Notch signaling pathway plays crucial roles in developmental processes such as tissue patterning, cell proliferation and cell fate determination (Bray, 2016). Malfunction of Notch signaling results in various malignant diseases in human, including neuropsychiatric diseases, metabolic disorders and multiple types of cancer (Salazar and Yamamoto, 2018). The core components and signal transduction routes of Notch signaling are highly conserved among the animal kingdom (Fortini, 2009). Named after the wing margin nicking phenotype observed in the Drosophila mutant, the Notch gene encodes a transmembrane protein which functions as signal receptor (Bray, 2016). Binding of Notch with ligand proteins Delta or Serrate, which are presented at the membrane of signal sending cells leads to a series of proteolytic cleavage of the Notch protein (Fortini, 2009). As a consequence, the Notch intracellular domain (NICD) is released and translocates into nucleus in the signal receiving cells (Kopan and Ilagan, 2009). NICD interacts with the transcription factor Suppressor of Hairless [Su(H)] and the co-activator Mastermind (Mam) to form a ternary complex. The Su(H)/NICD/Mam complex recognizes specific cis-regulatory regions and activates transcription of Notch target genes. In the absence of signal input, Su(H) recruits co-repressors and inhibits the expression of Notch targets (Kopan and Ilagan, 2009).

The Notch signaling is tightly controlled by auxiliary factors that modulate the expression, stability and activity of the core components (Fortini, 2009). Recent studies have revealed that protein ubiquitination is extensively involved in the regulation of Notch signaling pathway (Le Bras et al., 2011; Weinmaster and Fischer 2011). Protein ubiquitination is a reversible post-translational modification catalyzed by four distinct enzymes. The E1 (Ub-activating) and E2 (Ub-conjugating) enzymes are responsible for activating and conjugating the ubiquitin (Ub) moiety, respectively. The E3 (Ub ligases) enzyme recognizes specific substrates and transfers Ub from E2 onto them. The deubiquitinating enzyme (DUB) removes Ub from substrate proteins to counteract the ubiquitination process (Grabbe et al., 2011). E2 enzymes are now considered as the main determinant for the topology of ubiquitin chains, which directs the ubiquitinated substrates towards distinct fates (Ye and Rape, 2009).

Multiple E3s and DUBs have been demonstrated to regulate Notch signaling during fly development (Moretti and Brou, 2013). In the signal sending cells, E3 ligases Neuralized (Neur) and Mind bomb (Mib1) promote mono-ubiquitination of the ligand proteins Delta and Serrate to facilitate their endocytosis (Yeh et al., 2000; Lai et al., 2001; Pavlopoulos et al., 2001; Itoh et al., 2003; Le Borgne and Schweisguth, 2003; Li and Baker, 2004; Lai et al., 2005). Ubiquitination and endocytosis of ligand proteins are required for initiation of signal transduction in various tissues (Yeh et al., 2001; Le Borgne et al., 2005; Pitsouli and Delidakis, 2005; Wang and Struhl, 2005; Skwarek et al., 2007; Miller and Posakony, 2018). In the signal receiving cells, Notch molecules are ubiquitinated by E3 ligases Nedd4 and Suppressor of deltex [Su(dx)] and targeted for lysosomal degradation to avoid ligand independent activation (Cornell et al., 1999; Mazaleyrat et al., 2003; Sakata et al., 2004; Wilkin et al., 2004; Dalton et al., 2011). The E3 ubiquitin ligase Deltex (Dx) was isolated as a positive regulator of Notch signaling which genetically and physically interacts with Notch (Xu and Artavanis-Tsakonas, 1990; Diederich et al., 1994; Matsuno et al., 1995; Matsuno et al., 2002). Subsequent studies reveal that Dx promotes ubiquitination and ligand independent activation of Notch through the endocytic machinery (Hori et al., 2004; Wilkin et al., 2008; Hori et al., 2011; Yamada et al., 2011). Interestingly, Dx is also capable of inhibiting Notch activation in certain developmental contexts (Mukherjee et al., 2005; Fuwa et al., 2006; Dutta et al., 2017). The E3 ligase cbl is found to target both Dl (Wang et al., 2010) and Notch (Bala Tannan et al., 2018) for degradation. The DUB enzyme Fat facets (Faf) enhances Delta endocytosis to promote Notch signaling during fly eye development (Cadavid et al., 2000; Chen and Fischer, 2000; Chen et al., 2002; Overstreet et al., 2004), while another DUB enzyme USP5 negatively regulates Notch signaling in the same tissue (Ling et al., 2017). Several other DUBs have been implicated in Notch signaling regulation during wing development, but their substrates are still elusive (Zhang et al., 2012).

To date, very little is known about the roles of E2 enzymes in Notch signaling. Here we report that the E2 enzyme UbcD1 (also known as effete) positively regulates Notch signaling activity in the signal receiving cells during Drosophila wing development. UbcD1 is a highly conserved class I E2 enzyme (Treier et al., 1992), which plays important roles in a broad spectrum of cellular and developmental events. UbcD1 participates in regulation of telomere behavior (Cenci et al., 1997; Cipressa et al., 2013), apoptosis (Ryoo et al., 2002; Yeh and Bratton, 2013), innate immunity (Chen et al., 2017), dendrite pruning (Kuo et al., 2006), oogenesis (Ohlmeyer and Schupbach, 2003; Chen et al., 2009), neuroblast proliferation (Li et al., 2014) as well as Hedgehog (Hh) signaling and fly wing patterning (Pan et al., 2017). Our study represents the first analysis for the role of UbcD1 in Notch signaling pathway, which will help to understand the functional complexity and diversity of UbcD1.

