Imp and Syp RNA-binding proteins govern decommissioning of Drosophila neural stem cells

Abstract

The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a 'decommissioning' phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex, causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than in other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together, our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages.

Keywords: Cell cycle exit; Mediator complex; Mushroom body; Neuroblast; RNA-binding protein.

Fig. 1.

Imp and Syp regulate non-MB NB decommissioning. (A-D″″) Ectopic Imp or Syp-depletion prolongs NB life into the adult. Composite confocal images of control (CTRL; A-A″″), Syp RNAi (B-B″″), Imp gain of function (GOF; C-C″″), and Syp/Imp-depleted (D-D″″) fly brains at specific developmental times, immunostained for GFP (green), Mira (magenta) and phospho-Histone H3 (pH3, blue). Transgenes were driven by dpnEE-GAL4 in the NBs of central brain. Yellow arrows indicate the MB NBs. Insets show the boxed areas at higher magnification. Scale bar: 50 µm (10 µm in inset). (E) Quantification of NB size in the anterior region of the fly brain (measured by the diameter of Mira-labeled NBs, mean±s.d., n=6 brains). (F) Syp depletion prolongs NB Imp expression. Representative confocal images of 8 h APF fly brains immunostained for Imp (magenta), GFP (green) and Dpn (blue) in control and Syp depletion conditions/experiments driven by dpnEE-Gal4. Scale bar: 10 µm. (G) Imp gain of function did not affect Syp expression. Representative confocal images of 8 h APF fly brains immunostained for Syp (magenta), GFP (green) and Dpn (blue) in control and dpnEE-Gal4-driven Imp gain-of-function conditions/experiments. In F and G, NBs with a maximum diameter at the given focal plane are circled. Scale bar: 10 µm.

Fig. 2.

Protracted Imp expression protects MB NBs from early pupal decommissioning. (A) Imp is continuously expressed in MB NBs at early pupal development. Representative confocal images of 8 h APF wild-type fly brain immunostained for GFP (green) and Imp (magenta). The green dashed line indicates the MB region (note high Imp levels); MB NBs (circled with blue dashed line) show protracted Imp expression. The yellow dashed line circles non-MB NBs (posterior NB, pNB) at the same focal plane, which are negative for Imp expression. Scale bar: 10 µm. (B) Quantification of the grayscale value for Imp immunostaining in the MB NBs and pNBs in 8 h APF wild-type flies. **P<0.01 (Student's t-test) (mean±s.d., n=6 brains). AU, arbitrary fluorescent intensity units. (C-E′) Imp depletion prematurely ended MB neurogenesis. Representative confocal images of 48 h APF fly brains immunostained for GFP (green), Dpn (magenta) and Pros (blue) in control (CTRL; C,C′), Imp depletion (D,D′) and Syp gain of function (GOF; E,E′) experiments/conditions. Transgenes were driven by GAL4-OK107; diameter of MB neuroblast at 48 h APF (mean±s.d., n=6 brains) are indicated at the bottom right of each panel. Scale bar: 20 µm.

Fig. 3.

Induction of transgenic Imp at late larval stage prolongs NB life. (A) Scheme for induction time course of transgenic Imp. Heat shock inactivated the temperature-sensitive GAL80 at specific developmental times, allowing dpnEE-GAL4 to drive Imp expression. Flies were continuously incubated at 29°C after heat shock and fly brains were processed 38 h after pupal formation (38 h APF). (B-H) Late larval induction of Imp prolongs NB life. Composite confocal images of fly brains, immunostained for GFP (green) and Dpn (magenta). Experimental conditions were control (CTRL; B), targeted transgenic Imp induction at 8 h (C), 12 h (D), 15 h (E), 18 h (F) and 24 h (G) before pupal formation (BPF), and flies lacking GAL80[ts], which allowed continuous Imp expression (H). Yellow arrows indicate the MB NBs. The number of NBs per brain lobe (mean±s.d., n=6) is indicated at the bottom right of each panel. Scale bar: 50 µm.

Fig. 4.

