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

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

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 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

Rbp6 is not widely expressed in stem cell populations at different developmental stages.

<p>Rbp6 antibody staining (red in all panels) was not detected in the lymph gland (A–A’), the source of hematopoietic stem cells, in intestinal stem cells (which are identified by the expression of Escargot (green, B–B’), or in proliferating neuroblasts of the third instar larval brain, which are marked by either Deadpan expression (green, C) or phospho-histone H3 expression (green, C’). Rbp6 expression was detected at low levels in the germline stem cells (asterix) and possibly the somatic stem cells (arrow) of the adult ovary (D–D’). Scale bars: 20 µm. br represents the brain lobe (A–A’).</p