Notch signaling is activated in newborn L4 neurons but not L5.

(A) Schematic of Notch signaling pathway.

(B-B”) Hey as a reporter of active Notch signaling is only expressed in newborn L4 neurons but no other lamina neurons at 15h APF. Here and below, scale bar: 10 µm, n≥5 brains. Dashed line delineates the boundary between Bsh+ L4 and Bsh+ L5 neurons.

(C, D) Hey is expressed prior to the activation of the secondary HDTFs Ap and Pdm3 at 15h APF. Dashed line delineates the boundary between Bsh+ L4 and Bsh+ L5 neurons.

(E-H) Using twin-spot MARCM, two sibling neurons generated by one progenitor are traced. RFP and GFP cells are either both L4 neurons (Bsh+Pdm3-) or both L5 neurons (Bsh+Pdm3+). N=4.

Dashed line outlines RFP+ and GFP+ cell bodies.

L1 neurons express Delta and activate Notch signaling in adjacent L4 neurons.

(A, A’) Svp+Zfh1+ L1 neurons are adjacent to both newborn L3 and L4 neurons. Newborn L3 neurons (Erm+) are localized strictly above (distal to) L1 neurons (Svp+Zfh1+) and L1 neurons are localized strictly above (distal to) L4 neurons. Here and below, scale bar: 10 µm, n≥5 brains. Dashed line delineates the boundary between L1 (Svp+Zfh1+) and L4 (Bsh+) cell bodies.

(B, B’) Delta is expressed in Zfh1+ L1 neurons which are adjacent to Bsh+ L4 neurons. Dashed line delineates the boundary between L1 (Zfh1+) and L4 (Bsh+) cell bodies.

(C-C”) Delta is also expressed in a subset of LPCs (Tll+). Dashed line highlights Delta+ cell bodies.

(D) Summary of A-C data; triangles represent Delta expression.

(E-F”) Delta-KD (27G05-Gal4, tubP-GAL80[ts], UAS-Delta-RNAi) results in loss of Delta and Hey expression in lamina. HRP labels the axons of the photoreceptors which represent the lamina column. Solid white line outlines the lamina and dashed line delineates the boundary between Delta + cells and Hey+ cells.

(G) Summary

Bsh without Notch signaling activates Pdm3 and specifies L5 neuronal fate.

(A-D) N-KD in lamina (27G05-Gal4, tubP-GAL80[ts], UAS-Notch-RNAi) shows Hey expression is absent, and Bsh only activates Pdm3 and specifies L5 neuronal fate during lamina neurogenesis (19h APF). Here and below, scale bar, 10 µm, n≥5 brains.

(E-J) N-KD in lamina shows loss of Ap+Pdm3-L4 neurons and gain of Pdm3+ L5 neurons at 3-4d APF. (I-J) Quantification (single optical section).

(K) Summary

Data are presented as mean ± SEM. Each dot represents each brain. n=6 brains in (I), (J).

***P<0.001, ns=not significant, unpaired t-test.

Bsh with Notch signaling activates Ap and specifies L4 neuronal fate.

(A-D) Ectopic expression of N-ICD in newborn L5 neurons (Bsh-Gal4>UAS-N-ICD) results in ectopic Hey and Ap activation and an increased number of L4 neurons at 19h APF. Here and below, scale bar, 10 µm, n≥5 brains.

(E-J) N-ICD results in loss of Pdm3+ L5 neurons and gain of Ap+ L4 neurons at 3-4d APF. (I-J) Quantification (single optical section).

(K) Summary.

Data are presented as mean ± SEM. Each dot represents each brain. n=6 brains in (I), (J).

***P<0.001, ns=not significant, unpaired t-test.

Notch activation and Bsh expression are mutually independent; Notch signaling without Bsh specifies L3 neuron type.

(A-F) Notch activation and Bsh expression are mutually independent. (A-C) N-KD in lamina (27G05-Gal4, tubP-GAL80[ts], UAS-Notch-RNAi) results in loss of Hey expression without affecting Bsh expression. (C) Quantification (single optical section). Here and below, scale bar, 10 µm, n≥5 brains. (D-F) Bsh-KD in lamina (27G05-Gal4>UAS-Bsh-RNAi) results in loss of Bsh expression without affecting Hey expression. (F) Quantification (single optical section).

