Spatial and temporal patterning of neuroblasts and models for their integration.

A. Patterning the neuroectoderm: Early embryo patterning genes that establish the A-P and D-V body axes, also pattern the neuroectoderm, located ventro-laterally in the embryo. A ‘filet’ preparation allows visualization of the patterned neuroectoderm with representative A-P gene expression shown in blue and pink, and D-V columns in light green, yellow and red. B. Spatial patterning: NBs arise in an orthogonal array from this patterned neuroectoderm, each acquiring a unique molecular identity. C. Temporal patterning. Each spatially distinct NB (here, NB5-6 and NB7-4) transitions through a series of temporal windows corresponding to sequentially expressed TTFs, Hb > Kr > Pdm > Cas > Grh. This allows NBs to generate different neurons over time. During the Hb temporal window (pink box), NB5-6 generates Chaise Lounge neuron while NB7-4 generates G neuron. By the end of the temporal cascade, each NB produces distinct lineages. D-F. Models for spatiotemporal integration. Two NBs (NB1, NB2) express different spatial transcription factors (STF1, STF2) but share the same TTF. (D) Direct regulation: STFs and the TTF co-bind accessible enhancers of neuron type–specific terminal selector (TS) genes in each NB. (E) Epigenetic regulation: STFs first bind and open chromatin in an NB-specific manner, creating sites of integration (SoIs) that are later bound by the TTF to activate TS enhancers. (F) Outcome: distinct TS combinations (TS1/TS2 in NB1, TS3 in NB2) specify lineage-specific, time-appropriate neurons.

NB5-6 Gsb and NB7-4 En TaDa profiling and lineage-specific chromatin accessibility.

A,B. TaDa strategy to profile NB5-6 Gsb and NB7-4 En occupancy and chromatin accessibility. In NB5-6, Lbe-k-Gal4 drives a UAS construct in which mCherry is encoded in the primary ORF and Dam:Gsb (blue) or DamOnly (green) in the secondary ORF. In NB7-4, the split-Gal4 line 19B03AD;18F07DBD similarly drives Dam:En (red) or DamOnly. High mCherry expression reports driver specificity, while translation of Dam fusions from the secondary ORF keeps Dam levels low, minimizing toxicity and preserving binding specificity. DamOnly profiles are used as a proxy for chromatin accessibility. C. NB5-6-specific DamOnly and Dam:Gsb bind reproducibly. Heatmap shows pairwise-correlation between NB5-6-specific DamOnly and Dam:Gsb replicates. Hierarchical clustering separates DamOnly and Dam:Gsb replicates, with higher Pearson correlation coefficient values within conditions than between them. D. NB7-4-specific DamOnly and Dam:En bind reproducibly. Heatmap shows pairwise correlations between NB7-4-specific DamOnly and Dam:En replicates, where hierarchical clustering separates DamOnly and Dam:En samples, with stronger correlations within than between conditions. E. Binding at known Gsb targets. Genome browser snapshots of 4 replicates of NB5-6-Dam:Gsb show binding at known Gsb targets- wg, gsb-n and prd (46–48). F. Binding at known En targets: Genome browser snapshots of 3 replicates of NB7-4-Dam:En show binding at en, beta-Tub60D, ptc (49, 50). G. Chromatin is differentially accessible between NB5-6 and NB7-4. Volcano plot showing sites differentially bound by DamOnly in NB5-6 and NB7-4. Differentially accessible loci with FDR ≤ 0.05 are shown in magenta, while those with FDR > 0.05 are shown in blue. This threshold yielded 1933 differential sites. H. Visualizing differentially accessible loci. Genome browser snapshots show examples of differentially accessible sites in NB5-6 (blue tracks) and NB7-4 (red tracks). For all browser snapshots, Data range 0-50. Genotypes for NB5-6-Gsb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Gsb. NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly. NB7-4-En occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:En. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly.

En binds accessible chromatin.