Imp interacts with mediator complex. (A-D′) Imp promotes Med12 expression in NBs. Representative confocal images of 4 h APF fly brains immunostained for GFP (green), Med12 (magenta) and Dpn (blue) in control (CTRL; A,A′), Syp RNAi (B,B′), Syp/Imp depletion (C,C′) and Imp gain of function (D,D′) driven by dpnEE-Gal4. Green dashed lines circle NBs with a maximum diameter at the given focal plane. Scale bar: 20 µm. (E) Quantification of the relative intensity of Med12 immunostaining in NBs of the anterior region of fly brains as shown in A-D (mean±s.d., number of sampled NBs is indicated for each experiments/condition from 6 brains). *P<0.05, **P<0.01 (Student's t-test). (F-G′) Imp promotes Med12 expression in MB NBs. Representative confocal images of 4 h APF fly brains immunostained for GFP (green), Med12 (magenta) and Dpn (blue) in control (CTRL; E) and after Imp depletion (F) driven by Gal4-OK107. Arrows indicate the MB NBs. Scale bar: 20 µm. (H) Quantification of Med12 immunostaining in the MB NBs (relative to optic lobe) as shown in F and G. *P<0.05 (Student's t-test) (mean±s.d., number of sampled NBs is indicated for each experiment/condition from 6 brains). (I) Med6, Med27 and Med31 mRNAs could be effectively pulled down with Imp. RT-qPCR (mean±s.e.m., n=3) of RNA immunoprecipitation from Imp::GFP and Oregon-R larval brain lysates shows the percent inputs of candidate mediator complex components that were recovered by anti-GFP immunoprecipitation. Note that Med12 was not significantly enriched. *P<0.05, **P<0.01 (Student's t-test comparing the recovery of each transcript to that of the non-binding rp49).

Fig. 5.

The mediator complex functions downstream of Imp to induce NB shrinkage. (A-B″″) Med12 depletion blocks the inhibition of NB shrinkage caused by persistent Imp expression. Composite confocal images of Syp and Med12 joint knockdown (A) and Imp gain of function combined with Med12 knockdown (B) driven by dpnEE-Gal4. Brains were dissected at specific developmental ages and immunostained for GFP (green), Mira (magenta) and phospho-Histone H3 (pH3, blue). Insets show the boxed areas at higher magnification. Scale bar: 50 µm (10 µm in inset). (C-E) The mediator complex acts downstream of Imp to regulate MB NB shrinkage. Representative confocal images of 48 h APF fly brains immunostained for GFP (green), Dpn (magenta) and Pros (blue) in Med12 depleted (C) and combined Imp-depleted with Med12 gain-of-function conditions (D) driven by Gal4-OK107. Diameter of MB NBs at 48 h APF (mean±s.d., n=6 brains) is indicated at the top right of each panel. Scale bar: 20 µm. (E) Mean MB NB diameter (±s.d., n=6 brains) of flies at different developmental stages with transgenic manipulations driven by GAL4-OK107. *P<0.05, **P<0.01 (Student's t-test).

Fig. 6.

Syp ensures NB exit by promoting Pros accumulation. (A-D′) Syp is required for the accumulation of Pros. Representative confocal images of 8 h APF control (CTRL; A), and 24 h APF CTRL (B), Syp RNAi (C) and Syp/Imp gain-of-function (D) fly brains driven by dpnEE-Gal4 and immunostained for GFP (green), Mira (magenta) and Pros (blue). The NBs are outlined with dashed lines. Scale bar: 10 µm. (E) Quantification of the grayscale value for NB Pros immunostaining as shown in A-D. **P<0.01 (Student's t-test) (mean±s.d., n=6 brains). (F,G) Pros induction terminated the long-lasting Syp-depleted neuroblasts. Composite confocal images of fly brains in which neuroblasts were depleted of Syp without (F) or with (G) Pros induction at early pupal development. Brains were immunostained for Dpn (green) and Pros (magenta). Scale bar: 50 µm. (H) Quantification of the number of neuroblasts (Dpn-positive cells) in fly brains (mean±s.d., n=6) as shown in F and G. **P<0.01 (Student's t-test). (I-I″) Ectopic Imp is dominant to Syp for NB decommissioning. Composite confocal images of Syp/Imp gain-of-function fly brains at specific developmental ages. Brains were immunostained for GFP (green), Mira (magenta) and phospho-Histone H3 (pH3, blue). Insets show the boxed areas at higher magnification. Scale bar: 50 µm (10 µm in inset).

Fig. 7.

Regulation of NB decommissioning by Imp/Syp RNA-binding proteins. Reduction of Imp prior to the ecdysone signaling during metamorphosis (indicated by the unfilled arrow) is permissive for the ecdysone receptor (EcR)- and mediator complex-mediated metabolic switch that triggers non-MB NB decommissioning. Syp then promotes Pros accumulation to terminate NB cycling by terminal differentiation.