(G-L) Notch signaling without Bsh specifies L3 neuron type. (G) Schematic of the genetics used to trace the morphology of Hey+ lamina neurons. (H, I) In controls, GFP+ neurons express the L4 marker Bsh (white arrowhead) at 19h APF. In Bsh-KD, Bsh expression becomes absent in lamina and GFP+ cells now express L3 markers Zfh1 and Erm (white arrowhead). (J-L) In controls, GFP+ cells express the L4 marker Ap (white arrowhead) and have L4 morphology (asterisk) at 2.5d APF. In Bsh-KD, GFP+ cells express L3 marker Erm (white arrowhead) and adopt L3 morphology (asterisk). (L) Quantification for (J) and (K) (single optical section).

(M-O) (M) Schematic of the genetics used to trace the morphology and presynaptic sites of Hey+ neurons. (N, O) In controls, Tomato+ neurons have L4 morphology and presynaptic sites (Brp) in lamina. In Bsh-KD, Tomato+ neurons adopt L3 morphology and connectivity which lacks presynaptic localization in lamina (Xu et al., 2019).

(P) Summary.

Data are presented as mean ± SEM. Each dot represents each brain. n=5 brains in (C), (F) and (L).

***P<0.001, ns=not significant, unpaired t-test.

The NotchON L4 has correlated open chromatin, Bsh-bound loci and transcription profile that is distinct from the NotchOFF L5.

(A) Schematic of the Targeted DamID method. Upon GAL4 induction, low-levels of either Dam or Bsh:Dam are expressed, allowing genome-wide open chromatin and Bsh binding targets to be identified.

(B) L4 and L5 neurons show distinct open chromatin regions (yellow), and L4-specific transcribed genes (red) are enriched in L4-distinct open chromatin regions.

(C) L4 and L5 neurons show distinct Bsh-bound loci (yellow), and L4-specific transcribed genes (red) are enriched in L4-distinct Bsh-bound loci. P values from Fisher’s exact test; odds ratios (OR) expressed as L4/L5 in all cases.

(D) Model.

Model; see the discussion section for details.

E(spl)-mγ is not expressed in lamina neurons.

(A-A”) E(spl)-mγ-GFP is expressed in LPC region but not lamina neurons. Erm labels L3; Svp labels L1; Bsh labels Bsh+ LPCs, L4 and L5. Solid white line outlines LPC region.

Svp is not required to initiate or maintain Delta expression in L1 neurons.

(A-D) In Svp-KD (27G05-Gal4>UAS-Svp-RNAi), there is a loss of Svp while Hey expression remains unaffected at 19h APF. (C-D) Quantification (single optical section). Here and below, scale bar, 10 µm, n≥5 brains.

(E-G) In Svp-KD (27G05-Gal4>UAS-Svp-RNAi), Delta expression remains unaffected at 19h APF.

(G) Quantification (single optical section).

Data are presented as mean ± SEM. Each dot represents each brain. n=5 brains in (C) and (D). n=6 brains for control and n=5 brains for Svp-KD in (G). ***P<0.001, ns=not significant, unpaired t-test.

27G05-Gal4 is inserted in between two Notch target genes; Bsh without Notch signaling activates Pdm3 and specifies L5 morphology.

(A) Schematic of 27G05-Gal4 insertion site in between two Notch target genes (E(spl)m4-BFM and E(spl)m5-HLH).

(B-C”) Bsh without Notch signaling activates Pdm3 and specifies L5 morphology. In N-KD (27G05- Gal4, tubP-GAL80[ts], UAS-Notch-RNAi), Bsh activates Pdm3 but not Ap and specifies L5 neuronal fate during lamina neurogenesis (19h APF). White line outlines Bsh+ lamina cells. Scale bar, 10 µm, n≥5 brains.

Bsh specifies L5 neuronal fate over L4 following Delta-KD.

(A-C) Delta-KD (27G05-Gal4, tubP-GAL80[ts], UAS-Delta-RNAi) in lamina shows the decrease of Ap+ Pdm3-L4 neurons and increase of Pdm3+ L5 neurons at 3-4d APF. (C) Quantification (single optical section). Scale bar, 10 µm.

