Molecular and functional analysis of Drosophila single-minded larval central brain expression

Abstract

Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. In this paper, we demonstrate that sim is expressed in 3 clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in 3 clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which we term PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim+ neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5’-flanking, intronic, and 3’-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim+ brain cells, including all sim+ neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim+ brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim+ brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim+ brain neurons. Sequence analysis of existing sim mutant alleles identified 3 likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate Sim that have an early role in neuronal migration and a later role in axonogenesis.

Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression

“…Expression is maintained in the midline primordium from stages 9–11 by two distinct autoregulatory enhancers, one adjacent to Pe (sim-3.7 , which also contains the above-mentioned initiation element) (Nambu et al, 1991; Wharton et al, 1994) and the other adjacent to the sim late promoter (Pl; sim-1.0) (Supplementary Fig. S1A; (Freer et al, 2011; Muralidhar et al, 1993). These enhancers together provide uniform expression in all midline cells from stages 5–11, as indicated by GFP protein accumulation throughout the midline primordium (Fig.…”

“…Drosophila strains used include: sim3.7-Gal4 (Xiao et al, 1996), UAS-tau-GFP (Wheeler et al, 2006), sim-3.7-Gal4 UAS-tau-GFP (Wheeler et al, 2006), sim-1.0-Gal4 (Freer et al, 2011), UAS-mCD8-GFP(LL6) (Lee and Luo, 1999), sim H9 (Hilliker et al, 1980), UAS-sim (Xiao et al, 1996 ), prd-Gal4 (BL 1947), Dl 3 (Uemura et al, 1989), UAS-Su(H).VP16 (Kidd et al, 1998), 12xSu(H)bs-lacZ (Go et al, 1998), rho-1.0-GFP (rho PE-A-GFP; kindly provided by J. Posakony), UAS-aop.Act (BL 5789), UAS-pnt.P1 (BL 869), TM3-ftz-lacZ (BL 3218), and nanos-phiC31;;attP2 (68A1-B2) (Groth et al, 2004). …”

Sim1a and Arnt2 contribute to hypothalamo-spinal axon guidance by regulating Robo2 activity via a Robo3-dependent mechanism

“…Downstream targets of Sim1 and Arnt2 include transcriptional regulators, as well as cell migration-related genes (Liu et al, 2003), which may contribute to HTS axon guidance independent of Robo3. In Drosophila, abrogation of sim leads to an axonal fasciculation phenotype (Freer et al, 2011), which differs from the mouse Sim1 or zebrafish sim1a loss-of-function defects. Thus, Sim1 may control different aspects of axonal pathfinding by regulating different axon guidance factors.…”

Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers

“…There are at least four distinct midline primordium enhancers at the sim locus, as indicated by in vivo-tested fragments (1.0, 0.7, R15F08, 1.6; Fig. 2H) (Freer et al, 2011;Manning et al, 2012;Muralidhar et al, 1993;Sandmann et al, 2007;Wharton et al, 1994). The MF data reveal a total of eight strong MF peaks (a, c-i).…”

“…The Gal4 lines simC2.3, simD2.1 and simE2.3 were described previously (Freer et al, 2011). FlyLight Gal4 enhancer lines were obtained from the Bloomington Drosophila Stock Center, and embryos were subjected to fluorescent ISH with a Gal4 probe or crossed to UAS-mCD8::GFP and immunostained with anti-GFP as previously described (Pearson and Crews, 2014;Wheeler et al, 2006).…”