A. En binding relative to Sites of Integration (SoIs). Top: Schematic representing NB7-4-SoIs, defined as loci that are Hb-bound (light pink drop) and accessible in NB7-4 (dark red DNA strand); loci corresponding to NB5-6-SoIs are Hb-free (hollow drop) and less accessible (light red DNA strand) in NB7-4. Bottom: NB5-6-SoIs are Hb-bound (light pink drop) and accessible in NB5-6 (dark blue DNA strand); loci corresponding to NB7-4-SoIs are Hb-free (hollow drop) and less accessible (light blue DNA strand) in NB7-4. Differential analysis identified 798 NB5-6-SoIs and 230 NB7-4-SoIs (FDR ≤ 0.01, FC ≥ 2, see Methods, Fig. S2). B-C En binding at SoIs. En binds NB7-4-SoIs in NB7-4 (B). No En binding is observed at NB5-6-SoIs in NB7-4 (C). Insets show heatmaps of En binding at corresponding loci, averaged across 3 replicates. D-F. En binding relative to genome-wide chromatin accessibility. Schematic shows uniquely accessible sites in NB7-4 (306 sites) in dark red, while those less accessible (118 sites) are shown in light red (FDR ≤ 0.01, FC ≥ 2, see Methods, Fig. S3). En binds accessible sites in NB7-4 (E). No En binding is detected at less accessible sites (F). Insets show heatmaps of En binding at the corresponding loci, averaged across 3 replicates. G-I. Chromatin accessibility in NB7-4 relative to genome-wide En-bound and En-unbound sites. Schematic representing En-bound sites (dark pink drops; 786 sites) and En-unbound sites (hollow drops; 1158 sites) (FDR ≤ 0.01, FC ≥ 2; see Methods, Fig. S4). Chromatin is accessible at En-bound sites in NB7-4 (H) and is less accessible at En-unbound sites (I). Insets show heatmaps of Dam signal from NB7-4 at the corresponding loci, averaged across 3 replicates. In all signal plots, solid lines indicate mean, coloured ribbons indicate 95% confidence interval. Genotypes for NB5-6-Hb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Hb. NB7-4-Hb occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb (41). NB7-4-En occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:En. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly.

Gsb binds both accessible and less accessible chromatin.

A. Gsb binding relative to Sites of Integration (SoIs). Top: Schematic representing NB5-6-SoIs, defined as loci that are Hb-bound (light pink drop) and accessible in NB5-6 (dark blue DNA strand); loci corresponding to NB7-4-SoIs are Hb-free (hollow drop) and less accessible (light blue DNA strand) in NB5-6. Bottom: NB7-4-SoIs are Hb-bound (light pink drop) and accessible in NB7-4 (dark red DNA strand); loci corresponding to NB5-6-SoIs are Hb-free (hollow drop) and less accessible (light red DNA strand). Differential analysis identified 798 NB5-6-SoIs and 230 NB7-4-SoIs (FDR ≤ 0.01, FC ≥ 2, see Methods, Fig. S2). Gsb binds NB5-6-SoIs (B) and at sites corresponding to NB7-4-SoIs, which are less accessible and Hb-free in NB5-6 (C). Insets show heatmaps of Gsb signal at the corresponding sites, averaged across 4 replicates. D-F. Gsb binding relative to genome-wide chromatin accessibility. Schematic represents DNA loci uniquely accessible (dark blue, 118 sites) and less accessible (light blue, 306 sites) sites in NB5-6 (FDR ≤ 0.01, FC ≥ 2, see Methods,Fig. S3). Gsb is bound at accessible sites in NB5-6 (E) and some Gsb binding is also seen at less accessible chromatin (F). Insets show heatmaps of Gsb signal at the corresponding loci, averaged across 4 replicates. G-I. Chromatin accessibility in NB5-6 relative to Gsb-bound and Gsb-unbound sites. Schematic representing Gsb-bound sites (blue drops, 1158 sites) and Gsb-unbound sites (hollow drops, 786 sites) (FDR ≤ 0.01, FC ≥ 2; see Methods, Fig. S4). Dam signal from NB5-6 mapped at Gsb-bound sites shows that chromatin is accessible at Gsb-bound sites in NB5-6 (H). At Gsb-unbound sites, chromatin is more accessible than at Gsb-bound loci (I). Insets show heatmaps of Dam signal from NB5-6 at the corresponding loci, averaged across 4 replicates. In all cases, solid lines indicate mean, coloured ribbons indicate 95% confidence interval. Genotypes for NB5-6-Hb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Hb. NB7-4-Hb occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb (41). NB5-6-Gsb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Gsb. NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly.

Co-binding with Hb marks sites of highest accessibility.

