Sequential initiation of HDTFs during lamina neurogenesis

(A-A’’) Tll is identified as an LPC marker, expressed complementary to Elav; Dac labels both Tll+ LPCs and Elav+ neurons. LF: lamina furrow. Here and below, scale bar: 10 µm, n≥5 brains.

(B-B’’’) Tll+ cells are localized both within the lamina columns and before the columns. Lamina columns (white dash circle) are outlined by the photoreceptor axons which is labeled by Chaoptin. n≥5 brains. LF: lamina furrow.

(C-C’’’) Bsh is expressed in Tll+ Elav-LPCs (white dash circle) as well as in Elav+ L4 and L5 neurons.

(D-D’’’) Ap is expressed in L4 neurons. Newborn L4 neurons are Bsh+ Elav+ Ap-(yellow line circle), while older L4 neurons are Bsh+ Elav+ Ap+ (white line circle).

(E-E’’’) Pdm3 is expressed in L5 neurons. Newborn L5 neurons are Bsh+ Pdm3-(yellow line circle), while older L5 neurons are Bsh+ Pdm3+ (white line circle).

(F-F’’) Bsh, Ap and Pdm3 expressions are maintained in adult.

(G) Schematic of lamina neuron development in early pupa.

(H) Summary.

Bsh activates Ap/Pdm3 expression and specifies L4/L5 neuronal fate

(A-B’’) Bsh-KD in LPCs (R27G05-Gal4>UAS-Bsh-RNAi) eliminates Bsh in LPCs and neurons in wandering L3 (white circle). Tll labels all LPCs. LF: lamina furrow. L3: third larval instar. Here and below, scale bar: 10 µm, n≥5 brains.

(C) Schematic of lamina neuron development at 3rd larval instar.

(D) Schematic of lamina neuron development from 1day pupae to adult.

(E-F’’) Bsh remains undetectable in the lamina of 3-4day pupa in Bsh-KD (R27G05-Gal4>UAS-Bsh-RNAi).

(G-K) Bsh-KD in LPCs removes most L4 (Bsh/Ap) (J) and L5 (Bsh/Pdm3) (I) neuron markers. The Ap expression in L5 is caused by the Gal4 driver line but is irrelevant here. (K) The number of Elav+ cells in single slice. n=5 brains in (I) and (J), n=7 brains in (K). L4 layer, yellow outline. L5 layer, white outline.

(L) Summary.

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

Bsh suppresses L1/L3 neuronal fate

(A-E) Bsh-KD in LPCs results in the ectopic expression of the L1 marker Svp and L3 marker Erm in L4/L5 cell body layers (circled). (C) Schematic of lamina neuron development from 1day pupae to adult. (D and E) Quantification of Erm and Svp expression. Here and below, scale bar, 10 µm.

(F-H) Bsh-KD in LPCs does not produce ectopic Bab2-positive neurons or glia in the L5 layer (circled). n≥5 brains. Genotype: R27G05-Gal4>UAS-Bsh-RNAi. (H) Quantification of Bab2 expression.

(I-L) Bsh-KD in LPCs results in ectopic Svp+ L1 neurons at the expense of Pdm3+ L5 neurons. Bsh GFP+ neurons marked with yellow arrowheads show L1 marker Svp expression in Bsh-KD while L5 marker Pdm3 expression in control. Genotype: Bsh-LexA>LexAop-GFP. (K and L) Quantification of Pdm3 and Svp expression.

(M-O) Bsh-KD transforms L5 neuron morphology to L1-like neuronal morphology. (M) Control L5 neurons have very few dendrites in the lamina neuropil. Genotype: R27G05-Gal4, Bsh-LexA>LexAop-GFP. (N) Control L1 neurons show bushy dendrites throughout the lamina. Genotype: svp-Gal4, R27G05-FLP>UAS-FRT-stop-FRT-myrGFP. (O) Bsh-KD transforms L5 neuron morphology to L1-like neuronal morphology. Genotype: R27G05-Gal4>UAS-Bsh-RNAi; Bsh-LexA>LexAop-GFP.

(P) Summary.

Data are presented as mean ± SEM. Each dot represents each brain. n=5 brains in (D), (E), (H), (K) and (L). *P<0.05, **P<0.01, ***P<0.001, ns=not significant, unpaired t-test.

Bsh represses Zfh1 to suppress L1/L3 neuronal fate

(A-A’’) Zfh1 is expressed in all LPCs and some lamina neurons at 12h APF. GFP labels all lamina cells. Elav labels lamina neurons. Yellow dash circle outlines LPCs and white dash circle outlines lamina neurons. Genotype: R27G05-Gal4>UAS-myrGFP. Here and below, scale bar, 10 µm. n≥5 brains.

