1. Stem Cells and Regenerative Medicine
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Sequential activation of transcriptional repressors promotes progenitor commitment by silencing stem cell identity genes

  1. Noemi Rives-Quinto
  2. Hideyuki Komori
  3. Cyrina M Ostgaard
  4. Derek H Janssens
  5. Shu Kondo
  6. Qi Dai
  7. Adrian W Moore
  8. Cheng-Yu Lee  Is a corresponding author
  1. Life Sciences Institute, University of Michigan, United States
  2. Department of Cell and Developmental Biology, University of Michigan Medical School, United States
  3. Invertebrate Genetics Laboratory, National Institute of Genetics, Japan
  4. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden
  5. Riken Center for Brain Science, Japan
  6. Division of Genetic Medicine, Department of Internal Medicine and Comprehensive Cancer Center, University of Michigan Medical School, United States
Research Article
Cite this article as: eLife 2020;9:e56187 doi: 10.7554/eLife.56187
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Identification of candidate regulators of type II neuroblast functional identity.

(A) A diagram of the type II neuroblast lineage showing the expression patterns of genes and Gal4 drivers used throughout this study. (B) Gene transcription profiles of brat-null brains transiently overexpressing Insb driven by a type II neuroblast Gal4. Supernumerary type II neuroblasts in brat-null brains transiently overexpressing Insb displayed patterns of gene transcription that are indicative of immature INPs undergoing INP commitment in wild-type brains. (C) A strategy to induce synchronized INP commitment in supernumerary type II neuroblasts in brat-null brains. Larvae carrying a UAS-insb transgene and a type II neuroblast Gal4 were collected and aged at 25°C. One third of larvae were shifted to 33°C at 126 or 135 hr after hatching to induce high levels of transient Insb expression, and the last one-third remained at 25°C serving as the source enriched for type II neuroblast-specific transcripts (time 0). (D–F) Images of brat-null brains transiently overexpressing Insb driven by a type II neuroblast Gal4 for 0, 9, or 18 hr. Transient overexpression of Insb first induced Erm and then Ase expression in supernumerary type II neuroblasts in brat-null brains. (G) Volcano plots showing fold-change of gene expression in brat-null brains transiently overexpressing Insb for 9 or 18 hr. (H) Tll expression pattern in the type II neuroblast lineage. The tll::GFP(Bac) transgene revealed the expression of endogenous Tll in type II neuroblasts but not in immature INPs and INPs. The following labeling applies to all images in this figure: yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP. Scale bar, 10 μm.

Figure 1—figure supplement 1
Time-course analysis of transient Insb overexpression in brat-null brains.

(A) Quantification of total type II neuroblasts (top) or INPs (bottom) per brat-null brain lobe that transiently overexpressed Insb driven by a type II neuroblast Gal4. Insb overexpression led to progressive decrease in supernumerary type II neuroblasts but increase in INPs in brat-null brains. (B) Images of Erm::V5 expression in a type II neuroblast lineage. Erm::V5 is strongly expressed in immature INPs and weakly in INPs. (C–E) Images of Tll::GFP expression in the larval central nervous system. Tll::GFP is expressed at high levels in type II neuroblasts, and much lower levels in type I neuroblasts in the ventral brain region and in the ventral nerve cord. The following labeling is applicable to all panels of images in this figure: white dashed lines separates the optic lobe from the brain; yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP; orange arrow: type I neuroblast. Bar graphs are represented as mean ± standard deviation.

Figure 1—figure supplement 1—source data 1

Quantification of total type II neuroblasts or INPs per brat-null brain lobe that transiently overexpressed Insb.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig1-figsupp1-data1-v2.xlsx
tll is necessary and sufficient for type II neuroblast functional identity.

