Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously

Drosophila Rbp6 is an orthologue of vertebrate Msi-1 and Msi-2, but does not function redundantly with dMsi to regulate germline stem cell behaviour

The vertebrate RNA-binding proteins, Musashi-1 (Msi-1) and Musashi-2 (Msi-2) are expressed in multiple stem cell populations. A role for Musashi proteins in preventing stem cell differentiation has been suggested from genetic analysis of the Drosophila family member, dMsi, and both vertebrate Msi proteins function co-operatively to regulate neural stem cell behaviour. Here we have identified a second Drosophila Msi family member, Rbp6, which shares more amino acid identity with vertebrate Msi-1 and Msi-2 than dMsi. We generated an antibody that detects most Rbp6 splice isoforms and show that Rbp6 is expressed in multiple tissues throughout development. However, Rbp6 deletion mutants generated in this study are viable and fertile, and show only minor defects. We used Drosophila spermatogonial germline stem cells (GSC’s) as a model to test whether Drosophila Msi proteins function redundantly to regulate stem cell behaviour. However, like vertebrate Msi-1 and Msi-2, Rbp6 and Msi do not appear to be co-expressed in spermatogenic GSC’s and do not function co-operatively in the regulation of GSC maintenance. Thus while two Msi family members are present in Drosophila, the function of the family members have diverged

Rbp6 and Msi do not function co-operatively to regulate GSC identity, but mis-expression of Rbp6 causes loss of germ cells through apoptosis.

<p>(A) Msi expression (red) in a <i>w¹¹¹⁸</i> third instar larval testis is nuclear and found in germ cells of all stages. (*) denotes the hub. (B) Rbp6 antibody expression (red) is cytoplasmically localised to the cyst cells of the <i>w¹¹¹⁸</i> third instar larval testis. (C) A representative confocal micrograph of a <i>Rbp6¹,msi¹/Rbp6²,msi²</i> 2 day old adult double mutant testis containing germ cells (red) of varying stages, from GSCs (*) to spermatocytes. CPCs (Zfh1, green) abut the hub (FasIII, blue) and take over positions where GSC’s have been lost (arrows) as a result of the <i>msi</i> mutation. No enhancement of the <i>msi</i> mutant phenotype was observed. (D–E) <i>nos^Gal4</i>;<i>UAS-Rbp6</i> mutant testes vary from containing no germ cells (red, D) to containing only large spermatocytes in close proximity to the hub (white arrow, E). Acridine Orange labelling of <i>w¹¹¹⁸</i> testes (green, F) and <i>nosGal4</i>;<i>UAS-Rbp6</i> mutant testes (green, G) reveal more cell death in mutant tissue. Scale bars: 20 µm.</p

Percentage amino acid identity between Mouse and <i>Drosophila</i> Musashi family proteins.

<p>Percentage amino acid identity between Mouse and <i>Drosophila</i> Musashi family proteins.</p

CG32169, also known as Rbp6, is a paralogue of dMsi and an orthologue of mammalian Msi proteins.

<p>(A) Genomic representation of CG32169 (Rbp6) on chromosome 3L (modified from Flybase). (B) CG32169 (Rbp6) has six transcripts. Exons encoding for RRM1 and RRM2 are coloured green and blue respectively, and coding start sites are indicated (red arrow). The region to which the RNA probe was designed is underlined. (C) Sequence alignment of CG32169 (Rbp6) isoform A with <i>d</i>Msi, mouse Msi-1 and Msi-2 using CLUSTALW. Single-letter amino acid codes are used. Alignments among the four proteins are highlighted by black boxes for identical amino acids, and by grey boxes for similar amino acids. The two RNA recognition motifs are noted with a green or blue line. Each RRM includes two highly conserved sequences designated RNP-1 and RNP-2.</p

Intron and Exon sizes of Rbp6 splice isoform A.

<p>Intron and Exon sizes of Rbp6 splice isoform A.</p

Phylogram of Msi family sequences.

