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Cooperative control of ecdysone biosynthesis in drosophila by transcription factors séance, ouija board, and molting defective

Genetics (2018) 208(2) 605-622
DOI: 10.1534/genetics.117.300268
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Abstract

Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C2 H2 zinc finger transcription factors—Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)—cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster. Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan, ouib, or mld can be rescued by the expression of neverland, spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila. Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.

Figures

  • Figure 1 Generation of séance (CG8145) mutant alleles by the CRISPR/Cas9 system. (A) The genomic structure of séance and surrounding genes. The data are derived from the FlyBase GBrowse website (http://flybase. org/cgi-bin/gbrowse2/dmel/?Search=1; name=FBgn0037617). Numbers indicate the nucleotide positions at the 85A9 cytological location of the chromosome 3R scaffold. Boxed arrows represent gene spans and their directions. séan is shown in magenta. Four other ZAD-zinc finger protein genes are shown in cyan. (B) RNA in situ hybridization of Drosophila embryo with a séanprobe. (C) Séan expression profile based on four time points during the L3 stage, data retrieved from our previous study (Ou et al. 2016). (D) A schematic representation of the séan gene showing the sgRNA target sites. Exons are shown as black boxes, the transcription initiation site as an arrow, and sgRNA target sites as green triangles. (E) Sequences of sgRNA target sites and deleted regions of three isolated séan alleles: séan33, séan60, and sean557. The 20-bp target sequence corresponding to each target site is indicated in orange, the neighboring 59-NGG (or 59-CCN on the other strand) PAM in green, and the cleavage site of Cas9 is shown as red characters. Deleted regions are indicated by hyphens. (F) Predicted protein structures of séan alleles. Séan33 and Séan60 are composed of 33 and 60 amino acids, respectively. Séan557 is four amino acids longer than the wild-type protein, but lacks the first zinc finger domain entirely and part of the second zinc finger domain with an in-frame inappropriate amino acid stretch (dotted line). Also, see Figure S2 in File S1. CRISPR, clustered regularly interspaced short palindromic repeats; L3, third instar; PAM, protospacer adjacent motif; sgRNA, single-guide RNA; VDRC, Vienna Drosophila RNAi Center; wt, wild-type; ZAD, zinc fingerassociated domain.
  • Table 1 Term enrichment for genes with differential expression in séance-RNAi ring gland samples
  • Figure 2 Larval lethality and developmental arrest phenotype of séance mutant larvae. (A–C) The survival rate and developmental progression of control (A and B) and séan mutant animals (C), each at N = 50. (D) Comparison of body size and developmental stage between control (right and middle) and séan33/séan60 mutants (left) at 108 hr AEL. Control animals developed into L3 larvae and adults (not shown), whereas séan mutants showed arrested development as L1 larvae. (E) Phenotypic comparison of séan557 mutants, séan-RNAi, and a rescue with séan-cDNA. séan557 mutants arrest as L1 and L2, whereas PG-specific expression of séan-RNAi (phm.séan-RNAi, VDRC #100854) results in L3 lethality. Homozygous séan557 mutants were rescued by PG-specific expression of séan557 cDNA. (F) Wholelarvae ecdysteroid quantification. Control and sean557 homozygous mutants were compared during L1 (6–18 hr after egg hatch) and L2 (12–18 hr after L1/L2 molt). PG-specific séan-RNAi (VDRC #100854) L3 was compared to that of control animals at 40–44 hr L3. At least three samples were tested per genotype in each data set, and each sample was tested in triplicate. Error bars represent SE. Percentages were normalized to control levels of each data set. For L1, N = 300 for each genotype; for L2, N = 90 for each genotype; and for L3, N = 24 for each genotype. AEL, after egg laying; L1, first instar; L2, second instar; L3, third instar; PG, prothoracic gland; RNAi, RNA interference; TRIP, Transgenic RNAi Project; VDRC, Vienna Drosophila RNAi Center.