(B-B’’) Zfh1 is expressed in Svp+ L1 and Erm+ L3 neurons at 1-2d APF; Svp and Erm are never coexpressed. White dash circle outlines L1 and L3 neurons and yellow line indicates the rough boundary between L1 and L3 cell bodies.

(C) Schematic of lamina neuron development from 1day pupae to adult.

(D-F) Zfh1-KD in LPCs results in a loss of Svp+ L1 and Erm+ L3 neurons. Quantification: the number of Erm+ or Svp+ cell bodies in single optical slice. Genotype: R27G05-Gal4>UAS-zfh1-RNAi.

(G-I) Bsh-KD in LPCs results in ectopic Zfh1 in L4/L5 layers. White circles label Bsh+ cell bodies in L4 layer in control. L5 layer, white outline. Quantification: the percentage of Bsh+ or Zfh1+ neurons in L5 layer. Genotype: R27G05-Gal4>UAS-bsh-RNAi.

(J) Summary.

Data are presented as mean ± SEM. Each dot represents each brain. n=6 brains in (F) and n=5 brains in (I). ***P<0.001, unpaired t-test.

Bsh:DamID reveals Bsh direct binding to L4 identity genes and pan-neuronal genes

(A) Schematic for TaDa method.

(B) 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.

(C) Bsh:Dam peaks in L4 (46h-76h APF) called by find_peaks and L4 scRNAseq data (48h APF; 60h APF)(Jain et al., 2022a) are combined (see method).

(D) Bsh:Dam shows strong signals in L4 identity genes and pan-neuronal gene. The Dam alone signal indicates the open chromatin region in L4 neurons. The y axes of Bsh:Dam signal represent log2 (Bsh:Dam/Dam) scores. Bsh peaks in L4 neurons were generated using find_peaks (FDR<0.01; min_quant=0.9) and peaks2genes.

(E) Summary

Bsh and Ap form a coherent feed-forward loop to activate DIP-β

(A-D) Bsh Crispr KO in postmitotic L4 neurons results in loss of DIP-β expression in the proximal lamina neuropil (arrow) at 3d APF. DIP-β expression is detected using anti-DIP-β antibody. Signal in the distal lamina is from non-lamina neurons, probably LawF. Significantly reduced DIP-β fluorescence intensity is observed in the proximal lamina (75%–100% distance, marked by red bar (C, D). ***P<0.001, unpaired t-test, n=8 brains, each line represents each brain, scale bar, 10 µm. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs.

(E-I) Bsh Crispr KO in L4 neurons results in a decrease of primary dendrite length and proximal synapse number in postmitotic L4 neurons of 1d adults. Here and below, white dash lines indicate the lamina neuropil, and yellow lines show the boundary between the distal and proximal lamina. The average number of Brp puncta in L4 neurons present within the distal or proximal halves of lamina cartridges. *P<0.05, ***P<0.001, ns= not significant, unpaired t-test, n=100 cartridges, n=5 brains, each dot represents one cartridge, data are presented as mean ± SEM. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs, UAS-myrGFP, UAS-RSR, Brp-rst-stop-rst-smFPV5-2a-GAL4.

(J-M) Ap RNAi KD in postmitotic L4 neurons results in loss of DIP-β expression in the proximal lamina neuropil (arrow) at 3d APF. Signal in the distal lamina is from non-lamina neurons, probably LawF. Significantly reduced DIP-β fluorescence intensity is observed in the proximal lamina (75%– 100% distance, marked by red bar (L, M). ***P<0.001, unpaired t-test, n=8 brains, each line represents each brain, scale bar, 10 µm. Genotype: R27G05-Gal4>UAS-ApRNAi.

(N-R) Ap-KD in L4 neurons results in an increase of primary dendrite length and proximal synapse number in postmitotic L4 neurons in 1d adults. The average number of Brp puncta in L4 neurons present within the distal or proximal halves of lamina cartridges. *P<0.05, ***P<0.001, ns= not significant, unpaired t-test, n=100 cartridges, n=5 brains, each dot represents one cartridge, data are presented as mean ± SEM. Genotype: 31C06-AD, 34G07-DBD>UAS-RSR, 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4, UAS-Ap-shRNA, UAS-myrGFP.

(S) Summary.

Bsh+ L4/L5 are required for normal visual sensitivity

(A) Schematic of the Fly Vision Box.

(B) Bsh-KD adult flies show reduced responses to a high-speed stimulus. Left: stimulus with different temporal frequency; right: stimulus with 5Hz temporal frequency, but with the indicated contrast level.

