1. Developmental Biology
  2. Neuroscience
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Neuroblast-specific open chromatin allows the temporal transcription factor, Hunchback, to bind neuroblast-specific loci

  1. Sonia Q Sen
  2. Sachin Chanchani
  3. Tony D Southall
  4. Chris Q Doe  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Oregon, United States
  2. Imperial College London, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e44036 doi: 10.7554/eLife.44036
8 figures, 1 table and 2 additional files

Figures

Spatial and temporal cues are integrated to generate neuronal diversity.

(A) Spatial and temporal patterning. (Left) As neuroblasts delaminate from the neuroectoderm, they experience spatial transcription factors (e.g. Gsb, En, Vnd, Ind, Msh shown) that gives each neuroblast a unique molecular identity. (Middle) TTFs are sequentially expressed in most neuroblasts to specify GMC/neuronal identity based on birth-order. (Right) The integration of spatial and temporal factors specifies lineage-specific neuronal identity. (B) Independent specification: in this hypothesis, STFs and TTFs bind genomic targets independently, and their combinatorial effect specifies distinct neuroblast identity. In this model, TTF targets are the same in different NBs. (C) Sequential specification: in this hypothesis, STFs act first to bias or restrict subsequent TTF genomic binding, leading to the production of different neurons from different neuroblasts. In this model, TTF targets are the different in different NBs (D–F) The TaDa and CaTaDa Materials and method. See text for details.

https://doi.org/10.7554/eLife.44036.003
Figure 2 with 1 supplement
Identification of Gal4 lines specifically expressed in NB5-6 or NB7-4.

(A) Left: schematic showing spatial positions of NB5-6 and NB7-4. Right: Immunostaining of stage nine embryos showing neuroblast-specific STF expression (En, Gsb) and common TTF expression (Hb). Genotype: en-Gal4/UAS-GFP. (B–D’) NB5-6-Gal4 is expressed in the NB5-6 lineage from stage 10 until the end of embryogenesis. Dan is present in NB5-6 through stage 12 (C’). (D’) Schematic of NB5-6 expression (green outlines) and Hb expression (purple), see text for details. Note that Gal4 expression is present during the Dan + Hb competence window. Genotype: lbe-K-Gal4/UAS-myr::GFP. (E–G’) NB7-4-Gal4 is expressed in the NB7-4 lineage from stage 10 until the end of embryogenesis. Dan is present in NB5-6 through stage 12 (F’). (G’) Schematic of NB7-4 expression (green outlines) and Hb expression (purple), see text for details. Genotype: R19B03AD/UAS-myrGFP; R18F07DBD/+. (H–I) NB5-6 early-born Chaise Lounge neurons. Lateral view, anterior, left. (H) Two segmentally repeated Chaise Lounge neurons labelled by MCFO (hs-FLP lbe-K-Gal4 UAS-MCFO); the Chaise Lounge neurons are Hb+ (inset). Note the ipsilateral projections. (I) Two segmentally repeated Chaise Lounge neurons in the EM reconstruction, where they are named A27k. Inset: outline of CNS with Chaise Lounge neurons shown. (J–K) NB7-4 early-born G neuron. (J) MARCM clone made with en-Gal4 labels most or all of the NB7-4 lineage, including the diagnostic Channel Glia (CG) which are only made by NB7-4 (Schmidt et al., 1997; Schmid et al., 1999). Note the G neuron axon arbors which project the most laterally in the connective and are both ascending and descending (red arrowheads). SPG, subperineurial glia. Dorsal view, anterior to left. (J) The G neuron in the EM reconstruction (red). The neuropil is outlined in gray. Note the lateral axon projection that is ascending and descending, and the cell body position contacting the neuropil. Also note the two small bilateral midline processes, which match those of the grasshopper G neuron (Raper et al., 1983).

https://doi.org/10.7554/eLife.44036.004
Figure 2—figure supplement 1
Expression pattern of NB5-6 and NB7-4 Gal4 lines.

