Drosophila ataxin 2-binding protein 1 marks an intermediate step in the molecular differentiation of female germline cysts

Abstract

In the Drosophila ovary, extrinsic signaling from the niche and intrinsic translational control machinery regulate the balance between germline stem cell maintenance and the differentiation of their daughters. However, the molecules that promote the continued stepwise development of ovarian germ cells after their exit from the niche remain largely unknown. Here, we report that the early development of germline cysts depends on the Drosophila homolog of the human ataxin 2-binding protein 1 (A2BP1) gene. Drosophila A2BP1 protein expression is first observed in the cytoplasm of 4-, 8- and 16-cell cysts, bridging the expression of the early differentiation factor Bam with late markers such as Orb, Rbp9 and Bruno encoded by arrest. The expression of A2BP1 is lost in bam, sans-fille (snf) and mei-P26 mutants, but is still present in other mutants such as rbp9 and arrest. A2BP1 alleles of varying strength produce mutant phenotypes that include germline counting defects and cystic tumors. Phenotypic analysis reveals that strong A2BP1 alleles disrupt the transition from mitosis to meiosis. These mutant cells continue to express high levels of mitotic cyclins and fail to express markers of terminal differentiation. Biochemical analysis reveals that A2BP1 isoforms bind to each other and associate with Bruno, a known translational repressor protein. These data show that A2BP1 promotes the molecular differentiation of ovarian germline cysts.

Fig. 1.

The CC00511 protein trap reports the expression of A2BP1. (A) A germarium stained for endogenous Nanos (green) and Bam (red). A cyst containing low levels of Nanos and Bam is outlined. (B) A germarium stained for Sxl (red) and Bruno (green). (C) Summary of the known expression patterns of available markers. The black line and question mark signify a hypothetical protein that bridges the expression of early and late markers during cyst differentiation. (D) A2BP1^CC00511 protein trap line stained for GFP (green), 1B1 (red) and DAPI (blue). GFP expression is detected in the cytoplasm of early multicellular germline cysts (bracket). (E) The RRM domains (red) of Drosophila A2BP1 and human A2BP1 are 90% identical. (F) Structure of the A2BP1 gene and transposon insertions in the region. Scale bars: 20 μm.

Fig. 2.

A2BP1 is expressed in a novel pattern in the germarium. (A) Germarium stained for A2BP1 (green), Bam (red) and Nanos (blue). A2BP1 is expressed in intermediate cysts that express low levels of Bam and Nanos. (B) Germarium stained for A2BP1 (green), Sxl (red) and DNA (blue). (C) Germarium stained for A2BP1 (green), Mei-P26 (red) and 1B1 (blue). (D-F) A2BP1 expression precedes high levels of expression of other differentiation markers such as Orb, Rbp9 and Bruno. (D) Germarium stained for A2BP1 (green), Orb (red) and DNA (blue). (E) Germarium stained for A2BP1 (green), Rbp9 (red) and 1B1 (blue). (F) Germarium stained for A2BP1 (green), Bruno (red) and 1B1 (blue). Scale bars: 20 μm. Arrows point to cysts that express high levels of A2BP1 and low levels of the indicated differentiation markers.

Fig. 3.

A2BP1 expression shows that different female sterile mutations block cyst differentiation at discrete steps. (A) Control, (B) bam^Δ86, (C) mei-p26^mfs1, (D) snf¹⁴⁸, (E) Rbp9²⁶⁹⁰ and (F) arrest^qb72 ovaries stained for A2BP1 (green), 1B1 (red) and DNA (blue). A2BP1 is not readily detected in bam^Δ86, mei-p26^mfs1 or snf¹⁴⁸ mutant ovaries but is expressed at normal levels in Rbp9²⁶⁹⁰ and arrest^qb72 mutant ovaries. Scale bars: 20 μm.

Fig. 4.

A2BP1 mutations disrupt normal germline cyst development. (A,E) Control, (B) A2BP1^CC00511, (C) A2BP1^f02600 and (D,F) A2BP1^f01889 hemizygous ovaries stained for VASA (green), 1B1 (red) and DNA (blue). The A2BP1^CC00511 mutation results in the formation of egg chambers with extra nurse cells. The A2BP1^f02600 allele displays both germ cell counting defects and a mildly tumorous phenotype. A2BP1^f01889 mutants exhibit a pronounced germline cystic tumor phenotype. (G) Negatively marked germline clones of the A2BP1^e03440 mutation stained for GFP (green), 1B1 (red) and DAPI (blue). Mutant germline cells (outlined with broken line) do not differentiate and form pseudo-egg chambers. Scale bars: 20 μm.

Fig. 5.

Disruption of A2BP1 blocks intermediate cyst differentiation. (A) Control and A2BP1^e03440 hemizygous germaria stained for Nanos (green) and Bam (red). (B) Control and A2BP1^e03440 hemizygous germaria stained for Sxl (red) and DNA (blue). (C) Control and A2BP1^e03440 hemizygous germaria stained for Mei-P26 (green), 1B1 (red) and DNA (blue). (D) Control and A2BP1^e03440 hemizygous germaria stained for Orb (green), Spectrin (Spc) (red) and DNA (blue). (E) Control and A2BP1^e03440 hemizygous germaria stained for Rbp9 (green), 1B1 (red) and DNA (blue). (F) Control and A2BP1^e03440 hemizygous germaria stained for Bruno (green), 1B1 (red) and DNA (blue). (G) Control and A2BP1^e03440 hemizygous germaria stained for C(3)G (green), Cyclin-A (CycA) (red) and DNA (blue). Scale bars: 20 μm.

Fig. 6.

A2BP1 interacts with Bruno but not with Rbp9. (A) Sp/+; A2BP1^f02600/A2BP1^e03440, (B) rbp9^Δ1/+; A2BP1^f02600/A2BP1^e03440and (C) arrest^qb72/+; A2BP1^f02600/A2BP1^e03440 ovaries stained for Vasa (green), 1B1 (red) and DAPI (blue). A null mutation in rbp9 does not enhance the weak A2BP1^f02600/A2BP1^e03440 phenotype. However, one copy of the arrest^qb72 mutation modifies the weak A2BP1 phenotype so that tumorous pseudo-egg chambers (arrows) are now observed. (D) Graph showing quantification of the phenotype. (E) (Top panel) Western blot of an A2BP1 immunoprecipitation probed for Bruno. (Bottom panel) Gel showing RNA depletion in the RNAse treated extracts. A2BP1 and Bruno physically interact even in the absence of RNA. (F) Western blot of immunoprecipitations using extracts from S2 cells expressing FLAG-tagged proteins and HA-tagged A2BP1. Scale bars: 20 μm.