Schematic in (A) shows sites in NB5-6 bound by Gsb only (blue drops, 2,322 sites), Hb only (light pink drops, 2,571 sites) and Gsb+Hb (1,568 sites). Co-bound sites were identified (see Methods). Schematic in (B) shows sites in NB7-4 bound by En only (dark pink drops, 2,151 sites), Hb only (light pink drops, 2,460 sites) and En+Hb (1,419 sites). Dam signal in NB5-6 at sites bound by Gsb only (C) and by Hb only (D) shows some accessibility, but Dam signal at Gsb+Hb co-bound shows most accessibility (E). Insets show heatmaps of NB5-6-Dam signal at the corresponding sites, averaged across 4 replicates. F. Dam signal in NB7-4 at sites bound by En only (F) and by Hb only (G) shows some accessibility, but Dam signal at En+Hb co-bound shows most accessibility (H). Insets show heatmaps of NB7-4-Dam signal at the corresponding sites, averaged across 3 replicates. Genotypes for NB5-6-Hb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Hb. NB7-4-Hb occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb (41). NB5-6-Gsb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Gsb. NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly. NB7-4-En occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:En. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly.

Gsb remodels chromatin when ectopically expressed.

(A) Schematic of the experimental protocol while assaying chromatin accessibility and Hb occupancy (light pink drop) in control (top) and Gsb misexpressed (bottom) NB7-4. Control NB-7-4 and its chromatin is denoted as a pink cell or DNA respectively, Gsb misexpressed NB-7-4 and its chromatin is similarly denoted as yellow. B-C. Chromatin accessibility changes in NB7-4 upon Gsb misexpression. B. Loci that are accessible in control NB7-4 (red) and lose accessibility upon Gsb misexpression (yellow). C. Loci that are less accessible in control NB7-4 (red), but gain accessibility upon Gsb misexpression (yellow). Insets show heatmaps of control NB7-4-Dam (red) and Gsb misexpressed NB7-4-Dam (yellow) at the corresponding loci, averaged across 3 replicates each. D-E. Whole embryo Gsb binding at the changed accessibility sites from B-C. Whole embryo Gsb TaDa signal at sites corresponding to those that either lose (D) or gain (E) accessibility in NB7-4 upon Gsb misexpression. F-G. Hb binding at the changed accessibility sites from B-C. Hb signal in control (green) and Gsb misexpressed (yellow) NB7-4. Insets show heatmaps of control NB7-4-Hb (green) and with Gsb misexpression (yellow) at the corresponding loci, averaged across 3 replicates each. H-I. Gsb mediated changes in chromatin accessibility at SoIs in NB7-4. H. Chromatin in control (red) and Gsb misexpressed (yellow) NB7-4 corresponding to NB5-6-SoIs remains unchanged. I. Reduced chromatin accessibility in Gsb misexpressed NB7-4 (yellow) relative to control (red) at sites corresponding to NB7-4-SoIs. J-K. Gsb mediated changes in Hb occupancy at SoIs in NB7-4. J. Hb binding in control (green) and Gsb misexpressed (yellow) NB7-4 corresponding to NB5-6-SoIs remains unchanged. K. Reduced Hb binding in Gsb misexpressed NB7-4 (yellow) relative to control (green) at sites corresponding to NB7-4-SoIs. Genotypes for NB5-6-Hb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Hb. NB7-4-Hb occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb (41). NB5-6-Gsb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Gsb. NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly. NB7-4-En occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:En. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly. NB7-4-chromatin accessibility with Gsb misexpression: hsGsb; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly. NB7-4-Hb occupancy with Gsb misexpression: hsGsb; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb.

Broad loss of Gsb function results in loss of NB5-6 identity and gain of NB3-5 identity.