  • Figure 3 Expression analysis of Halloween genes and feeding rescue experiment in séan mutant larvae. (A) Rescue studies for séan33/séan60 larvae. Mutant animals fed 20-hydroxyecdysone (20E) and 7-dehydrocholesterol (7DC) developed into third-instar (L3) larvae, whereas animals reared on cholesterol- and ethanol-containing food (vehicle control) remained first-instar (L1) larvae. Bar, 1 mm. (B) The survival rate and developmental progression of séan33/séan60 mutant animals by oral administration of sterols and ecdysteroids (each N = 60). (C) Relative expression levels [quantitative PCR (qPCR)] of Halloween genes compared to those of controls (dotted line = 1) and various backgrounds of séanmutant or séan-RNAi (RNA interference) (phm.séan-RNAi, Vienna Drosophila RNAi Center #100854). BRGC, the brain-ring gland complex. Error bars indicate SEM. * P , 0.05 and ** P , 0.01 with Student’s t-test (black columns) and 95%C.I.s (all other columns). (D) Relative expression levels (qPCR) of Halloween genes in nvd-depleted prothoracic gland (PG). Expression levels were normalized to controls (dotted line = 1). nvd-RNAi was driven by the Feb36-GAL4 driver, and the BRGCs were dissected from late L3 larvae, collected at 44 hr after the second-instar (L2)/L3 molt. Error bars indicate 95% C.I.s. (E) Immunostaining of the PG cells from control and séan mutant L1 larvae at 36 hr after egg laying with antibodies against Phm (magenta) and Nvd (green). Bar, 25 mm. (F) Expression of nvd-cDNA in a sean557 mutant background rescued L1/L2 lethality to adulthood. L2 pupae form in rare cases using this genetic background; these animals attempt pupariation directly from L2 larvae.
  • Table 2 Rescue of séance mutants with PG-specific expression of a neverland cDNA
  • Figure 4 Transcriptional activity of Séan and Mld for the upstream element of nvd. (A) Schematic representation of the location of the element (2113 to 293) in the D. melanogaster nvd promoter region responsible for Séan-dependent transcriptional activation. Numbers indicate the distance from the transcription start site (+1, underlined) of nvd, which is based on FlyBase data (http://flybase.org/reports/FBgn0259697.html). The box indicates the 15-bp Séan-Mld response element. (B) The 15-bp element marked by the box in A exhibits a striking similarity to the Ouib response element in the spok promoter (15 bp). The bold letters and black lines indicate matching bases in the alignment between the element in the nvd promoter and the Ouib response element. (C) Luciferase reporter assay with plasmids containing the series of upstream elements of nvd. Numbers indicate the distance from the transcription start site of nvd. The white box indicates the Séan-Mld response element. The gray box represents the nvd CDS. Reporter activities of progressive deletion constructs are shown on the right (each at N = 3). The GFP expression plasmid was used as a negative control. (D) Luciferase reporter assay with plasmids containing the 9-bp transversion mutation in the 2113 to 293 region of the 300-bp upstream element of nvd (each at N = 3). The GFP expression plasmid was used as a negative control. Error bars indicate SEM. *** P , 0.005 using Student’s t-test with Bonferroni correction. CDS, coding sequence; WT, wild type.
  • Figure 5 Transcriptional activity of Ouib and Mld for the upstream element of spok. (A) Luciferase reporter assay using the expression of ouib and mld along with plasmids containing the series of upstream elements of spok. Numbers indicate the distance from the translation, but not transcription, start site (+1) of spok, as a transcription start site for spok has not been defined. The numbering style of this study is exactly the same as that of Komura-Kawa et al. (2015). The white box indicates the Ouib response element (ORE). The gray box represents the spok coding region. The inset is an enlarged view of the transcriptional activity of Ouib, Mld, and GFP for the upstream element of spok. Reporter activities of progressive deletion constructs are shown on the right (each at N = 3). The GFP expression plasmid was used as a negative control. (B) Quantitative PCR analysis to measure expression levels of endogenous spok expression in S2 cells transfected with various expression constructs, each at N = 3. (C) Luciferase reporter assay (each at N = 3) with the mld expression plasmid and luc plasmids containing the series of upstream elements of spok. (D) Luciferase reporter assay (each at N = 3) with séan, ouib, and/or mld expression plasmids, and luc plasmids containing the upstream elements of spok (the +331 to +32-bp region; 301 bp) and nvd (the +300 to +1-bp region; 300 bp). The GFP expression plasmid was used as a negative control. Error bars indicate SEM. * P , 0.01 and *** P , 0.005 using Student’s t-test with Bonferroni correction. Fluc/Rluc, firefly luciferase/Renilla luciferase.
  • Table 3 Joint PG-specific expression of spo and nvd rescues mld mutants

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Uryu, O., Ou, Q., Komura-Kawa, T., Kamiyama, T., Iga, M., Syrzycka, M., … Niwa, R. (2018). Cooperative control of ecdysone biosynthesis in drosophila by transcription factors séance, ouija board, and molting defective. Genetics, 208(2), 605–622. https://doi.org/10.1534/genetics.117.300268

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