(C) Bsh-KD adult flies show reduced phototaxis to both dim and bright lights. Relative intensity was used: 15 and 200 for dim and bright green respectively; 20 and 100 for dim and bright UV respectively.

(D) Bsh-KD adult flies show larger responses towards bright UV illumination. For lower UV levels, flies walk towards the green LED, but walk towards the UV LED at higher UV levels.

Data are presented as mean ± SD, with individual data points representing the mean value of all flies in each tube. For control (mcherry-RNAi), n=18 groups of flies (tubes), for Bsh-KD, n=16 groups of flies, run across 3 different experiments. Each group is 11-13 male flies. *P< 0.05, unpaired, two-sample t test controlled for false discovery rate.

(E) Model. Left: In wild type, Zfh1+ LPCs give rise to L1 and L3 neurons, whereas Zfh1+Bsh+ LPCs give rise to L4 and L5 neurons. The lineage relationship between these neurons is unknown. Right: Bsh KD results in transformation of L4/L5 into L1/L3 which may reveal a simpler, ancestral pattern of lamina neurons that contains the core visual system processing arrangement.

(F) Summary.

R27G05-GAL4 is turned on in all LPCs and turned off in lamina neurons.

(A-A’’’) R27G05-Gal4 drives GFP expression in all LPCs and lamina neurons (white dash circle) which are labeled by Dac at 96 hours after larval hatching (ALH). Hth labels LPCs next to lamina furrow (Piñeiro et al., 2014). Here and below, scale bar, 10 µm.

(B, C) R27G05-Gal4 is turned off in lamina neurons. Activating R27G05-Gal4 at 29C at the beginning of wandering L3 for 20 hours (by inactivating Gal80ts) reveals continued expression of GFP; in contrast, activating R27G05-Gal4 at 29C at 1d APF for 20 hours reveals little GFP, showing that R27G05 is not expressed in pupal stages containing postmitotic lamina neurons. Genotype: R27G05-Gal4>UAS-myrGFP, tubP-Gal80[ts].

(D, E) Although R27G05-Gal4 is turned off by 1d APF, GFP persists in lamina neuron cell bodies until 66h APF and in their neurites until 75h APF. Genotype: R27G05-Gal4>UAS-myrGFP.

Bsh-KD or Bsh-KO in LPCs affects L4/L5 neuronal fate.

(A-D) Bsh-KD in LPCs removes most L4 (Bsh/Ap) and L5 (Bsh/Pdm3) neuron markers of 3day adult flies. The Ap expression in L5 is caused by the Gal4 driver line but is irrelevant here. (C and D) The number of Pdm3+ (C) or Ap+/Pdm3-(D) cell bodies in single slice in control (A) and Bsh-KD (B). Here and below, scale bar, 10 µm, white circle outlines the region of L4 and L5 cell bodies. (E-I) Bsh-KO in LPCs (27G05-Gal4>UAS-Cas9, UAS-Bsh-sgRNAs) decreases Ap and Pdm3 expression in the lamina of 3-4day old pupa. (G-I) The number of Bsh+ (G), Pdm3+ (H) or Ap+/Pdm3-(I) cell bodies in single slice in control (E) and Bsh-KO (F).

Data are presented as mean ± SEM. Each dot represents each brain. n=6 brains in (C) and (D), n=5 brains in (G), (H), and (I). Each dot represents each brain. ***P<0.001, unpaired t-test.

Bsh-KD in L4 neurons results in loss of detectable Bsh between 2d and 3d APF.

(A-C) Bsh expression is detected at 2d APF following Bsh-KO in L4 neurons. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs.

(D-F) Bsh expression is absent at 3d APF following Bsh-KD in L4 neurons. Genotype: 31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs. GFP labels L4 neurons. White arrowhead indicates L4 cell body lacking Bsh expression.

Data are presented as mean ± SEM. ns= not significant, ***P<0.001, unpaired t-test, n= 6 brains; each dot represents one brain. Scale bar, 10 µm.

Bsh is not required in L4 neurons to maintain L4 neuronal fate.

(A-B’’) L1 marker Svp and L2 marker Bab2 are not detected in L4 cell bodies when knocking out Bsh in L4 (31C06-AD, 34G07-DBD>UAS-Cas9, UAS-Bsh-sgRNAs). Here and below, L4 is labeled by GFP (31C06-AD, 34G07-DBD> UAS-myrGFP). White outline, L4 cell body. Scale bar, 10 µm. (C-D’’) L3 marker Erm is not detected in L4 cell bodies when knocking out Bsh in L4.

(E-F’’) Bsh becomes absent in most L4 while Ap expression remains normal when knocking out Bsh in L4.