(A) Lbe-K-Gal4 (NB5-6-Gal4) expression is specific to NB5-6 and its progeny from stage 10-stage 16, followed by the addition of salivary gland expression (arrowheads) at stage 17. Genotype: Lbe-K-Gal4/UASmyrGFP (B) R19B03AD; R18F07DBD split line (NB7-4-Gal4) is specific to NB7-4 from stage 10 to the end of embryogenesis. In 6% of hemisegments, the R19B03AD; R18F07DBD line is expressed in the NB5-6 lineage (arrowheads). Genotype: R19B03AD/UAS-myrGFP; R18F07DBD/+.

https://doi.org/10.7554/eLife.44036.005
Figure 3 with 3 supplements
Generation of a functional, non-toxic Dam:Hb fusion protein.

(A) Low level Dam:Hb expression is non-toxic. Control 1, sca-gal4/sca-gal4; control 2, sca-gal4 UAS-HA::UPRT (Miller et al., 2009); Dam, sca-gal4 UAS-LT3-Dam; Dam:Hb, sca-gal4 UAS-LT3-Dam:Hb, Dam:Hb, Da-gal4 UAS-LT3-Dam:Hb (n = 300 for each genotype). (B) Dam:Hb retains Hb function and can induce ectopic Eve+ U neurons. Anterior up; midline, dashed line. Left hemisegment shows a single ectopic Eve+ neuron (yellow) to comprise six total U neurons, whereas the right hemisegment has the normal five U neurons. Below, quantification. Wild type (y w) represents 68 hemisegments from six embryos; Dam:Hb (da-Gal4 UAS-LT3-Dam:Hb, second ORF) represents 8 of 232 hemisegments from 15 embryos with an ectopic U neuron. ELs, Eve lateral neurons. (C) Dam:Hb binding is reproducible. Left, three biological replicates of genomic binding sites showing high Pearson correlation coefficients. Right, Dam:Hb binding over 1341 kb on chromosome IV is highly similar in all three biological replicates. Genotype da-Gal4 UAS-LT3-Dam:Hb in stage 17 embryos. Data range: −2.84–7.07. (D–G) Dam:Hb-bound loci correlate with Hb ChIP loci. (D) Alignment of Dam:Hb and Hb ChIP binding sites over 766 kb of genomic DNA near the Hb locus, where Hb is known to bind. Data range for Hb ChIP: −1.01–6.23; Data range for Dam:Hb: −2.63–5.3. (E) Alignment of Dam:Hb and Hb ChIP binding sites at the Krüppel (Kr) locus. Data range for Hb ChIP: −1.66–9.04; Data range for Dam:Hb: −0.63–5.68. (F) Dam:Hb peaks for three replicates (blue, cyan, yellow) are correlated with Hb ChIP signal. Plot shows the Hb ChIP signal ±10 kb of the center of all the peaks identified by Dam:Hb analysis in the three replicates. (G) Dam:Hb signal is enriched at sites of Hb ChIP binding (blue), but not that of Bcd (cyan) or Ftz (yellow). Plot shows the Dam:Hb signal ±5 kb of the center of all the peaks identified by ChIP-chip analysis.

https://doi.org/10.7554/eLife.44036.006
Figure 3—figure supplement 1
Dam:Hb and Hb-ChIP show similar binding at known Hb target genes.

op row: Hb ChIP-seq data (Bradley et al., 2010). Bottom row: Dam:Hb data showing the ratio of Dam:Hb binding over Dam alone binding. Dam:Hb enriched binding (blue) and Dam only enriched binding (red). Note the similarity of Hb occupancy as reported by the two techniques at these nine known targets of Hb (Lyne et al., 2007). Data range for Hb ChIP: −2.45–9.04; Data range for Dam:Hb: −0.93–6.24.

https://doi.org/10.7554/eLife.44036.007
Figure 3—figure supplement 2
Dam:Hb and Hb-ChIP binding is correlated.