(A) A diagram showing the expression patterns of genes in the type I and II neuroblast lineages. (B) Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-tllRNAi transgene driven by a type II neuroblast Gal4. Knocking-down tll function reduced the number of type II neuroblasts from 8 to 4 per brain lobe. (C–D) Images of brains that overexpressed a UAS-tllRNAi transgene driven by a type II neuroblast Gal4. Knocking-down tll function led to premature differentiation in type II neuroblast as indicated by reduced cell diameter and ectopic Ase expression but did not affect the maintenance of type I neuroblasts. (E) Quantification of total type II neuroblasts per brain that overexpressed a UAS-tll transgene driven by an INP Gal4. tll overexpression in INPs led to supernumerary type II neuroblast formation. (F–G) Images of brains that overexpressed a UAS-tll transgene driven by an INP Gal4. (H–I) Images of Sp1::GFP(Bac) brains. Sp1::GFP is detected in most cells in all type II neuroblast lineages that are exclusively located in the dorsal brain region, and is detected in few neurons in type I neuroblast lineages in the ventral brain region. (J) Images of the ventral region of Sp1::GFP(Bac) brains that overexpressed a UAS-tll transgene driven by a pan-neuroblast Gal4 (Wor-Gal4). Tll overexpression transforms type I neuroblasts (Ase+Sp1::GFP-) in the ventral brain region into type II neuroblasts (Ase-Sp1::GFP+). (K) Quantification of total ventral type I neuroblasts per brain lobe that overexpressed a UAS-tll transgene driven by a pan-neuroblast Gal4. Tll overexpression transforms 66% of type I neuroblasts in the ventral brain region into type II neuroblasts. (L–M) Images of the thoracic segments on the ventral nerve cord of Sp1::GFP(Bac) larvae that overexpressed a UAS-tll transgene driven by a pan-neuroblast Gal4 (Wor-Gal4). Tll overexpression transforms type I neuroblasts in the thoracic segments into type II neuroblasts. (N–O) Images of dorsal and ventral regions of erm::V5 brains. Erm::V5 is detected in immature INPs in type II neuroblast lineages but is undetectable in type I neuroblast lineages in the ventral region of larval brain. (P) Images of erm::V5 brains that overexpressed a UAS-tll transgene driven by a pan-neuroblast Gal4. Tll overexpression induced type I neuroblasts in the ventral-medial region of the brain to generate supernumerary type II neuroblasts interspersed with Erm::V5+ immature INPs. The following labeling applies to all images in this figure: white dashed line separates the optic lobe from the brain; yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP; orange arrow: type I neuroblast. Br: brain. NC: nerve cord. Scale bar, 10 μm. Bar graphs are represented as mean ± standard deviation. p-values: ***<0.005.

Figure 2—source data 1

Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-tllRNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig2-data1-v2.xlsx
Figure 2—source data 2

Quantification of total type II neuroblasts per brain that overexpressed a UAS-tll transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig2-data2-v2.xlsx
Figure 2—source data 3

Quantification of total ventral type I neuroblasts per brain lobe that overexpressed a UAS-tll transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig2-data3-v2.xlsx
Figure 3 with 1 supplement
Ham is a novel regulator of INP commitment.

(A) Quantification of total type II neuroblasts per ermhypo brain lobe that overexpressed a UAS-RNAi transgene driven by a type II neuroblast Gal4. Knocking-down ham function consistently enhanced the supernumerary type II neuroblast phenotype in ermhypo brains. (B) A diagram summarizing the lesions in ham alleles. The molecular lesions in hamSk1 and hamSK4 alleles were not independently verified. (C–D) Ham expression in wild-type or hamSK1 homozygous brains. Ham was detected in immature INPs and INPs in wild-type brains, but was undetectable in hamSK1 homozygous brains. (E) Quantification of total type II neuroblasts per ham-mutant brain lobe. hamSK1 homozygous but not ham1 homozygous brains displayed a supernumerary type II neuroblast phenotype. (F–H) Images of ham single mutant or ham,erm double mutant brains. The heterozygosity of erm alone had no effect on type II neuroblasts, but enhanced the supernumerary type II neuroblast phenotype in hamSK1 homozygous brains. (I) Quantification of total type II neuroblasts per brain lobe of the indicated genotypes. erm and ham function synergistically to prevent supernumerary type II neuroblast formation. (J) Quantification of total type II neuroblasts per erm,ham double heterozygous brain lobe that overexpressed a UAS-ham transgene driven by an INP Gal4. Overexpressing Ham in INPs rescued the supernumerary type II neuroblast phenotype in erm,ham double heterozygous brains. The following labeling applies to all images in this figure: white dashed line separates the optic lobe from the brain; yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast. Scale bar, 10 μm. Bar graphs are represented as mean ± standard deviation. p-values: ***<0.005. ns: not significant.

Figure 3—source data 1

Quantification of total type II neuroblasts per ermhypo brain lobe that overexpressed a UAS-RNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig3-data1-v2.xlsx
Figure 3—source data 2

Quantification of total type II neuroblasts per ham-mutant brain lobe.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig3-data2-v2.xlsx
Figure 3—source data 3

Quantification of total type II neuroblasts per brain lobe of the indicated genotypes.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig3-data3-v2.xlsx
Figure 3—source data 4

Quantification of total type II neuroblasts per erm,ham double heterozygous brain lobe that overexpressed a UAS-ham transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig3-data4-v2.xlsx
Figure 3—figure supplement 1
Ham functions synergistically with Erm to suppress supernumerary type II neuroblast formation.