A. Control embryo with no Gsb knockdown, showing Ap-positive neurons born from NB5-6 (cyan circle), MP2 (orange circle) and NB4-3 (yellow circle). B. Test embryos with Gsb knockdown show missing Ap-positive neurons from NB5-6 (cyan asterisks). Scale bar 10µ. C. Schematic summarizing Tv neurons (Ap+Eya), born from NB5-6, dMP2 neurons (Ap-positive) born from MP2, and dAp neurons (Ap+Eya) born from NB4-3. D-F Tv neurons are a cluster of 4 Ap+ neurons born from NB5-6 in each thoracic hemisegment. These neurons express both Ap (D) and Eya (E). G-I. Upon Gsb knockdown, Tv neurons are absent (cyan asterisk). J-L. Every hemisegment has one dMP2 neuron born from MP2 (yellow dashed circle). There is also a pair of dAp neurons (orange dashed circle) born from NB4-3. dMP2 neuron also expresses Eya but dAp neurons do not. M-O. In test embryos with Gbs knockdown, dAP neuron (from NB4-3, yellow dashed circle) remains unaffected. However, there is an increase in the dMP2 neurons (orange dashed circle)-there are 2 pairs of Ap+ neurons here instead of one. Together (A-O) show a loss of NB5-6-like identity and a concomitant gain in MP2-like identity. NB4-3 lineage remains unaffected. Scale bar in insets 5µ. P. Control embryo showing Ems expression. Q. Upon broad knockdown of Gsb, there is an increase in Ems expression (green dashed box indicates an example of increased Ems). Scale bar 10µ. The decrease in thoracic Eya and Ap expression is quantified in (R) and (S). The gain in Ems expression has been quantified in (T). Each point represents a single hemisegment; boxplots indicate median and interquartile range. Statistical significance was assessed using two-tailed Mann–Whitney U tests. Significance levels are indicated as: *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. Genotype for control embryos: Vasa Cas9/+;;. For test embryos: Vasa Cas9/+; gsb-guideRNA/+.

Broad misexpression of Gsb results in loss of NB3-5 identity, but minimal gain of NB5-6 identity.

A. (Top) Schematic illustrating heat shock protocol used to induce broad Gsb misexpression at NB delamination (red arrowhead). (Bottom) Both NB3-5 and NB5-6 arise in the Msh domain; however, only NB5-6 normally expresses Gsb. NB3-5 identity is assessed using R59E09>GFP and Ems; NB5-6 identity is assessed using Lbe>GFP, Eya, and Ap. B. Control embryos with NB3-5 lineages labelled by R59E09>GFP. Insets show wild-type expression of Eya, Ems and Ap. C. Test embryos with broad Gsb misexpression shows some missing NB3-5 lineages (yellow asterisks). Insets show a concomitant loss of Ems (white asterisk); expression of Eya and Ap largely remains unaffected. D. Control embryos with NB5-6 lineage labelled by Lbe>GFP. Insets show wild-type expression of Eya, Ems and Ap. E. Test embryos with broad Gsb misexpression show ectopic Lbe>GFP expression (yellow arrowheads). Insets show a concomitant gain of Eya, but not Ap, and loss of Ems. The loss of NB3-5 identity is not always accompanied by a gain in NB5-6 identity. (F–J) Quantification of lineage markers under control vs test conditions. Each point represents a single hemisegment; boxplots show median and interquartile range. Quantification of GFP, Ems, Eya and Ap is shown separately in thoracic as well as abdominal segments. 4% of thoracic hemilineages showed gain of eya accompanied by loss of 3-5 lineage and its marker ems (n=48). 27% of thoracic and 38% of abdominal hemisegments showed loss of 3-5 lineages along with loss of ems (n=48). Statistical significance was assessed using two-tailed Mann–Whitney U tests. Significance levels are indicated as: *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. Genotypes for both control embryos (no heat shock) and test embryos (with heat shock): hsGsb/w-; Lbe-k-Gal4, UAS-mCD8-GFP and hsGsb/w-;;R59E09,UAS-myrGFP.

Resource Table: List of fly lines used.

Resource Table-Antibodies Used

Verification of TaDa Tools.

