Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fly Strains and Genetics
2.2. Immunohistochemistry
2.3. DNA Constructs and Generation of Transgenic Lines
2.4. Generation of Df(2R)Hey7552/6656 Mutant
2.5. CRISPR-Cas9 Induced Deletions in the Hey Locus
- Adults surviving the injection were mated individually with yw67c23(wt) flies.
- 2.
- A total of 10-15 F1 progeny [yw;CR//+ or yw;+//+] from each of the positive P crosses were mated individually with Df(2R)Hey7552/6656[w+]//CyOwglacZ(w−) flies. DNA from each of the mated F1s was extracted after they had generated an adequate number of offspring (F2) and was used in the PCR screen for the CR event (positive F1).
- 3.
- Selection against the Df(2R)Hey7552/6656[w+] chromosome among the progeny of the positive F1s. Individual (w−) F2 progeny (yw; CR//CyOwglacZ or yw; +//CyOwglacZ) were mated with Df(2R)Hey7552/6656[w+]//CyOwglacZ(w−) flies. DNA from each of the mated F2s was extracted after they had generated an adequate number of offspring (F3) and was used in the PCR screen for the CR event (positive F2).
- 4.
- Selection against Df(2R)Hey7552/6656[w+] phenotype among the progeny of the positive F2s and inter-se cross of F3 yw; CR//CyOwglacZ virgin female and male siblings in order to establish the CRISPR deletion mutant line.
3. Results
3.1. Identification of Two Enhancers from the Hey Locus
3.2. Both HeyUPFL and HeyIN2 Enhancers Are Active in the Embryonic CNS
3.3. HeyUPFL and HeyIN2 Enhancers Support Hey Expression in the Developing Midgut
3.4. HeyUPFL and HeyIN2 Enhancers Are Active in Larval CNS
3.5. Notch-Dependence of HeyUPFL and HeyIN2 Regulatory Elements
3.5.1. Notch Deficient Background
3.5.2. Deletion/Mutational Analysis
3.6. Functional Analysis of Hey Gene Using Genome Engineering
3.6.1. Generation of a Null Hey Allele
3.6.2. Generation of Lines with Deletions of the Su(H) Binding Sites from Hey Enhancers
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Study of the Hey Upstream Region |
---|
attp40: HeyUPFL-nbgal |
attp40: HeyUPFL-n GFP |
attp40: HeyUP2+3SH-nGFP |
attp40: HeyUPNSH-nGFP |
Study of the Hey intron 2 |
attp40: HeyIN2-nGFP |
attp2: HeyIN2-n bgal |
attp40: HeyIN2-n bgal |
attp40: Heyin2mSALL-nGFP |
attp40: Heyin2mSALL-nlacZ |
Hey mutants |
Df(2R)Hey7552/6656/CyO twistG > UASGFP |
HeyUPcr |
HeyIN2cr |
HeyUPcrIN2cr/CyO,wglacZ |
HeyUPcrIN2cr/CyO,twistG > UASGFP |
Name | Sequence (5′ to 3′) | |
---|---|---|
| ||
FOR Heyup | CAGAATTCCGGCGGCTTTGTAACCTCCACTTTGCG | [−2289 to −2264] |
REV Heyup | CTGGTACCGAGGCAGATGCAGTTGGCGGCTAG | [+356 to +380] |
heyPf-E1 | GGAATTCGTGTGTATGTGTGTGCGGC’ | [−2119 to −2101] |
UPFip | GCGGAATTCCCTCCACTTTGCGTCCAGCGATA | [−2275 to −2252] |
UPRip | GCGGGTACCTTGACTATAATGGAGCCCCGTATCAA | [−1656 to −1631] |
UPMB-F | GCGGAATTCCCTCCACTTTGCGTCCAGCGATA | [−1895 to −1871] |
CDS-F | CTGAGCTCATGGATCACAACATGCACGTCAATG | [+481 to +496] |
heyIr-K1 | GCGGTACCCTGCAACAAGATACGAGGAGG | [+2089 to +2109] |
heyIf-X1 | CGCTCTAGAATTACCAAGCCCACTTGAGC | [+1376 to +1395] |
| ||
1st Su(H)-NruI | CTAAACGCAAGCCGCTGTCGCGAACAATTGAGTAACG | |
2nd Su(H)-StuI | GTTGTCCCGAACAGGGTCGAATTGAGGCCTAATTATGATTGC | |
3rd Su(H)-SmaI | CTCGAATCTCGGGGCTCCCGGGGCTCAGTAGC | |
4th Su(H)-ApaLI | GGGTTGTTCTTTATGCTCGTCGCGTGCACCCAGCTATTTTAATTG | |
| ||
f07552GP fwd | GGTGCCACCATGAACCAAACT | upstream of WH f07552 (+) |
f06656GP REV | TCGCGCACACGATAAGTCAC | Hey exon1(−) [+308 to +328] |
Tn1 REV | TCCAAGCGGCGACTGAGATG | WH5′ plus-left primer [31] |
Tn2 FWD | CCTCGATATACAGACCGATAAAAC | WH3′ plus-right primer [31] |
| ||
INT2 5′gRNA | CTTC GAGCAAATAGAGGGTTAACT AAAC AGTTAACCCTCTATTTGCTC | |
INT2 3′gRNA | AAAC GAACTGGGATTAGACAGCTC CTTC GAGCTGTCTAATCCCAGTTC | |
UP 5′gRNA | CTTC GTGCGGGTCGTGCGCCGGAA AAAC TTCCGGCGCACGACCCGCAC | |
UP 3′gRNA | CTTC GCTTGATTTGGGGTGGGTTC AAAC GAACCCACCCCAAATCAAGC |
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Monastirioti, M.; Koltsaki, I.; Pitsidianaki, I.; Skafida, E.; Batsiotos, N.; Delidakis, C. Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities. Genes 2024, 15, 1071. https://doi.org/10.3390/genes15081071
Monastirioti M, Koltsaki I, Pitsidianaki I, Skafida E, Batsiotos N, Delidakis C. Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities. Genes. 2024; 15(8):1071. https://doi.org/10.3390/genes15081071
Chicago/Turabian StyleMonastirioti, Maria, Ioanna Koltsaki, Ioanna Pitsidianaki, Emilia Skafida, Nikolaos Batsiotos, and Christos Delidakis. 2024. "Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities" Genes 15, no. 8: 1071. https://doi.org/10.3390/genes15081071