(A) Quantification of total type II neuroblasts per brain lobe of the indicated genotypes. erm,ham double heterozygous brains displayed the supernumerary type II neuroblast phenotype. (B) Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-hamRNAi transgene driven by a pan-neuroblast Gal4. Knock-down of ham function in type II neuroblasts led to a mild supernumerary neuroblast phenotype. Bar graphs are represented as mean ± standard deviation. p-values: **<0.05, ***<0.005. ns: not significant.

Figure 3—figure supplement 1—source data 1

Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-hamRNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig3-figsupp1-data1-v2.xlsx
Ham suppresses INP reversion by repressing gene transcription.

(A–B) Images of wild-type or hamSK1 homozygous type II neuroblast mosaic clones. Supernumerary neuroblasts (−15 μm) in hamSK1 homozygous clones were always located far from the parental neuroblast (0 μm), and were surrounded by Ase+ cells. (C) Quantification of total neuroblasts per ham1 or hamSK1 homozygous type II neuroblast clone. hamSK1 homozygous clones contained supernumerary neuroblasts but ham1 homozygous clones did not. (D) A diagram summarizing UAS-ham transgenes used in this study. (E) Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-ham transgene driven by a type II neuroblast Gal4. Overexpressing full-length Ham or HamΔC-ZF led to premature differentiation in type II neuroblasts. (F) Quantification of total type II neuroblasts per hamSK1 homozygous brain lobe that overexpressed various UAS-ham transgenes driven by an INP Gal4. Overexpressed full-length Ham, HamΔC-ZF or ERD::HamN-ZF rescued the supernumerary neuroblast phenotype in hamSK1 homozygous brains. (G) Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-VP16::hamN-ZF transgene driven by a type II neuroblast Gal4. Overexpressing VP16::HamN-ZF led to supernumerary type II neuroblast formation. The following labeling applies to all images in this figure: yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast. Scale bar, 10 μm. Bar graphs are represented as mean ± standard deviation. Ppvalues: ***<0.005. ns: not significant.

Figure 4—source data 1

Quantification of total neuroblasts per ham1 or hamSK1 homozygous type II neuroblast clone.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig4-data1-v2.xlsx
Figure 4—source data 2

Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-ham transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig4-data2-v2.xlsx
Figure 4—source data 3

Quantification of total type II neuroblasts per hamSK1 homozygous brain lobe that overexpressed various UAS-ham transgenes.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig4-data3-v2.xlsx
Figure 4—source data 4

Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-VP16::hamN-ZF transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig4-data4-v2.xlsx
Erm- and Ham-mediated repression renders tll refractory for activation by Notch.

(A) A diagram depicting our hypothesis that ectopic activation of tll in INPs leads to supernumerary type II neuroblasts in erm,ham double heterozygous brains. (B) Quantification of total type II neuroblasts per brain lobe that was erm,ham double heterozygous or erm,ham,tll triple heterozygous. Heterozygosity of tll suppressed the supernumerary neuroblast phenotype in erm,ham double heterozygous brains. (C–D) Images of erm,ham double heterozygous brains that carries a tll::GFP(BAC) transgene. Tll::GFP becomes ectopically expressed in INPs and supernumerary type II neuroblasts (*) in erm,ham double heterozygous brains. (E) Quantification of total type II neuroblasts erm,ham double heterozygous brain lobe that carried one copy of the tll::GFP(BAC) transgene. One copy of the tll::GFP(BAC) transgene was sufficient to enhance the supernumerary type II neuroblast phenotype in erm,ham double heterozygous brains. (F–H) Images of type II neuroblasts that mis-expressed a UAS-erm or UAS-ham transgene driven by a pan-neuroblast Gal4. Erm or Ham mis-expression drastically reduced Tll::GFP expression in type II neuroblasts. (I) Quantification of Tll::GFP expression relative to Dpn expression in type II neuroblasts that mis-expressed a UAS-erm or UAS-ham transgene driven by a pan-neuroblast Gal4. Erm mis-expression reduced Tll::GFP expression in type II neuroblasts before the onset of Ase expression, whereas Ham mis-expression reduced Tll::GFP expression in type II neuroblasts after the onset of Ase expression. (J) Quantification of total type II neuroblasts per wild-type or hamSK1 homozygous brain lobe that overexpressed a UAS-NRNAi transgene driven by an INP Gal4. Knocking-down Notch function in INPs suppressed the supernumerary type II neuroblast phenotype in hamSK1 homozygous brains. (K) Quantification of total type II neuroblasts per erm or ham heterozygous brain lobe that overexpressed a UAS-Nintra transgene driven by an INP Gal4. Overexpressing Nintra in INPs more efficiently induced supernumerary neuroblasts in erm,ham double heterozygous brains than in erm or ham heterozygous brains. The following labeling is applicable to all panels of images in this figure: yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP; *: supernumerary type II neuroblasts. Scale bar, 10 μm. Bar graphs are represented as mean ± standard deviation. p-values: **<0.05, ***<0.005. ns: not significant.

