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

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Abstract

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. © 2012 Siddall et al.

Figures

  • Figure 1. CG32169, also known as Rbp6, is a paralogue of dMsi and an orthologue of mammalian Msi proteins. (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
  • Figure 2. Phylogram of Msi family sequences. 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 insectspecific 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 RRMcontaining protein, Hrb27c, was used as an outgroup. Mm: Mus musculus (mouse), Hs: Homo sapiens (human), Gg: Gallus gallus (chicken), Tg: Taeniopygia guttata (zebra finch), Dr: Danio rerio (zebra fish), Sk: Saccoglossus kowalevskii (acorn worm/hemichordate), Nv: Nasonia vitripennis (jewel wasp), Tc: Tribolium castaneum (red flour beetle), Dm: Drosophila melanogaster (fruit fly), Da: Drosophila ananassae (fruit fly), Dg: Drosophila grimshawi (fruit fly), Dmo: Drosophila mojavensis (fruit fly), Ce: Caenorhabditis elegans (nematode), Cb: Caenorhabditis briggsiae (nematode), Ta: Trichoplax adhaerens (placozoan), Sj: Schistosoma japonicum (blood fluke). doi:10.1371/journal.pone.0049810.g002
  • Table 1. Intron and Exon sizes of Rbp6 splice isoform A.
  • Table 2. Percentage amino acid identity between Mouse and Drosophila Musashi family proteins.
  • Figure 3. Expression of Rbp6 mRNA throughout development. (A–A’’) Rbp6 mRNA is detected in the prothoracic gland precursor cells in the embryo. Rbp6 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. doi:10.1371/journal.pone.0049810.g003
  • Figure 4. Expression of Rbp6 protein throughout development in w1118 and Rbp6 mutant tissue. (A) Schematic of Rbp6 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 w1118 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 Rbp62 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 Rbp61 (D–D’’) and Rbp63 (E–E’’) mutant tissue. Scale bars: 10 mm. doi:10.1371/journal.pone.0049810.g004
  • Figure 5. Rbp6 is not widely expressed in stem cell populations at different developmental stages. 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 mm. br represents the brain lobe (A–A’). doi:10.1371/journal.pone.0049810.g005
  • Figure 6. Rbp6 is not required for spermatogenesis. (A) Cartoon representation of the apical region of the Drosophila 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 [10]). (B) Confocal image of a w1118 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) Rbp61/Rbp63 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 Rbp61/Rbp63 mutant testis. Cysts appear to be generated normally in Rbp6 mutant testes. (E) A Rbp61/Rbp63 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 Rbp61/Rbp63 testis shows one haploid nucleus (white arrow) and one mitochondrial derivative (green arrow), each approximately the same diameter. Scale bars: 20 mm. doi:10.1371/journal.pone.0049810.g006

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Siddall, N. A., Kalcina, M., Johanson, T. M., Monk, A. C., Casagranda, F., Been, R. P., … Hime, G. R. (2012). 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. PLoS ONE, 7(11). https://doi.org/10.1371/journal.pone.0049810

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