Left: Increased Hb ChIP signal at Dam:Hb peaks. Right: Increased Dam:Hb signal at Hb ChIP peaks. Heatmap shows the Hb ChIP/Hb TaDa signal ±10 kb of the center of all the peaks. Each row in the heatmap corresponds an individual peak, and the signal around that peak.

https://doi.org/10.7554/eLife.44036.008
Figure 3—figure supplement 3
Hb binding motifs are enriched at Dam:Hb bound loci.

Hb DNA-binding motif (Stanojević et al., 1989) is enriched in the top 1000 Dam:Hb peaks. Analysis using the Homer suite of tools (Heinz et al., 2010). Comparable results were obtained when all Dam:Hb peaks were used.

https://doi.org/10.7554/eLife.44036.009
Figure 4 with 1 supplement
Dam:Hb has distinct genomic binding sites in NB5-6 and NB7-4 lineages.

(A,B) Dam:Hb binding in the NB5-6 lineage and the NB7-4 lineage is reproducible. (A) Three biological replicates of Dam:Hb in each neuroblast lineage are shown, with high Pearson correlation coefficients within each neuroblast replicate, and low correlation coefficients between each neuroblast. (B) Dam:Hb binding over 1,341 kb on chromosome IV is qualitatively similar between lineages. Data range: −3.49–8.71. (C–F) Differential binding data showing Dam:Hb binds different loci in NB5-6 versus NB7-4. (C) A binding affinity heatmap (scaled) showing reads at loci differentially occupied by Dam:Hb in NB5-6 and NB7-4. Loci (rows) are shown for biological replicates of both neuroblasts with greater densities of Dam:Hb binding in darker colours. Note that sites with higher counts in the three NB7-4 replicates (top right) are depleted in the three NB5-6 replicates (top left), and vice versa. (D) Volcano plot showing differentially occupied loci that are FDR ≤ 0.01 in magenta, FDR > 0.01 in blue, and those that have a fold change of less than two in grey. This threshold corresponds to 718 loci in NB5-6 and 504 loci in NB7-4. Genome-wide Hb-bound loci in both neuroblasts were analysed for differential analysis using DiffBind (Ross-Innes et al., 2012) with DESeq2 and edgeR and two independent peakcallers with similar results. These plots show DESeq2 results with the MACS2 peak caller (Zhang et al., 2008). (E,F) The top five enriched Dam:Hb-bound loci are shown for NB5-6 (blue track in F) versus NB7-4 (green tracks in G) lineages. The black bars represent the loci identified as differentially bound in the analysis. Data range: −1.9–3.96. For all panels, NB5-6 genotype: NB5-6-Gal4 UAS-LT3-Dam:Hb or UAS-LT3-Dam. NB7-4 genotype: NB7-4-Gal4 UAS-LT3-Dam:Hb or UAS-LT3-Dam.

https://doi.org/10.7554/eLife.44036.010
Figure 4—figure supplement 1
Dam:Hb shows similar binding at loci expressed in both NB5-6 and NB7-4.

Kr, pdm2 and zfh2 are expressed in both NB5-6 and NB7-4 and show similar Dam:Hb binding profiles. Black bars, enriched binding observed in one of the three replicates. Data range: −2.44–9.57.

https://doi.org/10.7554/eLife.44036.011
Figure 5 with 1 supplement
Dam only binding shows differential open chromatin landscapes in NB5-6 and NB7-4 lineages.

(A–C) Dam binding is reproducible and correlates with DNAse I sites. (A) Three biological replicates are shown, with high Pearson correlation coefficients. (B) Dam binding is enriched at DNAse I hypersensitive peaks. (C) Dam binding over 1,533 kb on chromosome 3R is similar in all replicates (red tracks), and similar to DNAseI hypersensitivity data (ochre tracks). Data range for Dam: 0–50; Data range for DNAseI: 0–150. Genotype: Da-Gal4/UAS-LT3-Dam. (D–E) Dam binding reveals different open chromatin domains in NB5-6 versus NB7-4. (D) Heat map showing Dam binding sites in NB5-6 have high Pearson correlation coefficients in two replicates, but note the low correlation coefficients between NB5-6 and NB7-4 replicates, showing that each neuroblast has different open chromatin landscapes. (E) Dam binds different loci in the NB5-6 lineage versus the NB7-4 lineage. MA plot showing 3656 loci enriched for Dam binding in the NB5-6 lineage (top) and 5084 loci enriched for Dam binding in the NB7-4 lineage (bottom).

https://doi.org/10.7554/eLife.44036.012
Figure 5—figure supplement 1
Dam only shows similar binding at loci expressed in both NB5-6 and NB7-4.