Figure 5—source data 1

Quantification of total type II neuroblasts per brain lobe that was erm,ham double heterozygous or erm,ham,tll triple heterozygous.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig5-data1-v2.xlsx
Figure 5—source data 2

Quantification of total type II neuroblasts erm,ham double heterozygous brain lobe that carried one copy of the tll::GFP(BAC) transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig5-data2-v2.xlsx
Figure 5—source data 3

Quantification of Tll::GFP expression relative to Dpn expression in type II neuroblasts that mis-expressed a UAS-erm or UAS-ham transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig5-data3-v2.xlsx
Figure 5—source data 4

Quantification of total type II neuroblasts per wild-type or hamSK1 homozygous brain lobe that overexpressed a UAS-NRNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig5-data4-v2.xlsx
Figure 5—source data 5

Quantification of total type II neuroblasts per erm or ham heterozygous brain lobe that overexpressed a UAS-Nintra transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig5-data5-v2.xlsx
Figure 6 with 1 supplement
Erm and Ham function through Hdac3 to prevent INPs from reverting to type II neuroblasts.

(A) Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-ham transgene driven by an INP Gal4. Overexpressing full-length Ham or HamΔC-ZF in INPs suppressed the supernumerary type II neuroblast phenotype in erm-null brains. (B) Quantification of total type II neuroblasts per ham heterozygous brain lobe that overexpressed a UAS-RNAi transgene driven by a type II neuroblast Gal4. Reducing activity of the indicated chromatin complex did not increase INP reversion into supernumerary type II neuroblasts in ham heterozygous brains. (C) Quantification of total type II neuroblasts per ham heterozygous brain lobe that overexpressed a UAS transgene driven by a type II neuroblast Gal4. Reducing Hdac3 activity increased INP reversion into supernumerary type II neuroblasts in ham heterozygous brains. (D) Quantification of total type II neuroblasts per erm heterozygous brain lobe that overexpressed a UAS-hdac3RNAi transgene driven by a type II neuroblast Gal4. Reducing Hdac3 activity increased INP reversion into supernumerary type II neuroblasts in erm heterozygous brains. (E–F) Images of tll::GFP(Bac) brains that overexpressed a UAS-hdac3RNAi transgene driven by a pan-neuroblast Gal4. Reducing Hdac3 activity in type II neuroblasts led to ectopic Tll::GFP expression in immature INPs and INPs. (G) Quantification of Tll::GFP expression relative to Dpn expression in INPs derived from type II neuroblasts that overexpressed a UAS-hdac3RNAi transgene. Reducing Hdac3 activity in type II neuroblasts led to ectopic Tll::GFP expression in INPs. The following labeling is applicable to all panels of images in this figure: yellow dashed line encircles a type II neuroblast lineage; white arrow: type II neuroblast; white arrowhead: Ase- immature INP; yellow arrow: Ase+ immature INP; yellow arrowhead: INP. Bar graphs are represented as mean ± standard deviation. p-values: **<0.05, ***<0.005. ns: not significant.

Figure 6—source data 1

Quantification of total type II neuroblasts per brain lobe that overexpressed a UAS-ham transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig6-data1-v2.xlsx
Figure 6—source data 2

Quantification of total type II neuroblasts per ham heterozygous brain lobe that overexpressed various UAS transgenes.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig6-data2-v2.xlsx
Figure 6—source data 3

Quantification of total type II neuroblasts per erm heterozygous brain lobe that overexpressed a UAS-hdac3RNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig6-data3-v2.xlsx
Figure 6—source data 4

Quantification of Tll::GFP expression relative to Dpn expression in INPs derived from type II neuroblasts that overexpressed a UAS-hdac3RNAi transgene.

https://cdn.elifesciences.org/articles/56187/elife-56187-fig6-data4-v2.xlsx
Figure 6—figure supplement 1
Ham overexpression in Ase+ immature INPs suppressed INP reversion in erm-null brains.

