Drosophila to Explore Nucleolar Stress

Abstract

Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.

Keywords: Drosophila; Nopp140; P bodies; neuroblasts; nucleolar stress; ribosomopathy.

Figure 1

Decrease in eye size due to nucleolar stress generated by the expression of Nopp140-specific RNAi in the eyeless (ey) pattern. ey-GAL4/CyO flies were crossed to homozygous UAS-C4 flies at 27–28 °C for efficient knockdown of Nopp140. The Nopp140-RNAi expressing flies (ey-GAL4 > UAS-C4) with straight wings were compared to parental types and their siblings with curly wings. Data are presented as the average eye area in µm² of all scored flies. The total number of eyes and the number of individual flies scored per genotype are listed at the bottom of the graph. Statistical significance was calculated by an unpaired Student’s t-test using GraphPad Prism 9.0.1 with p values < 0.0001 indicated as (***).

Figure 2

Eye phenotypes caused by nucleolar stress resulting from RNAi depletion of nucleolar phosphoprotein of 140 kDa (Nopp140) in the ey expression pattern (ey-GAL4 > UAS-C4). Photographs representing eye size and morphology are presented by genotype: parental genotypes were (A) ey-GAL4/CyO and (B) flies homozygous for UAS-C4, the transgene encoding shRNA targeting the 5′ end of Nopp140 transcripts [29]; (C) UAS-C4/CyO sibling progeny did not express RNAi and, thus, served as controls; and (D–F) ey-GAL4/UAS-C4 siblings expressed RNAi. Eye phenotypes in ey-GAL4/UAS-C4 progeny were variable. Bar = 200 µm for all images.

Figure 3

Activation of the JNK pathway in Nopp140-/- knockout larvae. (A) The left-hand western blot shows activation of c-Jun N-terminal kinase (JNK) as detected by an antibody against phospho-JNK in KO121 Nopp140-/- larval extracts versus wild type and WH-/- larval extracts. WH- refers to the original fly line containing the pBac element (f04633) with the WH- orientation and located in the 3′ coding sequence of P5CDH1 positioned immediately downstream of Nopp140 and used to delete the Nopp140 gene (see [31]). The right-hand western blot shows accumulation of pro-apoptotic Hid compared to parental controls. JNK phosphorylates AP-1 which then activates the hid gene. Companion Coomassie stained gels are included below each Western blot as loading controls. (B) While JNK kinase increased in KO121 Nopp140-/- larvae compared to similarly aged (2nd instar) wild type (w¹¹¹⁸) controls, RT-PCRs showed, as expected, that JNK transcript levels were not significantly different between KO121 Nopp140-/- and wild type (w¹¹¹⁸). Expression of puc is a commonly used indicator of JNK kinase activity. Abundance of autophagy transcripts Atg1, Atg18.2, and Atg8a were also elevated. Data are presented as average RT-PCR products of individual transcript levels compared to that of Actin5C. Error bars represent mean ± standard deviation of at least three independent determinations and statistical significance was calculated by a paired Student’s t-test using GraphPad Prism 9.0.1 with p values < 0.05 are indicated (*) and with p values < 0.005 are indicated (**). (C) Representative agarose gels show Actin5C (as a standard control), JNK, and puc RT-PCR products. Four independent experiments were performed per gene, and PCR reactions were performed in triplicate.

Figure 4

Ultrastructural examination of midgut cells from KO121 Nopp140-/- and wild type larvae. (A) The midgut cell cytoplasm from a 3–4-day old KO121 Nopp140-/- larva lacked ribosomes but contained numerous electron dense granules each consisting of a 35–45 nm diameter core with ribosomes tethered to their periphery. A mitochondrion is on the left. (B) For comparison, the cytoplasm of a wild type midgut cell contained copious ribosomes. The nuclear envelope occupies the bottom portion of the image. (C) The cytoplasm of a 6-day old KO121 Nopp140-/- larval midgut cell showed only the cores with very few ribosomes left in the cell. Many of the core granules appeared in clusters. (D) For comparison, a wild type midgut cell. The nucleus occupies the upper right-hand portion of the image. (C,D) The insets provide high magnification views of 290 nm square areas.

Figure 5

Nucleolar stress-induced midgut granules contain Processing (P) body markers: Maternal expression at 31B (Me31B) and Decapping Protein 1 (DCP1). GFP-Me31B was a GFP protein trap fusion expressed from the endogenous Me31B gene promoter. DCP1 was detected by immuno-fluorescence. (A–D) As a positive control, GFP-Me31B and DCP1 colocalized in P bodies present in wild type larval gonads. (E–H) As a negative control wild type midgut caecum contained the two P body markers, but they were diffusely distributed throughout the cells with no apparent formation of P bodies under non-stress conditions. ((I–L, M–P), respectively) The KO121 Nopp140-/- midgut caecum and Malpighian tubules displayed colocalization of GFP-Me31B and DCP1 in the cytoplasmic granules supporting their identity as putative P bodies. Bar = 10 µm for all images.

Figure 6

Mushroom body neuroblasts (MB NBs) displayed resilience amid nucleolar stress. In larval brains (A,E,I) anti-coilin labeled coilin within nuclei, (B,F,J) DAPI labeled nuclear DNA, (C,G,K) anti-Deadpan labeled nuclei of only the neuroblasts (NBs), and (D,H,L) a half hour pulse of EdU labeled S-phase cells. (A,E) In wild type and Nopp140+/- larval brains, coilin localized to Cajal bodies (CBs) in neurons (white arrow heads) but throughout the non-nucleolar nucleoplasm in NBs as identified by Deadpan labeling (C,G). S-phase NBs and ganglionic mother cells (GMCs) throughout the brain were EdU positive in both wild type (D: w¹¹¹⁸, n = 15) and Nopp140+/- (H, n = 12) larvae. In the one J11 Nopp140-/- larval brain shown here (L, n = 9), two of the four MB NBs (white arrows) and the MB lineage GMCs (yellow arrows) were labeled with EdU. (K) Anti-Deadpan labeled all four MB NBs (white arrows) and confirmed the identity of the EdU positive MB GMCs by their proximity to the MB NBs. (I) In the J11 Nopp140-/- brain, the MB NBs (white arrows) maintained a wide expanse of coilin throughout the non-nucleolar nucleoplasm, while other NBs had comparatively less nucleoplasmic coilin but showed now discernable CBs (white arrow heads). Bar = 25 µm for all images.

Figure 7

Our current working model of nucleolar stress in Drosophila induced by the loss of ribosome biogenesis factors. Various cell stress responses are tissue specific, but all are predicted to be JNK-dependent. Autophagy was visible by TEM in the Drosophila gut epithelium upon loss of NS1 and NS2 [27,28] while apoptosis occurred in diploid imaginal wing disc cells upon loss of Nopp140 [30]. This report showed that upon complete loss of Nopp140 by gene deletion, JNK was activated leading to the up-regulations in puc, Hid, Atg1, Atg18.2, and Atg8a. We know that activated JNK regulates the formation of P bodies in mammalian cells by phosphorylating DCP1 [59]. We have yet to establish this JNK-DCP1 link in Drosophila and to link the activation of JNK to nucleolar stress via several different upstream kinases.