Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

Abstract

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an ‘Orange’ protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.

bHLH-O proteins are crucial for Drosophila neuroblast self-renewal and mediate Notch-induced overproliferation

“…Despite the near extinction of E(spl)m and m8 expression, no obvious effect was seen in Notch pathway mutant type I lineages in the larva: they consisted of one Dpn-positive NB, 2-5 GMCs and several neurons, as noted before (Almeida and Bray, 2005;Monastirioti et al, 2010;San-Juán and Baonza, 2011). Instead, type II clones were recovered less efficiently and showed reduced proliferation (supplementary material Fig.…”

“…Primary antibodies were rabbitanti--gal (Cappel); rat-anti-Elav 7E8A10 (DSHB); rabbit-anti-GFP (Minotech); mouse-anti-GFP (Molecular Probes); guinea-pig-anti-Hey (Monastirioti et al, 2010); rat-anti-Dpn (Boone and Doe, 2008); rabbitanti-Ase (a gift from A. Jarman, University of Edinburgh, UK); mouseanti-Pros MR1A (DSHB); and mouse-anti-CycE (a gift from H. Richardson, Peter MacCallum Cancer Centre, Melbourne, Australia).…”

“…The role of Notch signalling (and bHLH-O proteins) in postembryonic neurogenesis in Drosophila is not well understood. Null clones for the pathway seem not to affect the proliferation of most RESEARCH ARTICLE bHLH-O and NB self-renewal NBs (Almeida and Bray, 2005;Monastirioti et al, 2010), whereas a more global knockdown reduces NB numbers (Wang et al, 2006) and eliminates type II lineages (Bowman et al, 2008). Conversely, overactivation of the pathway causes significant NB hyperplasia, accompanied by loss of differentiated cells (Bowman et al, 2008;Wang et al, 2006;Weng et al, 2010), although it is not clear whether this hyperplasia arises from all NBs or only from the more sensitive type II variety.…”

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

“…Immunohistochemistry was performed as previously described (Baumgardt et al, 2009). Primary antibodies were: guinea pig anti-Hey [1:500; provided by C. Deldiakis (Monastirioti et al, 2010)]; guinea pig anti-Deadpan (1:1000; provided by J. B. Skeath); rabbit anti-phospho-histone H3 Ser10 (pH3) (1:250; Upstate/Millipore, Billerica, MA, USA); rabbit anti--gal (1:5000; ICN-Cappel, Aurora, OH, USA); rabbit anti-cleaved Caspase 3 (1:100; Cell Signaling Technology, Danvers, MA, USA); chicken anti--gal (1:1000; Abcam, Cambridge, UK); rabbit anti-GFP (1:500; Molecular Probes, Eugene, OR, USA); guinea pig antiCol (1:1000), guinea pig anti-Dimm (1:1000), chicken anti-proNplp1 (1:1000) and rabbit anti-proFMRFa (1:1000) (Baumgardt et al, 2007); rat anti-Grh (1:1000) (Karlsson et al, 2010); rabbit anti-Nab [1:1000; provided by F. Díaz-Benjumea (Terriente Felix et al, 2007)]; rabbit anti-Cas [1:250; provided by W. Odenwald (Kambadur et al, 1998)]; rat mAb anti-GsbN [1:10; provided by R. Holmgren (Buenzow and Holmgren, 1995)]; mouse mAb anti-Dac dac2-3 (1:25), mAb anti-Antp (1:10), mAb anti-Pros MR1A (1:10), mAb anti-Eya 10H6 (1:250) and mAb anti-NICD (1:10) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA); rabbit anti-Inscutable [1:1000; provided by F. Yu and W. Hongyan, Temasek Lifesciences Laboratory (TLL), National University of Singapore, Singapore)]; rat anti-Miranda (1:100; provided by F. Matsuzaki, Center for Developmental Biology, RIKEN, Kobe, Japan); and guinea pig anti-Numb (1:1000; provided by J.…”

“…In addition, we assayed Notch pathway activation in an indirect manner by analyzing the expression of well-known Notch target genes: members of the HES (Hairy, Enhancer of Split) family of bHLH transcription factors (reviewed by Bray, 2006), as well as the HES-related Notch target Hey (Monastirioti et al, 2010). Hey has recently been found to be expressed in many 'A' cells (Notch-activated sibling neurons and/or glia) and to depend upon Notch signaling for its expression (Monastirioti et al, 2010). In line with this, we found that postmitotic cells in the early part of the lineage express Hey (Fig.…”