Formation of wings made insects the first group of animals that gained the ability to fly during evolution (Shimmi et al., 2014). The shape, size and venation patterns of insect wings are highly stereotyped and species specific, and these traits are widely used in biology researches ranging from species identification, organ development to evolutionary modelling (Parchem et al., 2007). Our knowledge of the genetic and molecular basis of insect wing development largely originates from studies in the model insect, Drosophila melanogaster (De Celis and Diaz-Benjumea, 2003). The Notch mutant likely represents one of the first recorded Drosophila mutations that affect wing development. Later studies demonstrate that the Notch signal pathway is highly conserved among the insects and regulates various developmental processes across different species. Notch signaling regulates wing margin formation in Drosophila hydei (Van Breugel and Langhout, 1983) and sheep blowfly (Davies et al., 1996; Chen et al., 1998), wing morphogenesis in silkworm (Sato et al., 2008; Ling et al., 2015) and pigment patterns in the butterfly wing (Reed, 2004; Reed and Serfas, 2004). Further studies indicate that Notch signaling is required for oogenesis in Blattella germanica (Irles et al., 2016) and locust (Song et al., 2019), reproductive constraint in the adult worker honeybee (Duncan et al., 2016), appendage development in silk worm (Liu, 2012) and camouflage patterns in caterpillars (Jin et al., 2020). Recent studies reveal crucial role of Notch signaling during body segmentation in insect species such as cockroaches (Pueyo et al., 2008; Chesebro et al., 2013) and silkworm (Liu, 2013). Whether Notch signaling regulates segmentation in cricket is still under debate (Kainz et al., 2011; Mito et al., 2011), but segmentation in Drosophila (Liao and Oates, 2017) and grasshopper (Dearden and Akam, 2000) is likely independent of Notch signaling. These studies highlight the important and diverse roles of Notch signaling, identification of new factors involved in Notch signal transduction will help us to better understand how it operates to control insect development.

Our data presented here suggests a novel role for UbcD1 as a positive regulator of the Notch signaling pathway during fly wing development. Previous studies have found that UbcD1 genetically interacts with the DUB Faf (Cadavid et al., 2000) and E3 ligase Neur (Lai et al., 2001), both of which regulate Dl endocytic trafficking during fly eye development. However, whether and how Dl protein and Notch signaling are affected in UbcD1 mutant eye disc cells have not been investigated (Cadavid et al., 2000; Lai et al., 2001). Furthermore, Faf is dispensable for fly wing development (Fischer-Vize et al., 1992). The E3 ligase Neur is essential for sensory precursors specification but not wing margin formation and other Notch signaling dependent processes during wing development (Yeh et al., 2000; Lai and Rubin, 2001). Therefore, UbcD1 is likely involved in Notch signaling regulation in multiple tissues and developmental contexts, targeting distinct signal molecules and transduction steps. Our genetic analysis suggests that UbcD1 functions presumably at early steps before the cleavage of full-length Notch protein, but also impacts later transduction events such as Notch trafficking and distribution in the developing wing. The molecular targets of UbcD1 and the exact mechanisms that how UbcD1 impacts Notch signaling still remains elusive.

Alternatively, UbcD1 might regulate Notch signaling indirectly through cellular processes such as endolysosomal trafficking. Our results indicate that UbcD1 is likely required for maturation of late endosomes and following steps towards lysosomal degradation. A crucial event during endosome maturation is Rab conversion, during which the early organizer Rab5 is replaced by the late organizer Rab7. Recent work identifies Dmon1, a member of the Sand1/Mon1 protein family, as a crucial factor for Rab conversion during fly wing development (Yousefian et al., 2013). In fly wing disc cells, LOF of Dmon1 results in reduced association of Rab7 with endosomes, enhancement of endolysosomal acidification and accumulation of Notch proteins (Yousefian et al., 2013). The high similarity of these LOF phenotypes indicate that UbcD1 might be involved in Rab conversion. The exact role of UbcD1 in the endolysosomal machinery remains an open question.

Given the broad cellular activities of UbcD1, it is not surprising to find that UbcD1 might regulate multiple signaling pathways during wing development. It has been shown that UbcD1 negatively regulates Hh signaling activation in the wing (Pan et al., 2017). When UbcD1 expression was inhibited by RNAi, down-regulation of Wg was observed in cells located at the D/V boundary due to disruption of Notch signaling transduction. In contrast, accumulation of Wg were found in UbcD1 RNAi cells at the edge of wing pouch (Figure 7E). The expression of Wg is regulated by signaling pathways other than Notch at this region. For example, in response to cell apoptosis, another cellular event that involves UbcD1 (Ryoo et al., 2002), the JNK pathway is sufficient to induce Wg expression in these cells (Ryoo et al., 2004). Whether and how UbcD1 is involved in these pathways during wing development awaits further investigation.