The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence.

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM

Negative-feedback regulation of proneural proteins controls the timing of neural precursor division

Neurogenesis requires precise control of cell specification and division. In Drosophila, the timing of cell division of the sensory organ precursor (SOP) is under strict temporal control. But how the timing of mitotic entry is determined remains poorly understood. Here, we present evidence that the timing of the G2-M transition is determined by when proneural proteins are degraded from SOPs. This process requires the E3 ubiquitin ligase complex, including the RING protein Sina and the adaptor Phyl. In phyl mutants, proneural proteins accumulate, causing delay or arrest in the G2-M transition. The G2-M defect in phyl mutants is rescued by reducing the ac and sc gene doses. Misexpression of phyl downregulates proneural protein levels in a sina-dependent manner. Phyl directly associates with proneural proteins to act as a bridge between proneural proteins and Sina. As phyl is a direct transcriptional target of Ac and Sc, our data suggest that, in addition to mediating cell cycle arrest, proneural protein initiates a negative-feedback regulation to time the mitotic entry of neural precursors

Reproduction disrupts stem cell homeostasis in testes of aged male Drosophila via an induced microenvironment.

Stem cells rely on instructive cues from their environment. Alterations in microenvironments might contribute to tissue dysfunction and disease pathogenesis. Germline stem cells (GSCs) and cyst stem cells (CySC) in Drosophila testes are normally maintained in the apical area by the testicular hub. In this study, we found that reproduction leads to accumulation of early differentiating daughters of CySCs and GSCs in the testes of aged male flies, due to hyperactivation of Jun-N-terminal kinase (JNK) signaling to maintain self-renewal gene expression in the differentiating cyst cells. JNK activity is normally required to maintain CySCs in the apical niche. A muscle sheath surrounds the Drosophila testis to maintain its long coiled structure. Importantly, reproduction triggers accumulation of the tumor necrosis factor (TNF) Eiger in the testis muscle to activate JNK signaling via the TNF receptor Grindelwald in the cyst cells. Reducing Eiger activity in the testis muscle sheath suppressed reproduction-induced differentiation defects, but had little effect on testis homeostasis of unmated males. Our results reveal that reproduction in males provokes a dramatic shift in the testicular microenvironment, which impairs tissue homeostasis and spermatogenesis in the testes

EcR regulation of BR-Z1 before and after the prepupa-to-pupa transition.

<p>(A–B′) DN-EcR-expressing clones (green) displayed BR-Z1 (red) downregulation at third-instar larval stages (A, A′) and upregulation 20–24 h APF (B, B′). (C–F) BR-Z1 (red) expression detected in wing discs of <i>en-GAL4>UAS-DN-EcR; Tub-GAL80^ts/+</i> 21–22 h APF. White arrowheads indicate anterior-posterior boundaries. BR-Z1 expression in the posterior compartment (bottom) was unaffected when animals were incubated at 18°C from larva to pupa (C), or at 18°C except during larva-to-prepupa transition (−4 to 3 h APF) at non-permissive temperatures (37°C for one hour and 29°C for six hours) (D). BR-Z1 levels were upregulated in pupae incubated at non-permissive temperatures 8–12 h APF (E) or 13–21 h APF (F). (G) A diagram showing the temperature regimen in animals shown in Figure C–F.</p

Temporal regulation of Sens expression by CSN4.

<p>(A–C) Sens (red) expression in wild-type wing discs at the PWM. The expression gradually declined from 6–8 h APF (A) to 16–18 h APF (B), and was below detection 20–22 h APF (C). (D–E′) Sens (red) levels at the PWM 6–8 h APF were identical between <i>CSN4^null</i> cells and neighboring <i>CSN4^null/+</i> cells (D, D′), and were upregulated in <i>CSN4^null</i> clones 16–18 h APF (E, E′).</p

The CSN suppresses Sens to inhibit neural differentiation of PWM bristles.

<p>(A, B) Overexpression of <i>sens</i> in <i>C96-GAL4</i>-driven <i>UAS-flag-sens</i> resulted in ectopic innervated bristles at the PWM of adult wings (A) and ectopic Hnt-positive cells at the PWM of wing discs 20–24 h APF (B). (C–F) Sens (red) expression was detected in <i>CSN4^null</i> clones (C, C′), <i>CSN5^null</i> clones (D, D′) and <i>CSN6</i> RNAi knockdown by <i>en-GAL4</i> (E, F) at the PWM 20–24 h APF. (G, H) Innervated bristles with sockets (arrowheads) appeared at the PWM of <i>neur-GAL4</i> double knockdown of <i>CSN1b</i> and <i>CSN7</i> (G), which were no longer detected when <i>sens</i> was also knocked down (H). (Insets in G, H) Enlarged figures showing single bristle and socket (arrowhead). (I, J) Double knockdown of <i>CSN1b</i> and <i>CSN7</i> by <i>neur-GAL4</i> resulted in accumulation of higher-level Hnt at the PWM, which was suppressed by <i>sens</i> knockdown (J). (K, K′) Ac (red) was not expressed in <i>CSN4^null</i> clones in wing disc 18–22 h APF. (L, L′) Ectopic Sens-positive cells were detected in PWM clones for <i>CSN4^null</i> in <i>sc^10-1</i> null mutant disc 20–24 h APF.</p

The CSN suppresses BR-Z1 expression after prepupa-to-pupa transition.

<p>(A–F′) BR-Z1 (red) expression in <i>CSN4^null</i> clones in wing discs from late third-instar larval to pupal stages 24–28 h APF. (A–C′) BR-Z1 expression was not affected in <i>CSN4^null</i> clones at late third larval instar (A, A′) and prepupal stages (B–C′). (D–F′) BR-Z1 levels in <i>CSN4^null</i> clones were upregulated at pupal stages. (G) Diagram showing the mean anti-BR-Z1 immunofluorescent intensity from 0 to 34 h APF. The dashed line represents the mean relative intensities of anti-BR-Z1/anti-H3 in wild-type <i>w¹¹¹⁸</i> discs. The solid line represents the relative mean intensity of anti-BR-Z1 in <i>CSN4^null</i> cells/neighboring <i>CSN4^null/+</i> cells. The arrow marks the starting point when the BR-Z1 expression was significantly upregulated in <i>CSN4^null</i> clones. Error bars represent the standard deviation (SD). Five <i>w¹¹¹⁸</i> wing discs were scored (N = 5) for each time point, and at least three <i>CSN4^null</i> wing discs were scored (N≥3) for each time point, except for 12–14 h APF (N = 2).</p

Association of CSN subunits and EcR in BR-Z1 repression.

<p>(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of <i>CSN2</i> RNAi driven by <i>en-GAL4</i> compared to <i>en-GAL4/+</i> control. <i>rp49</i> levels served as internal controls. (B–C′) <i>EcRE-lacZ</i> (red) expression was upregulated in <i>CSN4^null</i> (B, B′) and <i>CSN5^null</i> (C, C′) clones 20–24 h APF, but unaffected in <i>CSN5^null</i> clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for <i>CSN5^null</i> and <i>DN-EcR</i> (green) were not further elevated compared to <i>CSN5^null</i> or <i>DN-EcR</i> single clones (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004760#pgen-1004760-g003" target="_blank">Figure 3B</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004760#pgen-1004760-g006" target="_blank">6B</a>).</p