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

Abstract

Larval tracheae of Drosophila harbor progenitors of the adult tracheal system (tracheoblasts). We showed previously that thoracic tracheoblasts arrest in the G2 phase of the cell cycle in an ATR-Checkpoint Kinase1(Chk1)-dependent manner prior to division and morphogenesis (Kizhedathu et al., 2018). Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 in cells that have ATR kinase activity. Wnt signalling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signalling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signalling, driving tracheoblast proliferation.

Comparative Transcriptomic Analysis of Riptortus pedestris (Hemiptera: Alydidae) to Characterize Wing Formation across All Developmental Stages

“…The wnt genes exhibit sequence homology from wnt in the mouse to wingless in Drosophila . In holometabolous insects, such as flies and butterflies, accurate patterning and development are regulated by a series of gene expression patterns in tissues at specific developmental stages [ 49 ]. For example, wingless is a morphogen that acts as a short-range inducer and a long-range organizer across development in Drosophila and participates in patterning of wing discs followed by specification of wing margin-specific patterns [ 50 ].…”

Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

“…Analysis of cell cycle phasing of tracheoblasts using FUCCI has shown that the cells are in the G1 phase at the time the embryo hatches into a larva and that the cells transition from G1-S-G2 in the first larval instar (L1). Tracheoblasts remain in G2 from the second larval instar (L2) through to mid third larval instar (L3) (32-40 h L3, ∼56 h) and divide rapidly thereafter ((Kizhedathu et al, 2018, 2020), Figure 1 A).…”

“…In contrast, the mechanisms for the activation of ATR/Chk1 during development, and the roles of these proteins therein, are less well understood. We reported recently that the ATR/Chk1 axis is co-opted during Drosophila development for inducing G2 arrest in progenitor cells (Kizhedathu et al, 2018, 2020). The current study was designed to shed light on the mechanism of activation of the ATR-Chk1 axis in this context, and to probe if it is distinct from the mechanism for DNA damage-induced activation.…”

“…First, arrested tracheoblasts phosphorylate Chk1 (phosphorylated Chk1, pChk1) in the absence of detectable DNA damage. Second, arrested tracheoblasts upregulate Chk1 expression in a Wnt signaling-dependent manner and high levels of Chk1 expression are essential for arrest (Kizhedathu et al, 2018, 2020). How the ATR/Chk1 pathway is activated in tracheoblasts, in the absence of DNA damage, was unclear from these studies.…”

“…G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al, 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry.…”