Conserved role for the Dachshund protein with Drosophila Pax6 homolog Eyeless in insulin expression

Abstract

Members of the insulin family peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. The Drosophila genome encodes seven insulin-like peptide genes, dilp1-7, and the most prominent dilps (dilp2, dilp3, and dilp5) are expressed in brain neurosecretory cells known as "insulin-producing cells" (IPCs). Although these dilps are expressed in the same cells, the expression of each dilp is regulated independently. However, the molecular mechanisms that regulate the expression of individual dilps in the IPCs remain largely unknown. Here, we show that Dachshund (Dac), which is a highly conserved nuclear protein, is a critical transcription factor that specifically regulates dilp5 expression. Dac was strongly expressed in IPCs throughout development. dac loss-of-function analyses revealed a severely reduced dilp5 expression level in young larvae. Dac interacted physically with the Drosophila Pax6 homolog Eyeless (Ey), and these proteins synergistically promoted dilp5 expression. In addition, the mammalian homolog of Dac, Dach1/2, facilitated the promoting action of Pax6 on the expression of islet hormone genes in cultured mammalian cells. These observations indicate the conserved role of Dac/Dach in controlling insulin expression in conjunction with Ey/Pax6.

Fig. 1.

dac is required for dilp5 expression in IPCs during the early larval period. (A) Dac is expressed in brain IPCs during the larval and adult stages. The dilp2-Gal4 > UAS-CD8-GFP flies were immunostained for GFP and Dac. (Left) Reconstructed cross-sections of the z-series. (Right) Single confocal images. (Scale bars, 50 μm.) (B) Developmental changes in the dilp5 expression level in dac mutants. E. early; L, late; M, middle. (C) The percent difference in dilp5 expression levels between the control and dac mutants, calculated from B. (D) Expression of Dilp5 in dac mutant IPCs. Control or dac-null mutant larvae were stained for GFP (green), Dilp5 (red), and nuclei (blue). (Scale bars, 10 μm.)

Fig. 2.

dac and ey are cell-autonomously required for dilp5 expression in IPCs. dilp5 or dilp2 transcripts were analyzed by qRT-PCR in flies of the indicated genotypes. (A) Developmental changes in the effects of dac RNAi (in the presence of Dcr-2). (B) ey mutants reduce dilp5 expression levels during the early larval period. (C) The effect of ey RNAi in IPCs (in the absence of Dcr-2). E, early; E-M, early-mid; M-L, mid-late. All the values are means and SD (n = 3). *P < 0.05; **P < 0.01 (Student's t test).

Fig. 3.

Dac and Ey synergistically promote dilp5 expression. (A and B) The expression of Ey (A) or Dac (B) was analyzed in IPCs of the indicated genotypes. The IPCs were identified by Dilp2 staining. Single confocal images are shown. (Scale bars, 10 μm.) (C) dac interacts genetically with ey. dilp5 transcripts were analyzed by qRT-PCR in flies of the indicated genotypes. (D) dilp5 expression was analyzed by qRT-PCR in S2 cells using a dilp5 genomic reporter. The coexpression of Dac and Ey synergistically promotes dilp5 transcription. (E) Luciferase activity was analyzed in S2 cells using the dilp5 promoter that controlled the expression in IPCs (6). (F) The interaction of Ey and Dac with the dilp5 promoter was analyzed by ChIP-qPCR. (Upper) GFP-tagged proteins were precipitated in the absence or presence of Flag-tagged Ey/Dac. (Lower) Input samples. All values are means and SD (n = 3–4). *P < 0.05; **P < 0.01 (Student's t test).

Fig. 4.

Dac interacts physically with Ey and with itself through different regions. (A) Coimmunoprecipitation of full-length Dac and Ey from transfected HEK293T cells. Flag-tagged proteins were analyzed in the immunoprecipitate (IP) of the GFP proteins. WB, Western blot. (B) (Upper) Dac interacts with the PD of Ey. (Lower) The domain structure and deletion constructs of Ey are shown. Numbers refer to amino acids. (C) The DD1 domain of Dac interacts with Ey, whereas the DD2 domain of Dac facilitates self-association. (D) EMSA showing the interaction of Ey and Dac with the biotin-labeled dilp5 promoter in vitro.

Fig. 5.

Mammalian Dach1/2 binds to Pax6 and controls Insulin expression. (A) Luciferase activity was analyzed in HEK293T cells by using a rat Insulin-1 or rat Glucagon promoter. (B) Coimmunoprecipitation of Dach1 with Pax6 from HEK293T cells expressing the indicated constructs. Flag-GAPDH was cotransfected as a negative control for the immunoprecipitation assays. (C) Knockdown of Pax6 and Dach1/2 in Rin-m cells. The expression levels of Pax6, Dach1, and Dach2 were suppressed by introducing siRNA, as assessed by qRT-PCR. Insulin-1 expression levels were analyzed in Rin-m cells that had been transfected with the indicated siRNA. (D) The interactions of Pax6 and Dach1 with the Insulin-1 promoter were analyzed in Rin-m cells by ChIP-qPCR. All values are means and SD (n = 3). *P < 0.05; **P < 0.01 (Student's t test).