(G-H’’) L5 marker Pdm3 is not detected in L4 cell bodies when knocking out Bsh in L4.

(I) The percentage of each lamina neuron marker in L4 neurons (GFP+). ***P<0.001, ns= not significant, unpaired t-test, n= 5 or 6 brains, each dot represents each brain, data are presented as mean ± SEM.

Loss of Bsh in LPCs transforms L5 neurons into L1 neurons.

(A-A’’) Bsh-LexA drives LexAop-myrGFP expression in Bsh+ LPCs and Bsh+ neurons. Tll labels lamina LPCs. White circle outlines GFP+ cells. Here and below, scale bar, 10 µm.

(B-B’’) Bsh-LexA drives LexAop-myrGFP expression in L5 neurons but not L4 at 20h APF. Older GFP cell bodies express L5 marker Pdm3. Ap labels L4 neurons. White circle outlines GFP+ cells. (C-D’’’) Bsh-KD results in L1 marker Svp ectopically expressed in GFP+ neurons but not GFP+ LPCs in Bsh-KD. GFP labels LPCs and L5 neurons in control (Bsh-LexA>LexAop-myrGFP); Tll labels LPCs. White line circle outlines GFP+ neurons. GFP+ LPCs are Tll positive (yellow arrowhead).

Ap-KD eliminates Ap initiation and maintenance in L4 neurons.

(A-C) Ap initiation in L4 neurons is eliminated at 19-20h APF when knocking down Ap (27G05-GAL4>UAS-Ap-RNAi). The top Bsh+ labels L4 and the bottom Bsh+ labels L5. Ap and Pdm3 are expressed in older L4 and older L5 neurons respectively. The Ap expression in L5 neurons is caused by the Gal4 driver line but is irrelevant here. Scale bar, 10 µm. (C) The percentage of Ap expression in L4 neurons (top Bsh+ row) in control (A) and Ap-KD (B).

(D-F) Ap expression remains undetectable in adult though Bsh expression remains normal. Bsh is unlikely able to reinitiate Ap expression if normal Ap initiation is lost. Scale bar, 10 µm. (F) The percentage of Ap expression (Ap+/Pdm3-) in L4 neurons (Bsh+/Pdm3-) in control (D) and Ap-KD (E).

Data are presented as mean ± SEM. Each dot represents each brain. n=6 brains in (C) and (F).

***P<0.001, unpaired t-test.

Ap is not required to suppress other lamina neuron markers in L4 neurons.

(A-H’’) L1 (Svp, Zfh1), L2 (Bab2), L3 (Erm, Zfh1) and L5 (Pdm3) markers are not expressed in L4 neurons (top Bsh+ row) at 3-4d APF when knocking down Ap (27G05-GAL4>UAS-Ap-RNAi). Scale bar, 10 µm.

(I) The percentage of each lamina neuron marker in L4 neurons (top row of Bsh+). ***P<0.001, ns= not significant, unpaired t-test, n= 6 brains, each dot represents each brain, data are presented as mean ± SEM.

Zfh1 expression and epistasis with Bsh.

(A-D) Zfh1-KD in LPCs significantly reduces Zfh1 expression in all LPCs and lamina neurons at 12h APF. White line circle outlines LPCs. Yellow line circle outlines lamina neurons. White dash circle outline Zfh1+ glia. Scale bar here and below, 10 µm. Genotype: R27G05-Gal4>UAS-Zfh1-RNAi. Quantification: Zfh1 signal of three cells bodies from each brain and normalized Zfh1 signal by setting the highest Zfh1 signal as 100.

(E-H) The total number of Ap+ and Pdm3+ cell bodies is decreased when knocking down Zfh1 in LPCs. The number of Bsh+ L4 and L5 neurons is decreased when knocking down Zfh1 in LPCs. Scale bar, 10 µm. (G) The total number of Ap+ and Pdm3+ cell bodies in single slice. (H) The number of Bsh+ L4 and L5 neurons in single slice. Genotype: R27G05-Gal4>UAS-Zfh1-RNAi.

(I-K) The number of lamina neurons is decreased when knocking down Zfh1 in LPCs. Scale bar, 10 µm. (K) The number of Elav+ cells in single slice. Genotype: R27G05-Gal4>UAS-Zfh1-RNAi.

(L-N) Zfh1 is ectopically expressed in GFP+ neurons. GFP (Bsh-LexA>LexAop-myrGFP) labels LPCs and L5 neurons in control. White line circle outlines GFP+ neurons. GFP+ LPCs are Tll+ (yellow arrowhead). Scale bar, 10 µm. (N) The percentage of Zfh1+ cells in GFP+ neurons (Elav+) in control (L) and Bsh-KD (M). We divided GFP+ neurons into three domains with equal width from the newborn to older neurons.