(A) Kr, pdm2 and zfh2 are expressed in both NB5-6 and NB7-4 and show similarly open chromatin at these loci. Black bars, enriched binding observed in all three replicates. Data range: 0–70. (B) Dam binding over 1,341 kb on chromosome IV in three biological replicates for each neuroblast lineage (NB5-6 lineage – blue tracks; NB7-4 lineage – green tracks), are qualitatively similar within lineages.

https://doi.org/10.7554/eLife.44036.013
Figure 6 with 1 supplement
Differential chromatin in the 5–6 and 7–4 neuroblast lineages is correlated with differential Hb occupancy.

(A–C) Dam:Hb binds within neuroblast-specific open chromatin. (A) Dam signal (open chromatin) in NB 5–6 (blue lines) and NB 7–4 (green lines) at loci where Dam:Hb binding is enriched in NB5-6 over NB7-4. Note that the chromatin is more open in NB5-6 than in NB7-4 at these loci. (B) Dam signal (open chromatin) in NB 7–4 (green lines) and NB 5–6 (blue lines) at loci where Dam:Hb binding is enriched in NB7-4 over NB5-6. Note that the chromatin is more open in NB7-4 than in NB5-6 at these loci. (C) Dam signal (open chromatin) at loci similarly occupied by Hb in both NB5-6 and NB7-4 lineages. (D) The top five Dam:Hb enriched loci in NB5-6 are in regions of NB5-6 open chromatin (blue tracks); however, in NB7-4 these loci are not in open chromatin (Dam; green tracks), and are not bound by Dam:Hb. Rows from top to bottom: genomic locus, Dam:Hb enrichment in NB5-6, Dam only enrichment in two replicates in NB5-6, Dam:Hb enrichment in NB7-4, and Dam only enrichment in two replicates in NB7-4. Data range for IP3K1, rut, pbl is 0–109; data range for CG13131 and mspo is 0–15. (E) The top five Dam:Hb enriched loci in NB7-4 are in regions of open chromatin in NB7-4 (green tracks); however, in NB5-6 these loci are not in open chromatin (Dam; blue tracks) and are not bound by Dam:Hb. Rows from top to bottom: genomic locus, Dam:Hb enrichment in NB5-6, Dam only enrichment in two replicates in NB5-6, Dam:Hb enrichment in NB7-4, and Dam only enrichment in two replicates in NB7-4. Data range for sqz, InR and en is 0–35; data range for lov and H15 is 0–20.

https://doi.org/10.7554/eLife.44036.014
Figure 6—figure supplement 1
Dam:Hb binding is biased towards regions of open chromatin.

Dam binding (open chromatin) is correlated with Dam:Hb binding in each neuroblast lineage. (A) Dam signal (open chromatin) of the two replicates of NB5-6 is plotted at the region of Dam:Hb occupancy in the NB5-6 lineage. (B) Dam signal (open chromatin) of the two replicates of NB7-4 is plotted at the region of Dam:Hb occupancy in the NB7-4 lineage. Note the correspondence of open chromatin (increased Dam signal) at the regions of Hb occupancy in both NB lineages. These results are consistent with preferential Hb binding at regions of open chromatin. (C) Schematic showing preferential binding of Dam:Hb at sites of open chromatin.

https://doi.org/10.7554/eLife.44036.015
Gsb binding is enriched at open chromatin and Dam:Hb bound loci in NB5-6, but not NB7-4.

