time as the tissue elongates, opening the question of the functional coordination between the mechanisms of segmental patterning and posterior growth. The study of these processes in different arthropods has played an important role in unravelling some of the molecular mechanisms of segment formation. However, the behavior of cells during elongation and how cellular processes affect this segmental patterning has been poorly studied. Cell proliferation together with cell rearrangements are presumed to be the major forces driving axis elongation in the red flour beetle Tribolium castaneum. However, there still no strong evidence about the role and distribution of cell proliferation within the embryo. In this study, we propose to address these questions by using whole embryo cultures and pharmacological manipulation. We show that considerable cell proliferation occurs during germband elongation, measured by incorporation of the nucleoside analog of thymidine 5-Ethynyl-2’-deoxyuridine, EdU. Moreover, proliferating cells appeared to be spread along the elongating embryo with a posterior bias at early segmentation. In addition, when we blocked cell division, treated germbands were always shorter than controls and in some cases not able to fully elongate, even when control embryos already started to retract and leg buds are evident. Finally, we found that the absence of cell proliferation has no apparent effect on segmental patterning, as evidenced by Tc-engrailed (Tc-en) gene expression.

Arthropod segmentation

“…The material for new segments is generally provided by a combination of cell division and convergent extension, butas in vertebratesthe relative contributions of these cell behaviours to axial elongation vary widely across species (Auman et al, 2017;Benton, 2018;Benton et al, 2016;Mito et al, 2011;Nakamoto et al, 2015;Steventon et al, 2016). Accordingly, although cell division may in some species be coordinated with segment addition, segment patterning processes do not appear to be mechanistically dependent on the cell cycle (Cepeda et al, 2017), aside from in special cases such as malacostracan crustaceans. This group exhibits a highly derived mode of segmentation in which patterning occurs through regimented asymmetrical divisions of rows of posterior cells (Scholtz, 1992).…”

Elongation during segmentation shows axial variability, low mitotic rates, and synchronized cell cycle domains in the crustacean, Thamnocephalus platyurus

“…It is interesting to speculate whether, in these cases, the anterior growth zone is the region of segment pre-patterning and thus cell are not cycling. By contrast, examination of Tribolium using EdU exposure showed no apparent regionally distinct incorporation within the growth zone [44].…”

“…We argue this mitosis contributes to normal elongation. This appears to be true, even for a number species that also use cell movement to elongate [12,44]. What is clear is that, except for malacostracans, no arthropods show a narrow zone of dedicated proliferative cells in the posterior growth zone that would be similar to what has been documented in leeches or some polychaetes [68,69].…”

“…Indeed, in a related anostracan, Artemia, Freeman [33] found the same general pattern in the trunk using morphological landmarks: more cells were in mitosis in the anterior trunk region than the posterior. In the few species in which mitosis has been examined during sequential segmentation [25,[44][45][46]; this study), mitosis in the already specified segments is extensive and no doubt contributes greatly to overall elongation. It is becoming clear that this overall elongation along the body leads to a false expectation of high mitosis in the growth zone and at the same time potentially obscures a low but real amount of posterior growth.…”

“…Nonetheless, for the vast array of arthropods, the phenomena responsible for posterior elongation remain unknown and understudied, especially compared with the exploration of patterning genes regulating segmentation. The general morphometric changes accompanying elongation have been studied systematically in two insects-Tribolium [12,44] and Oncopeltus [25]-both of which show a limited amount of growth. Here, we used careful staging to track growth in larvae of the crustacean Thamnocephalus, which appear to have a more obvious amount of posterior growth since they add most of their segments post-hatching.…”