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A Homeobox Transcription Factor Scarecrow (SCRO) Negatively Regulates Pdf Neuropeptide Expression through Binding an Identified cis-Acting Element in Drosophila melanogaster

  • Sudershana NairEmail author
  • Jae Hoon Bahn
  • Gyunghee Lee
  • Siuk Yoo
  • Jae H. ParkEmail author
Article

Abstract

In Drosophila, transcriptional feedback loops contribute to intracellular timekeeping mechanisms responsible for daily rhythms. Pigment-dispersing factor (PDF) is the major neuropeptide produced by latero-ventral neurons (LNvs) that function as a central pacemaker for circadian locomotor activity rhythms. PDF synchronizes other clock neurons thereby playing an essential role in the maintenance and coordination of circadian locomotor rhythms. However, the underlying molecular mechanism of the LNvs-specific Pdf expression is not well understood. Here, using Pdf promoter-bashing experiment, we identified a cis-acting Pdf regulatory element (PRE) that is sufficient for driving Pdf expression in the LNvs. We have also identified a homeobox transcription factor, scarecrow (SCRO), as a direct binding factor to PRE. Furthermore, transgenic expression of scro in the clock neurons abolished Pdf expression and circadian locomotor activity rhythms, and such repressive function requires DNA-binding homeodomain, but none of the other conserved domains. scro is predominantly expressed in the optic lobe and various clusters of cells in other areas of the central nervous system. A homozygous scro-null mutant generated by CRIPSR is lethal during embryonic and early larval development, suggesting that scro plays a vital role during early development.

Keywords

Clock Circadian rhythms Pigment-dispersing factor Neuropeptide Nkx2.1 Scarecrow 

Notes

Acknowledgments

We thank Michael Rosbash (Brandeis Univ.) and Amita Sehgal (U. Penn) for the transgenic stocks.

Funding Information

This work is supported in part by an NIH grant (MH66197) and Univ. of Tennessee Hunsicker Research Incentive award.

Supplementary material

12035_2020_1874_MOESM1_ESM.pdf (2.8 mb)
ESM 1 Fig. S1 Alignment of PRE-like sequences from different Drosophila species. Identical nucleotides are indicated by asterisks. The 13-bp tandem repeats are indicated in red. (Abbr. Dm, Drosophila melanogaster; Ds, D. simulans; De, D. erecta; Da, D. ananassae; Dp, D. pseudoobscura; Dv, D. virilis). Fig. S2 CRISPR/Cas9-induced mutation of scro. A schematic illustrating a deletion allele of scro (scroΔ1) generated by two guide RNAs. Vertical arrows in the exon-2 (E2) and exon-3 (E3) indicate gRNA target sites, and horizontal arrows under each exon are PCR primers used for the screening of mutants. Sequencing result shows a frame-shifted open reading frame (residues indicated by red capitals). Premature stop codon (TAA) is indicated by *. Fig. S3 Replica images of Fig. 2e and 2f. Yellow arrowheads indicate PDF neurons detected by co-expression of GFP and nRFP. Open arrowheads denote random nRFP signals that are not associated with Pdf-Gal4 activity. Fig. S4 Intact PDF neurons in response to scro expression. Pdf-Gal4, UAS-lacZ double transgenic line was crossed to a UAS-scro, and F1 larval CNS was stained with anti-β-gal and anti-PDF as indicated. Fig. S5 PDF-immunoreactivity in scroΔ1 mutant brain of first instar larvae. (left) scroΔ1/+. Four LNs were detected in most samples (n=7). (right) scroΔ1/Δ1. One-to-four LNs were detected (n=6). (PDF 2914 kb)

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Copyright information

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Authors and Affiliations

  1. 1.Department of Biochemistry and Cellular and Molecular BiologyUniversity of TennesseeKnoxvilleUSA
  2. 2.Department of GeneticsAlbert Einstein College of MedicineNew YorkUSA
  3. 3.Department of Integrative Biology and PhysiologyUniversity of California Los AngelesLos AngelesUSA
  4. 4.Department of Life SciencesYeungnam UniversityGyeongsanRepublic of Korea

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