A. Design of TaDa and CATaDa tools: The ubiquitous Da-Gal4 driver expresses mCherry from the primary ORF and either DamOnly (green) or Dam:TF (yellow) from the secondary ORF. High mCherry expression reports driver specificity, while translation of Dam fusions from the secondary ORF keeps Dam levels low, minimizing toxicity and preserving binding specificity. B. Whole-embryo DamOnly and Dam:Gsb bind reproducibly: Heatmap shows pairwise Pearson correlations between DamOnly and Dam:Gsb replicates. Hierarchical clustering separates DamOnly and Dam:Gsb samples, with stronger correlations within than between conditions. C. Binding at known Gsb targets: Genome browser snapshots of log2 ratio files (Dam:Gsb/DamOnly) are shown (3 replicates), Dam:Gsb in blue positive tracks, DamOnly in orange negative tracks. Replicates show occupancy at wg (46), gsb-n (47), prd (48). Data range −3.520-7.11. Pink bars indicate peaks, colour intensity indicates peak score. D. TaDa-Gsb correlates with published Gsb ChIP-seq data: Dam:Gsb signals are enriched at Gsb-ChIP peaks but not at Twi-ChIP or Bcd-ChIP sites (43). E. Motif enrichment at Dam:Gsb sites: de novo motif analysis at Dam:Gsb peaks reveals a fused Paired–Homeobox motif. Together (B-E) show that Dam:Gsb reproducibly and specifically reports Gsb occupancy in the chromatin. F. Whole-embryo DamOnly and Dam:En bind reproducibly: Heatmap shows pairwise-correlation between replicates of DamOnly and Dam:En. Hierarchical clustering shows that DamOnly and Dam:En replicates cluster separately, with stronger correlations within than between conditions. G. Binding at known En targets: Genome browser snapshots of log2 ratio files (Dam:En/DamOnly) are shown (3 replicates), Dam:En in blue positive tracks, DamOnly in orange negative tracks. Replicates show occupancy at en (49), beta-tub60D (50) and ptc (49). Data range −3.520-7.11. Pink bars indicate peaks, colour intensity indicates peak score. H. Motif enrichment at Dam:En sites: See entry at Rank 5, p-value 1e-17. Together, (F-H) show that Dam:En reproducibly and specifically reports En binding in chromatin. Genotypes for embryo-wide Gsb occupancy: w-;; Da-Gal4/UAS-LT3-Dam:Gsb. For embryo-wide En occupancy: w-;; Da-Gal4/UAS-LT3-Dam:En. For embryo-wide chromatin accessibility: w-;; Da-Gal4/UAS-LT3-DamOnly.

NB-specific sites of integration (SoIs) are differentially accessible (41).

A. Schematic representing NB5-6-SoIs (798 sites), defined as loci that are Hb-bound (light pink drop) and accessible in NB5-6 (dark blue DNA strand), but remain Hb-free (hollow drop, dotted light-pink outline) and less accessible in NB7-4 (light red DNA strand). Conversely, NB7-4-SoIs (230 sites) are Hb-bound (light pink drop) and accessible in NB7-4 (dark red DNA strand), while the corresponding loci in NB5-6 are Hb-free (hollow drop, dotted light-pink outline) and less accessible (light blue DNA strand) (FDR ≤ 0.01 and FC≥2). B-D. At NB5-6-SoIs, Hb signal from NB5-6 (dark grey) is higher than that from NB7-4 (light green) (B). Conversely, at NB7-4-SoIs, Hb signal from NB7-4 is higher than that from NB5-6 (C). At shared Hb-bound loci (1140 sites, Hb-bound in both NBs with ≥50% overlap), Hb binding signal from both NBs is similar. 3 Hb replicates each from NB5-6 and NB7-4 have been averaged. Solid lines indicate mean; coloured ribbons indicate 95% confidence intervals. Insets show heatmaps of Hb signal from NB5-6 and NB7-4 at the corresponding loci, averaged across replicates. E-G. At NB5-6-SoIs, Dam signal shows chromatin is accessible in NB5-6 (blue) but less accessible in NB7-4 (red) (E). At NB7-4-SoIs, Dam signal shows chromatin is more accessible in NB7-4 than in NB5-6 (F). At shared Hb-bound sites, chromatin is similarly accessible in both NBs (G). Together (B-G) validates computationally determined SoIs. 4 replicates of Dam from NB5-6 have been averaged, as have 3 replicates of Dam from NB7-4. Solid lines indicate mean, coloured ribbon indicates 95% confidence interval. Insets show heatmaps of Dam from NB5-6 and NB7-4 at the corresponding loci, averaged across replicates. Genotypes for NB5-6-Hb occupancy: w-; Lbe-k-Gal4/+; +/UAS- Dam:Hb. NB7-4-Hb occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:Hb (41). NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly.

Validation of NB-specific accessible sites.

