Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila

Abstract

Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. Here we investigate the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). We report that tracheoblasts pause in G2 for ~48–56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). We find that mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24–32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. We propose that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.

Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

“…These tracheoblasts remain quiescent through most of larval life and rekindle a mitotic program prior to pupariation. Tracheoblasts in the Dorsal Trunk (DT) in Tr2 enter larval life in G1, transition from G1 to S to G2 in the first larval instar (L1) and remain arrested in G2 from second instar (L2) till 32–40 hr into third larval instar (L3) whereupon they re-enter mitosis ( Figure 1A ; Kizhedathu et al, 2018 ). These cells express high levels of Chk1 mRNA and phosphorylated Chk1 (pChk1) during the period in which they are paused in G2 and downregulate levels of Chk1 mRNA and pChk1 upon mitotic re-entry.…”

“…Since perturbations in Wnt signaling led to precocious proliferation akin to the loss of Chk1, next we probed the role of Wnt pathway in Chk1 activation (phosphorylated Chk1, pChk1). pChk1 levels are high in tracheoblasts in L2 and early L3 and significantly lower at 32–40 h L3 ( Kizhedathu et al, 2018 ). pChk1 immunostaining in Btl-TCF RNAi at L2 showed that the levels of pChk1 were dramatically reduced in comparison to wild type ( Figure 2A , n = 6–8 tracheae per condition per experiment, n = 3).…”

“…We have previously reported that arrested tracheoblasts, and the tracheae they comprise, grow significantly during the period in which the cells are arrested in G2. Premature exit from arrest resulting from the knockdown of Chk1 retards growth and results in a reduction in the overall size of Tr2 DT ( Figure 6A , Kizhedathu et al, 2018 ). In light of the role of Wnt signaling in the regulation of Chk1 , we examined how Wnt mutants impact the growth of the tracheae.…”

“…Tracheoblasts arrest in G2 for a period of ~56 hr during larval life and then enter mitosis at the onset of pupariation. The tracheoblasts, and the tracheae they comprise, grow in size while arrested (increase ~11 fold in volume) and undergo rapid size-reductive divisions thereafter (cell division time ~10 hr)( Kizhedathu et al, 2018 ). Contrary to other models of developmental G2 arrest in Drosophila, tracheoblasts express all the drivers necessary for G2-M and utilize a different mechanism for G2 arrest.…”

“…Contrary to other models of developmental G2 arrest in Drosophila, tracheoblasts express all the drivers necessary for G2-M and utilize a different mechanism for G2 arrest. We have shown previously that tracheoblasts co-opt the ATR (mei-41)/Chk1(Grapes) DNA damage checkpoint pathway for the induction of G2 arrest ( Kizhedathu et al, 2018 ). Loss of ATR/Chk1 results in tracheoblasts initiating mitoses precociously.…”