<p>Vertebrates contain two Msi paralogues, Msi2 (red box) and Msi1 (yellow box). A single orthologue found in the hemichordate (Msih_Sk) is closely aligned with the vertebrate sequences. The Rbp6 clade (blue box) of Msi proteins is insect-specific but also more closely aligned with the vertebrate proteins than the single orthologues found in nematodes. The Msi clade (orange box) is a more highly derived insect-specific group of proteins. Single representatives of the Msi family can be found in Trichoplax and Schistosoma. The RRM-containing protein, Hrb27c, was used as an outgroup. Mm: <i>Mus musculus</i> (mouse), Hs: <i>Homo sapiens</i> (human), Gg: <i>Gallus gallus</i> (chicken), Tg: <i>Taeniopygia guttata</i> (zebra finch), Dr: <i>Danio rerio</i> (zebra fish), Sk: <i>Saccoglossus kowalevskii</i> (acorn worm/hemichordate), Nv: <i>Nasonia vitripennis</i> (jewel wasp), Tc: <i>Tribolium castaneum</i> (red flour beetle), Dm: <i>Drosophila melanogaster</i> (fruit fly), Da: <i>Drosophila ananassae</i> (fruit fly), Dg: <i>Drosophila grimshawi</i> (fruit fly), Dmo: <i>Drosophila mojavensis</i> (fruit fly), Ce: <i>Caenorhabditis elegans</i> (nematode), Cb: <i>Caenorhabditis briggsiae</i> (nematode), Ta: <i>Trichoplax adhaerens</i> (placozoan), Sj: <i>Schistosoma japonicum</i> (blood fluke).</p

Expression of <i>Rbp6</i> mRNA throughout development.

<p>(A–A’’) <i>Rbp6</i> mRNA is detected in the prothoracic gland precursor cells in the embryo. <i>Rbp6</i> mRNA is also detected in the third instar prothoracic gland (B–B’), the third instar larval brain (C–C’), the ovarioles of the adult ovary (D–D’) and in the adult testis (E–E’). S-Sense, A-Antisense.</p

Rbp6 is not required for spermatogenesis.

<p>(A) Cartoon representation of the apical region of the <i>Drosophila</i> testis. GSCs (red) are located next to a group of somatic hub cells (HC, pale blue). Each GSC is surrounded by a somatic stem cell called a cyst progenitor cell (CPC, yellow). The GSC daughter, called the gonialblast (GB, pink), undergoes four rounds of mitosis to generate a cyst of 16 interconnected spermatogonial germ cells (SGCs, dark blue) (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049810#pone.0049810-Siddall2" target="_blank">[10]</a>). (B) Confocal image of a <i>w¹¹¹⁸</i> adult testis stained with a germ cell marker (Vasa, red) and a marker that accumulates on somatic cell membranes (Dcad2, green). GSCs are labelled (*) in the merged image, hub is labelled with an arrow. (C) <i>Rbp6¹/Rbp6³</i> mutant testis have normal numbers of Vasa-labelled GSCs (*) surrounding the hub (arrow). (D). A cyst of 8 interconnected germ cells (Vasa, red, white dotted line) connected by the spectrin-rich fusome (1B1 (adducin-related), green) in a <i>Rbp6¹/Rbp6³</i> mutant testis. Cysts appear to be generated normally in <i>Rbp6</i> mutant testes. (E) A <i>Rbp6¹/Rbp6³</i> mutant testis with a displaced hub (Dcad2, green) have normal numbers of GSCs (*) abutting the hub (arrow). (F) A phase contrast micrograph of onion-stage spermatids in a <i>Rbp6¹/Rbp6³</i> testis shows one haploid nucleus (white arrow) and one mitochondrial derivative (green arrow), each approximately the same diameter. Scale bars: 20 µm.</p

Expression of Rbp6 protein throughout development in <i>w¹¹¹⁸</i> and <i>Rbp6</i> mutant tissue.

<p>(A) Schematic of <i>Rbp6</i> transcripts. The peptide synthesized for the generation of the Rbp6 antibody was derived from the exonic sequences depicted in yellow, thus the possibility remains that splice isoforms A, C, D, E and F could be detected with this antibody. The deletions generated in our study span different regions of Rbp6 (depicted at the bottom of A). (B) Rbp6 expression (red) in tissue dissected from <i>w¹¹¹⁸</i> flies shows that Rbp6 is expressed posterior to the morphogenetic furrow (arrow) in third instar larval eye discs (B), in the cytoplasm of somatic cyst cells (arrow) in the adult testis (B’; hub is denoted by *), in the cytoplasm of cells in the third instar ring gland (B’’), in the non-proliferative cells of the third instar brain lobe (B’’’; dividing cells are labelled with Phosphohistone H3 (green)), and in the oocyte and nurse cells of the adult ovary (arrow; B’’’’–B’’’’’). In <i>Rbp6²</i> mutant tissue, no antibody expression was detected in the third instar ring gland (C’’) or the oocyte of the ovary (arrow; C’’’’), but was detectable in the eye disc (C), adult testis (C’) and larval brain lobe (C’’’). Rbp6 antibody expression (red) was undetectable in both Rbp6<i>¹</i> (D–D’’) and <i>Rbp6³</i> (E–E’’) mutant tissue. Scale bars: 10 µm.</p