(A–B) Images of erm-null brains that overexpressed a UAS-ham transgene driven by an INP Gal4. Ham overexpression in INPs suppressed the supernumerary type II neuroblast phenotype in erm-null brains. Scale bar, 10 μm.

A proposed model for the regulation of type II neuroblast functionality.

We propose that Erm and Ham recruits Hdac3 to silence tll during INP commitment, preventing re-activation of Notch signaling in INPs from triggering Tll expression in wild-type brains. The tll locus remains in an activatable state in erm- or ham-null brains, and re-activation of Notch signaling in INPs triggers aberrant Tll expression driving INP reversion to supernumerary type II neuroblasts.

Tables

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Antibodyanti-GFP
(Chicken polyclonal)
Aves Labs, INC.Cat#GFP-1020,
RRID:AB_2307313
IF(1:2000)
Antibodyanti-V5
(Mouse monoclonal)
ThermoFisher ScientificCat#R960-25,
RRID:AB_2556564
IF(1:500)
Antibodyanti-Ase
(Rabbit polyclonal)
Weng et al., 2010 doi: 10.1016/j.devcel.2009.12.007.IF(1:400)
Antibodyanti-Hamlet (Rabbit polyclonal)Eroglu et al., 2014 doi: 10.1016/j.cell.2014.01.053.IF(1:50)
Antibodyanti-Dpn
(Rat monoclonal)
Lee et al., 2006a doi: 10.1038/nature04299.clone 11D1BC7.14IF(1:2)
AntibodyAlexa Fluor 488 AffiniPure Anti-Chicken IgY (IgG) (H+L)
(Donkey polyclonal)
Jackson Immuno
Research Laboratories, INC.
Cat#703-545-155,
RRID:AB_2340375
IF(1:500)
AntibodyAlexa Fluor 647 AffiniPure anti-Rat IgG (H+L)
(Goat polyclonal)
Jackson Immuno Research Laboratories, INC.Cat#112-605-167
RRID:AB_2338404
IF(1:500)
Antibodyanti-Mouse IgG (H+L) Highly Cross-Adsorbed
Secondary Antibody, Alexa Fluor 488
(Goat polyclonal)
ThermoFisher
Scientific
Cat#A-11029,
RRID:AB_2534088
IF(1:500)
Antibodyanti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Goat polyclonal)ThermoFisher ScientificCat#A-11034,
RRID:AB_2576217
IF(1:500)
Antibodyanti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Goat polyclonal)ThermoFisher ScientificCat#A-11035,
RRID:AB_2534093
IF(1:500)
OtherRhodamine PhalloidinThermoFisher ScientificCat#R415IF(1:100)
Genetic reagent (D. melanogaster)brat11/CyO, Actin-GFPLee et al., 2006b doi: 10.1016/j.devcel.2006.01.017.
Genetic reagent (D. melanogaster)w1118; Df(2L)Exel8040/CyOBloomington
Drosophila Stock Center
BDSC: 7847
FlyBase: FBst0007847;
RRID:BDSC_7847
FlyBase symbol: Df(2L)Exel8040/CyO
Genetic reagent (D. melanogaster)y1, M{vas-int.Dm}ZH-2A w*; M{UAS-insbFL-myc}ZH-86FbKomori et al., 2018 doi: 10.1101/gad.320333.118.
Genetic reagent (D. melanogaster)Wor-Gal4(II)Lee et al., 2006a doi: 10.1038/nature04299.
Genetic reagent (D. melanogaster)Wor-Gal4(III)Weng et al., 2010 doi: 10.1016/j.devcel.2009.12.007.
Genetic reagent (D. melanogaster)y1, w*; P{tubPGAL80} LL10, P{neoFRT}40A/CyOBloomington Drosophila Stock CenterBDSC: 5192
FlyBase: FBst0005192;
RRID:BDSC_5192
FlyBase symbol: y1, w*; P{tubPGAL80} LL10, P{neoFRT}40A/CyO
Genetic reagent (D. melanogaster)P{hsFLP}1, P{tubP-GAL80}LL1, w*,
P{neoFRT}19A; P{UAS-mCD8::GFP.L}LL5
