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. The 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. The 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. The 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. The 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+). The 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. A solid white line outlines the lamina and a 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 Bsh+ lamina neurons are mainly Pdm3+ L5 neurons, though the number of Bsh+ lamina neurons remains unaffected 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 shows Bsh+ lamina neurons are mainly Ap+ L4 neurons, though the number of Bsh+ lamina neurons remains unaffected 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 the lamina. In Bsh-KD, Tomato+ neurons adopt L3 morphology and connectivity, which lacks presynaptic localization in the 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 the LPC region but not in lamina neurons. Erm labels L3; Svp labels L1; Bsh labels Bsh+ LPCs, L4 and L5. A solid white line outlines the 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). The 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 increase of Pdm3+ L5 neurons at the expense of L4 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-distinct 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.