Data are presented as mean ± SEM. Each dot represents each brain. n=5 brains. **P<0.01, ns=not significant, unpaired t-test.

Temporally-restricted activation of Notch signaling by 27G05- Gal4 enables Bsh to specify L4 neuronal fate over L5.

(A) Schematic of the genetics for temporally-restricted activation of Notch signaling.

(B-C”) Activation of Notch signaling from 0h APF (affected region circled) results in ectopic Hey expression at 19h APF. Here and below, scale bar, 10 µm, n≥5 brains.

(D-G) Activation of Notch signaling from 0h APF (affected region circled) results in no change to Bsh expression, loss of Pdm3+ L5 neurons and gain of Ap+Pdm3-L4 neurons at 3-4d APF. (F and G) Quantification (single optical section).

Data are presented as mean ± SEM. Each dot represents each brain. ***P<0.001, ns=not significant, unpaired t-test.

Bsh-Gal4 is expressed in newborn L5 neurons; Bsh does not activate L5 marker Pdm3 when Notch signaling is activated in newborn L5 by Bsh-Gal4 and UAS-N-ICD.

(A-B”) Bsh-Gal4 is expressed in newborn L5 neurons. (A-A”) GFP+ cells (white line circle; Bsh-Gal4>UAS-myrGFP) label L5 neurons which are in the bottom (proximal) row of Bsh+ cells during lamina neurogenesis (13h APF). Here and below, scale bar, 10 µm, n≥5 brains. (B-B”) GFP+ cells (white line circle; Bsh-Gal4>UAS-myrGFP) label Pdm3+ newborn L5 and early born L5 neurons during lamina neurogenesis (16h APF).

(C-D”) Bsh does not activate L5 marker Pdm3 when Notch signaling is activated in newborn L5 by Bsh-Gal4 and UAS-N-ICD. Pdm3 expression is initiated in L5 neurons in control but not in N-ICD (Bsh-Gal4>UAS-N-ICD).

Bsh-Gal4 activation of Notch signaling in newborn L5 neurons specifies L4-like morphology and presynaptic sites.

(A) Schematic of the genetics used to trace the morphology and presynaptic site (Brp) of Hey+ neurons.

(B-E) In controls, Tomato+ neurons in lamina have L4 morphology and presynaptic sites labeled by Brp-V5. In Bsh-KD, Tomato+ cells, which trace the endogenous and ectopic Hey+ neurons, have L4-like morphology and presynaptic sites labeled by Brp-V5 in the lamina. Scale bar, 10 µm. n≥5 brains.

6-60-Gal4 used for TaDa experiment is specifically expressed in L5 neurons at 46h-76h APF.

(A) Schematic of the genetics used to perform targeted Dam ID experiments (see Figure 6).

(B-B”) 6-60-Gal4, which is expressed at 46h-76h APF, drives GFP expression in all Pdm3+ L5 neurons and weak GFP expression in some Ap+ L4 neurons. Scale bar, 10 µm. n≥5 brains.

(C) Both Dam and Bsh:Dam show high Pearson correlation coefficients among their three biological replicates, with lower Pearson correlation coefficients between Dam and Bsh:Dam. Heat map is generated using the multiBamSummary and plotCorrelation functions of deepTools.

L4-disinct open chromatin regions are more likely to contain the Su(H) binding motif; L5-specific transcribed genes are correlated with L5-distinct open chromatin regions but not L5-distinct Bsh-bound loci.

(A) L4 and L5 neurons show distinct open chromatin regions (yellow), and the Su(H) binding motif (blue) is more likely to be present in L4-distinct open chromatin regions.

(B) L4 and L5 neurons show distinct open chromatin regions (yellow), and L5-specific transcribed genes (red) are enriched in L5-distinct open chromatin regions.

(C) L4 and L5 neurons show distinct Bsh-bound loci (yellow); L5-specific transcribed genes (red) and L5-distinct Bsh-bound loci do not show correlation. P values from Fisher’s exact test; odds ratios (OR) expressed as L4/L5 in all cases.