Data are presented as mean ± SEM. Each dot represents a brain. n=5 brains per genotype. **P<0.01,

***P<0.001, unpaired t-test.

UAS-Bsh:Dam expressed by 31C06-Gal4 is functional and specifically expressed in L4 neurons; Bsh:Dam shows binding peak in ap locus but not pdm3 or zfh1 in L4 neurons.

(A) Schematic of the system used to restrict Bsh:Dam expression spatially to L4 (using the 31C06-Gal4 driver) and temporally to 46-76h APF (using the temperature-sensitive Gal4 inhibitor, Gal80ts; red T-bar). Note high expression of GFP from the first ORF (thick green arrow), and low expression of Bsh or Bsh:Dam following readthrough of the stop codons prior to the second ORF (thin green arrow).

(B-B’’) 31C06-Gal4, Gal80ts, UAS-Bsh:Dam is expressed in Ap+ L4 neurons when conditionally expressed from 46h-76h APF. Scale bar, 10 µm.

(C-E) Bsh:Dam retains Bsh function and can partially rescue Ap and Pdm3 expressions in Bsh-KO flies (Bsh-KO; R27G05-Gal4>UAS-myrGFP-Bsh:Dam). Note that Bsh:Dam expression from the second ORF is extremely low to avoid the toxicity and is not detected by immunostaining. White circle outlines the cell body area of lamina neurons. (E) Quantification of the number of Ap+ or Pdm3+ or Bsh+ cell bodies within z stack of 2.88 µm in control (C-C’’) and Bsh:Dam (D-D’’). Scale bar, 10 µm. Data are presented as mean ± SEM. Each dot represents a brain. Control: n=5 brains; Bsh:Dam: n=4 brains. ns= not significant, **P<0.001, unpaired t-test.

(F) Bsh:Dam shows strong signals in ap gene but not pdm3 or zfh1 gene in L4 neurons during synapse formation window. The Dam alone signal indicates the open chromatin region in L4 neurons. The y axes of Bsh:Dam signal represent log2 (Bsh:Dam/Dam) scores. Bsh peaks in L4 neurons were generated using find_peaks (FDR<0.01; min_quant=0.9) and peaks2genes.

DIP-β expression is disrupted when knocking down Ap in L4 neurons.

(A-C) Ap-KD (31C06-AD, 34G07-DBD>UAS-RSR, 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4, UAS-Ap-shRNA; see methods for an explanation of this genotype) significantly reduced Ap expression in L4 neurons at 2d APF. White line circle outlines lamina neuron cell bodies. Scale bar, 10 µm. (C) Quantification of Ap signal in the cell bodies of L4 neurons in control (A) and Ap-KD

(B). We averaged the Ap signal of three cells bodies from each brain and normalized Ap signal by setting the highest Ap signal as 100. ***P<0.001, unpaired t-test, n= 6 brains, each dot represents each brain, data are presented as mean ± SEM.

(D-F’) DIP-β expression is disrupted in L4 neurons in Ap-KD (31C06-AD, 34G07-DBD>UAS-RSR and 79C23-S-GS-rst-stop-rst-smFPV5-2a-GAL4) and UAS-Ap-shRNA. Note that DIP-β signal in the proximal lamina belongs to L4 in control (arrow, L4), whereas DIP-β signal in the distal lamina belongs to unknown cells. Scale bar, 10 µm.

(E-G) Quantification of DIP-β fluorescence intensity along the long axis of lamina cartridges (see white lines in (D) and (F)) from the distal dash line to the proximal dash line. Significantly reduced fluorescence intensity is observed in the proximal lamina (75%–100% distance, marked by red bar in (E) and (G)) of Ap-KD flies compared to control. ***P<0.001, unpaired t-test, n=5 brains, each line represents each brain.

Bsh-KD in LPCs results in loss of Bsh in the adult fly

(A-A’) Control flies have Bsh+ neurons in the adult L4 neurons (arrow). Genotype: w+, R27G05-Gal4>UAS-mcherry-RNAi, tubP-Gal80[ts]. Neuropil marker Brp (blue), Bsh neurons (green). Scale bar, 10 µm.

(B-B’) Bsh-KD in LPCs results in loss of Bsh+ neurons in adult L4 neurons. Neuropil marker Brp (blue), there are no Bsh+ neurons, although there is background staining (green). Scale bar, 10 µm. Genotype: w+, R27G05-Gal4>UAS-Bsh-RNAi, tubP-Gal80[ts].