Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos

Abstract

In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. Here we show that hypomorphic alleles of Notchbelonging to the N facet class -reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated morphogenetic cell motility process. We demonstrate that during OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the EGFR, to fine-tune the activity of Egfr signaling. Consistently, the loss-of-function defects of N facet alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam dependent transcription. IntroductionDrosophila eye development serves as a paradigm for many developmental patterning processes and the dissection of the associated signaling pathways (Cagan and Ready, 1989a;Roignant and Treisman, 2009;Tomlinson and Ready, 1987;Wolff and Ready, 1991). The Drosophila eye consists of ~800 highly regularly arranged ommatidia, or facets, with each consisting of 8 photoreceptor (R-cell) neurons (R1-R8), arranged into a precise invariant trapezoidal pattern, and 12 accessory (cone, pigment, and bristle) cells (Tomlinson and Ready, 1987;Wolff and Ready, 1991). During larval stages, the eye develops from an imaginal disc, which is initially composed of identical pluripotent precursor cells. As a wave of cell proliferation and differentiation (referred to as morphogenetic furrow, MF) moves across the disc from posterior to anterior, it leaves regularly spaced preclusters of differentiating cells in its wake that will mature into ommatidia (Cagan and Ready, 1989a;Roignant and Treisman, 2009;Tomlinson and Ready, 1987;Wolff and Ready, 1991). At the 5-cell precluster stage, several patterning steps are apparent in addition to R-cell induction and differentiation, one being the differential specification of the two cells within the R3/R4 pair, which breaks the initial symmetry of the precluster. This differential R3/R4 specification requires the Wnt-Frizzled (Fz)/Planar Cell Polarity (PCP) pathway and its interplay with and asymmetric upregulation of Notch (N)signaling (Blair, 1999;Cooper and Bray, 1999;Fanto and Mlodzik, 1999;Mlodzik, 1999;Strutt and Strutt, 1999). This cell fate induction step is followed by the rotation of the ommatidial precluster, referred to as ommatidial rotation, towards the dors...

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

“…We have previously reported that Notch signaling in R3/R4 pairs is critical to coordinate OR via Notch-signaling feeding into its transcriptional regulatory mechanism(s) (Koca et al, 2019). Our studies suggested that Notch signaling in R4 directly promotes the transcription of aos, encoding an inhibitory ligand to EGFR, and thus fine-tunes the critical EGFR signaling activity (Gaengel and Mlodzik, 2003) within the OR process (Koca et al, 2019).…”

“…To understand whether the contrasting apical localization pattern of dAbl between R3/R4 is important for OR, we aimed to perturb dAbl function and localization specifically in cells of the R3/R4 pair. To this end, we employed the md0.5-Gal4 driver which is initially active in both R3/R4 precursors and later becomes specifically upregulated in R4 as a result of Notch-mediated R4 specification (Cooper and Bray, 1999;Koca et al, 2019). md0.5-Gal4 mediated knockdown (KD) of dAbl, within R3/R4 pairs led to a significant aberration in the rotation pattern of ommatidial clusters as compared to wild-type, with clusters generally rotating faster (Figure 4A-B,D; Suppl.…”

“…We have previously reported that Notch signaling in R3/R4 pairs is critical to coordinate OR via Notch-signaling feeding into its transcriptional regulatory mechanism(s) (Koca et al, 2019). Our studies suggested that Notch signaling in R4 directly promotes the transcription of aos, encoding an inhibitory ligand to EGFR, and thus fine-tunes the critical EGFR signaling activity (Gaengel and Mlodzik, 2003) within the OR process (Koca et al, 2019). Here, we show that in addition to its interaction with the EGFR signaling pathway, Notch signaling regulates OR via its apical junctional recruitment of dAbl in R4, linking Notch activity here to the non-canonical Abl-mediated Notch pathway and associated local cellular processes.…”

“…For example, it is established that Fz/PCP signaling feeds into cadherin-based cell adhesion machinery through downstream effectors to regulate the OR process (Mirkovic et al, 2011). Furthermore, cytoskeletal reorganization of ommatidial cells is coordinated with adhesion remodeling to drive the OR process downstream of several signaling pathways, including Fz/PCP, EGFR, and Notch signaling (Brown and Freeman, 2003;Fiehler and Wolff, 2007;Gaengel and Mlodzik, 2003;Koca et al, 2019;Winter et al, 2001). Despite this knowledge, actual mechanistic insights into the OR process remain largely elusive.…”

How do the Fat–Dachsous and core planar polarity pathways act together and independently to coordinate polarized cell behaviours?

“…These include signalling by the small GTPases RhoA and Rac, upstream of Rho kinase [36,43–45] and modulation of E-cadherin and N-cadherin expression via the kinase Nemo [46–49]. Notch activity in the R4 cell also contributes to rotation by regulating EGF signalling [46,50–53].…”