Bloomington Drosophila Stock CenterBDSC: 5134
FlyBase: FBst0005134;
RRID:BDSC_5134_
FlyBase symbol: P{hsFLP}1, P{tubP-GAL80}LL1, w*,
P{neoFRT}19A; P{UAS-mCD8::GFP.L}LL5
Genetic reagent (D. melanogaster)y1 w*; PBac{y[+mDint2] w[+mC]=tll EGFP.S}VK00037Bloomington Drosophila Stock CenterBDSC: 30874
FlyBase: FBst0030874;
RRID:BDSC_30874
FlyBase symbol: y1 w*; PBac{y[+mDint2] w[+mC]=tll EGFP.S}VK00037
Genetic reagent (D. melanogaster)Erm-Gal4 (II)Pfeiffer et al., 2008 doi: 10.1073/pnas.0803697105.
Genetic reagent (D. melanogaster)Erm-Gal4 (III)Pfeiffer et al., 2008 doi: 10.1073/pnas.0803697105.
Genetic reagent (D. melanogaster)tllRNAi: y1 sc* v1 sev21; P{TRiP.HMS01316}attP2Bloomington Drosophila Stock CenterBDSC: 34329
FlyBase: FBst0034329;
RRID:BDSC_34329
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS01316}attP2
Genetic reagent (D. melanogaster)M{UAS-tll.ORF-VN}ZH-86FbFlyORFF004752 FBst0502964;
RRID:FlyORF_ F004752
FlyBase symbol: M{UAS-tll.ORF-VN}ZH-86Fb
Genetic reagent (D. melanogaster)Ase-Gal80 (II)Neumüller et al., 2011 doi: 10.1016/j.stem.2011.02.022.
Genetic reagent (D. melanogaster)erm1/CyO, Act-GFPWeng et al., 2010 doi: 10.1016/j.devcel.2009.12.007.
Genetic reagent (D. melanogaster)erm2/CyO, Act-GFPWeng et al., 2010 doi: 10.1016/j.devcel.2009.12.007.
Genetic reagent (D. melanogaster)UAS-ermWeng et al., 2010 doi: 10.1016/j.devcel.2009.12.007.
Genetic reagent (D. melanogaster)PBac{erm-flag4C(g)}VK33Janssens and Lee, 2014 doi: 10.1242/dev.106534.
Genetic reagent (D. melanogaster)DRNAi: y1 v1; P{TRiP.JF02115}attP2Bloomington Drosophila Stock CenterBDSC: 26217
FlyBase: FBst0026217;
RRID:BDSC_26217
FlyBase symbol: y1 v1; P{TRiP.JF02115}attP2
Genetic reagent (D. melanogaster)AseRNAi: y1 sc* v1 sev21; P{TRiP.HMS02847}attP2Bloomington Drosophila Stock CenterBDSC: 44552
FlyBase: FBst0044552;
RRID:BDSC_44552
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS02847}attP2
Genetic reagent (D. melanogaster)hamRNAi: y1 v1; P{TRiP.JF02270}attP2Bloomington Drosophila Stock CenterBDSC: 26728
FlyBase: FBst0026728;
RRID:BDSC_26728
FlyBase symbol: y1 v1; P{TRiP.JF02270}attP2
Genetic reagent (D. melanogaster)hamRNAi: y1 sc* v1 sev21; P{y[+t7.7] v[+t1.8]=TRiP.HMS00470}attP2Bloomington Drosophila Stock CenterBDSC: 32470
FlyBase: FBst0032470;
RRID:BDSC_32470
FlyBase symbol: y1 sc* v1 sev21; P{y[+t7.7] v[+t1.8]=TRiP.HMS00470}attP2
Genetic reagent (D. melanogaster)OpaRNAi: y1 sc* v1 sev21; P{TRiP.HMS01185}attP2/TM3, Sb1Bloomington Drosophila Stock CenterBDSC: 34706
FlyBase: FBst0034706;
RRID:BDSC_34706
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS01185}attP2/TM3, Sb1
Genetic reagent (D. melanogaster)w1118; Df(2L)Exel7071/CyOBloomington Drosophila Stock CenterBDSC: 7843
FlyBase: FBst0007843;
RRID:BDSC_7843
FlyBase symbol: w1118; Df(2L)Exel7071/CyO
Genetic reagent (D. melanogaster)hamSKI, FRT40A/CyOThis paperA new hamlet mutant fly line
Genetic reagent (D. melanogaster)P{w[+mW.hs]=GawB}elav[C155],
P{w[+mC]=UAS-mCD8::GFP.L}Ptp4E[LL4],
P{ry[+t7.2]=hsFLP}1, w[*]
Bloomington Drosophila Stock CenterBDSC: 5146