(A) Gsb ChIP-chip signal at the regions of Dam-bound (open) chromatin; note the enrichment in NB5-6 (blue lines) but not NB7-4 (green lines). The number of peaks used is 20,838 and 18,201 for the NB5-6 reps and 29,817 and 31,080 for NB7-4. (B) Gsb ChIP-chip signal at the regions of Dam:Hb bound loci; note the enrichment in NB5-6 (blue lines) compared to NB7-4 (green lines). The number of peaks used is 504 and 718 in the two NBs, respectively. (C) Monte Carlo analysis shows that the average enrichment of Gsb signal around the actual NB5-6 loci (red line) is significantly higher than the distribution of average signal calculated for a similar number of random loci (1000 iterations, black line).

https://doi.org/10.7554/eLife.44036.016
Sequential specification integrates spatial and temporal cues to generate diversity in Drosophila embryonic NB lineages.

Transient expression of spatial factors in the neuroectoderm (e.g. Gsb in row 5) establishes lineage-specific chromatin landscapes (e.g. NB5-6 lineage). Subsequently, TTFs (e.g. Hb) in the NB can access different genomic targets to regulate different genes in spatially distinct NB lineages. This results in the specification of different neural fates in different NB lineages.

https://doi.org/10.7554/eLife.44036.017

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Drosophila melanogaster)UAS-LT3-DamA BrandNAUAS drives mCherry from 1st cistron and Dam from 2nd cistron
Strain, strain background (D. melanogaster)engrailed-Gal4A BrandNAExpressed in row 6 and 7 neuroblasts
Strain, strain background (D. melanogaster)R19B03AD;R18F07DBDG RubinNAExpression in NB7-4 lineage from stage 9
Strain, strain background (D. melanogaster)Lbe(K)-Gal4S ThorNAExpression in NB5-6 lineage from stage 9; salivary gland at stage 17
Strain, strain background (D. melanogaster)UAS-LT3-Dam:HbThis paperNAUAS drives mCherry from 1st cistron and Dam:Hb from 2nd cistron
Strain, strain background (D. melanogaster)Sca-Gal4Y HiromiNAExpressed in all NBs
Strain, strain background (D. melanogaster)hsFLP;;UAS-MCFOA NernNAMCFO (multi-colored-flip-out) line
Strain, strain background (D. melanogaster)MARCM stockT LeeNAFor clonal analysis of NB7-4 lineage
Strain, strain background (D. melanogaster)UAS-HA:UPRTDoe labNAControl transgene
Strain,
strain
background
(D. melanogaster)
Da-Gal4BDSC55850 homozygous on III
Antibodychicken anti-GFP
(polyclonal)
Abcam (Eugene, OR)ab13970(1:1000)
Antibodymouse anti-en
(monoclonal)
DSHB (Iowa City, IA) 4D9(1:50)
Antibodyrabbit anti-Dan
(polyclonal)
Doe labNA(1:1000)
Antibodyrabbit anti-Hb
(polyclonal)
Doe labNA(1:400)
Antibodyrabbit anti-Eve
(polyclonal)
Doe labSC1320A(1:500)
Antibodyrat anti-Gsb
(monoclonal)
Holmgren Lab1:1 10E10/16F2(1:10)
Antibodymouse
anti-mCherry
(polyclonal)
Clonetech632543(1:500)
Antibodyrabbit
anti-V5::549
(polyclonal)
Rockland600-442-378(1:400)
Antibodymouse
anti-HA::488
(monoclonal)
Cell signaling2350S(1:200)
Antibodyrat anti-Ollas::650
(monoclonal)
NovusNBP1-06713(1:200)
AntibodySecondary
antibodies
(polyclonal)
Thermofisher
(Eugene, OR)
(1:400)

Additional files

Supplementary file 1

Hb enriched loci in NB5-6 lineage and NB7-4 lineages.

The genome coordinates for Hb enriched loci in NB5-6 and NB7-4. Columns show, from left to right: chromosome arm, start of fragment, end of fragment, concentration, concentration in NB5-6, concentration in NB7-4, fold enrichment (NB5-6/NB7-4), p-value, and False Discovery Rate (FDR).

https://doi.org/10.7554/eLife.44036.018
Transparent reporting form
https://doi.org/10.7554/eLife.44036.019

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