A. Schematic showing uniquely accessible (dark blue) and less accessible (light blue) loci in NB5-6. B. Schematic showing uniquely accessible (dark red) and less accessible (light red) loci in NB7-4. C-E. Dam signal at differentially accessible chromatin: At sites uniquely accessible in NB5-6 but less accessible in NB7-4 (118 sites), Dam signal from NB5-6 (blue) is higher than that from NB7-4 (red) (C). At sites uniquely accessible in NB7-4 but less accessible in NB5-6 (306 sites) Dam signal from NB7-4 is higher than from NB5-6 (D). At sites accessible in both NBs (2287 sites, Dam-bound in both NBs with ≥50% overlap), Dam signal from both NBs are similar. Together, (C-E) validate computationally determined NB-specific accessible sites. 4 replicates of Dam from NB5-6 have been averaged, as have 3 replicates of Dam from NB7-4. Solid lines indicates mean, coloured ribbons indicate 95% confidence interval. Insets show heatmaps of Dam signal from NB5-6 and NB7-4 at the corresponding loci, averaged across replicates. Genotypes for NB5-6-chromatin accessibility: w-; Lbe-k-Gal4/+; +/UAS-DamOnly. NB7-4-chromatin accessibility: w-; 19B03[AD]/+; 18F07[DBD]/UAS-DamOnly.

Validation of computationally determined STF-bound and -unbound sites.

A. Schematic representing Gsb-bound (blue drops) and -unbound sites (hollow drops, dotted blue outline). B. Schematic representing En-bound (pink drops) and -unbound sites (hollow drops, dotted pink outline). Sites defined as ‘Gsb-bound’ in NB5-6 are ‘En-unbound’ in NB7-4 (1158 sites). Conversely, sites that are ‘En-bound’ in NB7-4 are ‘Gsb-unbound’ in NB5-6 (786 sites) (FDR ≤ 0.01 and FC ≥2). C-E. STF signal at STF-bound/unbound sites: At Gsb-bound/En-unbound sites, Gsb signal from NB5-6 (blue) is higher than En signal from NB7-4 (pink) (C). At Gsb-unbound/En-bound sites, En signal from NB7-4 is higher than Gsb signal from NB5-6 (D). At sites STF-bound in both NBs (740 sites, NB5-6-Gsb-bound and NB7-4-En-bound peaks, with ≥50% overlap), Gsb signal from NB5-6 is similar to En signal from NB7-4. Together (C-E) validate computationally determined STF-bound/unbound sites. 4 replicates of Gsb from NB5-6 have been averaged, as have 3 replicates of En from NB7-4. Solid lines indicates mean, coloured ribbons indicate 95% confidence interval. Insets show heatmaps of Gsb from NB5-6 and En from NB7-4 at the corresponding loci, averaged across replicates. Genotypes for NB5-6-Gsb occupancy: w-; Lbe-k-Gal4/+; +/UAS-Dam:Gsb. NB7-4-En occupancy: w-; 19B03[AD]/+; 18F07[DBD]/UAS-Dam:En.

Validation of heat shock regime for Gsb misexpression.

A. Schematic showing heat shock protocol for sample collection. Embryos were collected (0h-1h AEL) and incubated at 25°C (control, no Gsb misexpression) or moved to 33°C (test, with Gsb misexpression) pre-delamination (Stage 7-8). B. Increased Gsb expression upon heat shock: qPCR results show increase in Gsb transcripts relative to wild-type CS embryos, under the heat shock protocol outlined in (A). C-D. Continued Gal4 expression upon Gsb misexpression: Crosses were set up to obtain embryos of the genotype hsGsb;19B03[AD]/UAS-mCD8-dsRed;18F07[DBD], with or without heatshock. (C-D) show that this NB7-4-specific Gal4 expression stays on under the heat shock protocol outlined in (A). White asterisks mark missing NB7-4 lineages. Scale bar 10µ.

Validation of gsb-guideRNA.

A. In control embryos (Vasa Cas9 > sgWhite), Vasa-Cas9 is directed to the white gene locus by the sgWhite guideRNA. FasII staining in these embryos shows intact axonal tracts. B. In test embryos (Vasa Cas9 > gsb gRNA), axonal tract defect is seen, consistent with loss of Gsb in embryos. C-D. Sanger sequencing chromatograms confirm Cas9-mediated editing at the gsb locus. Sequencing chromatograms of embryos expressing Vasa-Cas9 and sgWhite control (C) or gsb-guideRNA (Edited sample, (D)) were analyzed using the Synthego ICE (Inference of CRISPR Edits) online tool. The predicted Cas9 cut site (dashed black line) corresponds to position 747 bp. Mixed peaks downstream of the cut site in the edited sample indicate indel formation at the gsb target locus, consistent with efficient CRISPR editing. The control trace shows clean base calls with no evidence of indels. ICE predicts 75% editing efficiency. Genotypes for control embryos: Vasa Cas9/+; sgWhite/+. For test embryos: Vasa Cas9/y[1]v[1]; gsb-guideRNA/+.