FlyBase: FBst0005146;
RRID:BDSC_5146
FlyBase symbol: P{w[+mW.hs]=GawB}elav[C155],
P{w[+mC]=UAS-mCD8::GFP.L}Ptp4E[LL4],
P{ry[+t7.2]=hsFLP}1, w[*]
Genetic reagent (D. melanogaster)w1118; P{w[+mC]=UAS-Dcr-2.D}2Bloomington Drosophila Stock CenterBDSC: 24650
FlyBase: FBst00024650;
RRID:BDSC_24650
FlyBase symbol: w1118; P{w[+mC]=UAS-Dcr-2.D}2
Genetic reagent (D. melanogaster)w*; P{w[+mC]=tubP- GAL8ts}2/TM2Bloomington Drosophila Stock CenterBDSC: 7017
FlyBase: FBst00024650;
RRID:BDSC_7017
FlyBase symbol: w*; P{w[+mC]=tubP- GAL8ts}2/TM2
Genetic reagent (D. melanogaster)ham1,FRT40A/CyoMoore et al., 2002 doi: 10.1126/science.1072387.
Genetic reagent (D. melanogaster)UAS-hamMoore et al., 2002 doi: 10.1126/science.1072387.
Genetic reagent (D. melanogaster)RNAi of Notch: y1, v1; P{y+t7.7v+t1.8=TRiP.HMS00001}attP2Bloomington Drosophila Stock CenterBDSC: 33611
FlyBase: FBst0033611;
RRID:BDSC_33611
FlyBase symbol: y1, v1; P{y+t7.7v+t1.8=TRiP.HMS00001}attP2
Genetic reagent (D. melanogaster)Oregon-R-CBloomington Drosophila Stock CenterBDSC: 5
FlyBase: FBst0000005;
RRID:BDSC_5
FlyBase symbol: Oregon-R-C
Genetic reagent (D. melanogaster)P{hsFLP}1, y1 w*; P{UAS- N.intra.GS}2/CyO; MKRS/TM2Bloomington Drosophila Stock CenterBDSC: 52008
FlyBase: FBst0052008;
RRID:BDSC_52008
FlyBase symbol: P{hsFLP}1, y1 w*; P{UAS- N.intra.GS}2/CyO; MKRS/TM2
Genetic reagent (D. melanogaster)y1; M{vas-int.Dm}ZH-2A w*;
M{UAS-ham∆C-ZF-myc}ZH-86Fb
This paperTransgene expressing Hamlet mutant form of the C-terminal zinc finger deletion version
Genetic reagent (D. melanogaster)y1; M{vas-int.Dm}ZH-2A w*; M{UAS-ERD::hamN-ZF-myc}ZH-86FbThis paperTransgene expressing Hamlet the N-terminal zinc finger fused with ERD transcriptional repression domain
Genetic reagent (D. melanogaster)y1; M{vas-int.Dm}ZH-2A w*;
M{UAS-VP-16::hamN-ZF-myc}ZH-86Fb
This paperTransgene expressing Hamlet the N-terminal zinc finger fused with VP16 transcriptional activatoin domain
Genetic reagent (D. melanogaster)cu1, tll49/TM3, P{ftz/lacC}SC1, Sb1, Ser1Bloomington Drosophila Stock CenterBDSC: 7093
FlyBase: FBst007093;
RRID:BDSC_7093
FlyBase symbol: cu1, tll49/TM3, P{ftz/lacC}SC1, Sb1, Ser1
Genetic reagent (D. melanogaster)st1 e1 tll1/TM3, Sb1Bloomington Drosophila Stock CenterBDSC: 2729
FlyBase: FBst002729;
RRID:BDSC_2729
FlyBase symbol: st1 e1 tll1/TM3, Sb1
Genetic reagent (D. melanogaster)hamSK4, FRT40A/CyOBloomington Drosophila Stock CenterBDSC: 34329FlyBase: FBst0034329;RRID:BDSC_34329FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS01316}attP2
Genetic reagent (D. melanogaster)hdac3RNAi: y1 sc* v1 sev21; P{TRiP.HMS00087}attP2Bloomington Drosophila Stock CenterBDSC: 34778
FlyBase: FBst0034778;
RRID:BDSC_34778
FlyBase symbol: hdac3RNAi: y1 sc* v1sev21; P{TRiP.HMS00087}attP2
Genetic reagent (D. melanogaster)Su(z)12RNAi: y1 sc* v1 sev21; P{TRiP.HMS00280}attP2/TM3, Sb1Bloomington Drosophila Stock CenterBDSC: 33402
FlyBase: FBst0033402;
RRID:BDSC_33402
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS00280}attP2/TM3, Sb1
