Inhibition of Daughterless by Extramacrochaetae mediates Notch-induced cell proliferation

Abstract

During development, the rate of cell proliferation must be constantly monitored so that an individual tissue achieves its correct size. Mutations in genes that normally promote tissue growth often result in undersized, disorganized and non-functional organs. However, mutations in genes that encode growth inhibitors can trigger the onset of tumorigenesis and cancer. The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studies that are focused on identifying the molecular mechanisms that underpin growth control. Here, we examine the mechanism by which the Notch pathway, a major contributor to growth, promotes cell proliferation in the developing eye. Current models propose that the Notch pathway directly influences cell proliferation by regulating growth-promoting genes such as four-jointed, cyclin D1 and E2f1. Here, we show that, in addition to these mechanisms, some Notch signaling is devoted to blocking the growth-suppressing activity of the bHLH DNA-binding protein Daughterless (Da). We demonstrate that Notch signaling activates the expression of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) transcription factor. Emc, in turn, then forms a biochemical complex with Da. As Emc lacks a basic DNA-binding domain, the Emc-Da heterodimer cannot bind to and regulate genomic targets. One effect of Da sequestration is to relieve the repression on growth. Here, we present data supporting our model that Notch-induced cell proliferation in the developing eye is mediated in part by the activity of Emc.

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

“…Emc protein levels seem to be less affected by N signaling than the emc enhancer traps that have been the main focus of other cell cycle studies (Bhattacharya and Baker, 2009; Spratford and Kumar, 2015). In particular, we detected little change in Emc or Da protein levels in clones mutant for Su(H) (this was reported as data not shown in a previous publication and is shown directly here)(Bhattacharya and Baker, 2011)(Figure 6A–H).…”

“…Most or all variation in Da levels in different tissues has been attributed to regulation by Emc levels (Bhattacharya and Baker, 2011). Recently, it was proposed that N regulates the eye cell cycle by upregulating emc , thereby inhibiting Da, which is proposed to inhibit the cell cycle, so that a barrier to cell cycle entry is removed in cells deficient for the Notch pathway (Spratford and Kumar, 2015). …”

“…Even though Emc protein levels seem to be less affected by N signaling than is emc RNA(Figure 6A–D), it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry (Spratford and Kumar, 2015). Contrary to this model, previous studies have reported that Da is in fact required for the SMW(Brown et al, 1996).…”

“…Genes with transcription patterns that correlate with the SMW have been identified (Firth and Baker, 2007), but only a small subset of such genes are cell cycle regulatory genes (Dimova et al, 2003; Rustici et al, 2004). Recently, Emc was suggested to be the target of Notch that regulates the SMW(Spratford and Kumar, 2015). Emc is the Drosophila Inhibitor of DNA binding (Id) protein homolog and a negative regulator of bHLH-mediated differentiation (Campuzano, 2001).…”

FLPing Genes On and Off in Drosophila

“…The over-expression clones are marked by the presence of GFP. Additional examples of loss-of-function clones, FLP-out clones, and MARCM clones that were generated with this protocol can be found within the following publications: (Anderson et al, 2012; Spratford and Kumar, 2013, 2015). …”