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

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.

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.