Genetic reagent (D. melanogaster)Su(var)3-3RNAi: y1 sc* v1 sev21; P{TRiP.HMS00638}attP2Bloomington Drosophila Stock CenterBDSC: 32853
FlyBase: FBst0032853;
RRID:BDSC_32853
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.HMS00638}attP2
Genetic reagent (D. melanogaster)Su(var)205RNAi: y1 sc* v1 sev21; P{TRiP.GL00531}attP40Bloomington Drosophila Stock CenterBDSC: 36792
FlyBase: FBti0146447;
RRID:BDSC_36792
FlyBase symbol: y1 sc* v1 sev21; P{TRiP.GL00531}attP40
Genetic reagent (D. melanogaster)UAS-Mi-2DNKovač et al., 2018 doi: 10.1038/s41467-018-04503-2.
Genetic reagent (D. melanogaster)UAS-Mi-brmDNHerr et al., 2010 doi: 10.1016/j.ydbio.2010.04.006.
Genetic reagent (D. melanogaster)In(1)wm4; Su(var)3–91/TM3, Sb1 Ser1Bloomington Drosophila Stock CenterBDSC: 6209
FlyBase: FBst0006209;
RRID:BDSC_6209
FlyBase symbol: In(1)wm4; Su(var)3–91/TM3, Sb1 Ser1
Genetic reagent (D. melanogaster)w1118; PBac{Sp1- EGFP.S}VK00033Bloomington Drosophila Stock CenterBDSC: 38669
FlyBase: FBst0038669;
RRID:BDSC_38669
FlyBase symbol: w1118; PBac{Sp1- EGFP.S}VK00033
Sequenced-based reagentHam_FEroglu et al., 2014 doi: 10.1016/j.cell.2014.01.053.PCR primersatagatcctttggccagcagac
Sequenced-based reagentHam_REroglu et al., 2014 doi: 10.1016/j.cell.2014.01.053.PCR primersagtactcctccctttcggcaat
Sequenced-based reagentAse_FKomori et al., 2014b doi: 10.7554/eLife.03502.PCR primersagcccgtgagcttctacgac
Sequenced-based reagentAse_RKomori et al., 2014b doi: 10.7554/eLife.03502.PCR primersgcatcgatcatgctctcgtc
Sequenced-based reagentD_FThis paperPCR primersgcggcggcggtcaacaat
Sequenced-based reagentD_RThis paperPCR primerstgcggcgtacagcgaagggt
Sequenced-based reagentErm_FEroglu et al., 2014 doi: 10.1016/j.cell.2014.01.053.PCR primersgttacggccaggcatcgggtcaa
Sequenced-based reagentErm_REroglu et al., 2014 doi: 10.1016/j.cell.2014.01.053.PCR primersgggccaggcgggattactcgtctc
Sequenced-based reagentPntP1_FKomori et al., 2014b doi: 10.7554/eLife.03502.PCR primersggcagtacgggcagcaccac
Sequenced-based reagentPntP1_RKomori et al., 2014b doi: 10.7554/eLife.03502.PCR primersctcaacgcccccaccagatt
Sequenced-based reagentDpn_FKomori et al., 2014b doi: 10.7554/eLife.03502.Komori et al., 2014bPCR primerscatcatgccgaacacaggtt
Sequenced-based reagentDpn_RKomori et al., 2014bPCR primersgaagattggccggaactgag
Recombinant DNA reagentpUAST-ham∆C-ZF-myc-attB (plasmid)This paperPlasmid DNA of a transgene expressing Hamlet mutant form of the C-terminal zinc finger deletion version
Recombinant DNA reagentpUAST-ERD::hamN-ZF-myc-attB (plasmid)This paperPlasmid DNA of a transgene expressing Hamlet the N-terminal zinc finger fused with ERD transcriptional repression domain
Recombinant DNA reagentpUAST-VP16::hamN-ZF-myc-attB (plasmid)This paperPlasmid DNA of a transgene expressing Hamlet the N-terminal zinc finger fused with VP16 transcriptional activatoin domain
Software, algorithmLAS AFLeica MicrosystemsRRID:SCR_013673
Software, algorithmImageJ 1.50 gNational Institute of HealthRRID:SCR_003070

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