Temporally dynamic antagonism between transcription and chromatin compaction controls stochastic photoreceptor specification in flies

Abstract

Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). We investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. The ss locus is in a compact state in undifferentiated cells. An early enhancer drives transcription in all R7 precursors, and the locus opens. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In Ss^ON R7s, ss is open and competent for activation by a late enhancer, whereas in Ss^OFF R7s, ss is compact, and repression prevents expression. Our results suggest that a temporally dynamic antagonism, in which transcription drives large-scale decompaction and then compaction represses transcription, controls stochastic fate specification.

Keywords: Ash2; Drosophila; Klumpfuss; Lid; Spineless; chromatin; enhancer; photoreceptor; retina; stochastic.

Figure 1.. ss controls R7 subtype specification

A) R7 subtype specification. Expression of Ss promotes the Rh4-expressing R7 fate. Absence of Ss yields the Rh3-expressing R7 fate. B) Wild-type retinas contain 33% Rh3/Ss^OFF R7s and 67% Rh4/Ss^ON R7s in a random pattern (left). ss protein null mutants contain only Rh3/Ss^OFF R7s and no Rh4/Ss^ON R7s (right). Scalebar=20 μm. C) ss gene locus. Black oval=exon; black arrow=promoter; red rectangle=silencer; green rectangle=enhancer; S1=silencer 1; S2=silencer 2; EE=early enhancer; LE=late enhancer. D) Schematized eye-antennal imaginal disc. Antenna is subdivided into the A1, A2, and A3/arista. A=anterior; P=posterior; MF=morphogenetic furrow. E-H) Schematized depiction of R7 maturation. Insets illustrate how cells proceed through development over time. Gray=undifferentiated cells/U; green=precursors/P; blue=differentiating cells/D; orange=R7.

Figure 2.. Two temporally distinct enhancers drive ss expression

For A-E) Ctrl=peripodial membrane; A=antennal cells; U=undifferentiated cells; P=precursors; D=differentiating cells; R7=R7s. Ant=anterior; Pos=posterior. A) ss RNA is expressed in antennal cells, precursors, and R7s. Gray=ss RNA. Scalebar=100 μm. B) Nascent ss RNA transcripts in a subset of R7s distinguished by sev>Gal4, UAS>GFP. Magenta=R7 reporter; gray=ss RNA; sold circles=Ss^ON R7s, dashed circles=Ss^OFF R7s. Scalebar=5 μm. C) Schematized eye antennal imaginal disc. D) % cells expressing ss. E) Schematized ss expression across time. Insets illustrate ss expression dynamics. F) ss gene locus and CRISPR deletion screen. Black oval=exon; black arrow=promoter; red rectangle=silencer; green rectangle=enhancer; blue line=0% Ss^ON R7s; orange line=25% Ss^ON R7s; gray line=50–68% Ss^ON R7s; S1=silencer 1; S2=silencer 2; EE=early enhancer; LE=late enhancer. For G, H) Green=reporter; magenta=Elav (neurons). G) The EE reporter is expressed in precursors. H) The LE reporter is expressed in R7s.

Figure 3.. Decreasing early ss expression decreases % Ss^ON R7s.

For A, E, I, M, Q) Truncated schematized ss locus. For B, F, J, N, R) ss RNA in the antenna. Gray=ss RNA. Scalebar=10 μm. For C, G, K, O, S) ss RNA in precursors. Gray=ss RNA. Scalebar=10 μm. For D, H, L, P, T) Adult Rh3/Ss^OFF and Rh4/Ss^ON expression in R7s. Scalebar=20 μm. A-D) WT. E-H) PΔ. I-L) EEΔ. M-P) LEΔ. Q-T) Animals with sin variant. For U-W, Z) Orange line=mean WT expression. n.s. denotes p>0.05; *** denotes p<0.0005; **** denotes p<0.0001. U) Quantification of ss in antennal cells. V) Quantification of ss in precursors. W) Quantification of % Ss^ON R7s. X) Ss/Tgo mechanism in WT and breakdown in tgo mutants. Y) ss RNA in precursors and a subset of R7s in tgo null mutant clones. Dashed line=clone boundary. GFP- = tgo null mutant; GFP+ = wild type. Gray=ss RNA; magenta=GFP; dashed line=clone boundary. Scalebar=10 μm.

Figure 4.. Derepressing early ss expression increases % Ss^ON R7s.

For A-V) U=undifferentiated cells; P=precursors; D=differentiating cells; R7=R7s. A) ss expression in precursors is extended into differentiating cells in klu null mutant clones. GFP- = klu null mutant; GFP+ = wild type. Gray=ss RNA; magenta=GFP; dashed line=clone boundary; arrows= ss RNA in differentiating cells. Scalebar=10 μm. For B, F, J, N, R, V) Orange line=mean WT ss expression. B) % Ss^ON R7s increases in klu null mutants, similar to previous studies (Anderson et al 2017). **** denotes p<0.0001. N=3. For C, G, K, O, S) Schematized ss locus. For D, H, L, P, T) ss RNA in undifferentiated cells, precursors, and differentiating cells. Scalebar=10 μm. For E, I, M, Q, U) Quantification of expression for D, H, L, P, T. For F, J, N, R, V) % Ss^ON R7s. C-F) WT. G-J) S1Δ. K-N) PRE12Δ. O-R) PRE2-EEΔ. S-V) ss^inv.

Figure 5.. Repression by the ss locus limits expression to a subset of R7s.

A) Schematic of the PR enhancer reporter construct and insertion sites (arrows). 1–4 inserted using CRISPR. 5* inserted using homologous recombination. B-C) Control insertion. Scalebar=20 μm. D-E) Insertion into ss. Scalebar=20 μm. F-G) *** denotes p<0.0005; **** denotes p<0.0001. F) % Ss^ON R7s with reporter expression. G) % Ss^OFF R7s with reporter expression.

Figure 6.. Dynamic chromatin compaction of the ss locus.

A) Schematic of DNA FISH probes used to label the upstream (blue), ss locus (green), and downstream (red) regions. For B-C) Left=image; right=schematized model. B) Ss^OFF cell with compact chromatin. C) Ss^ON cell with open chromatin. For D-J) Quantification. Ctrl=peripodial membrane cells; A=antennal cells; U=undifferentiated cells; P=precursors; D=differentiating cells; R7=R7s. Black circle=ss^ON cell; gray circle=ss^OFF cell; white rectangle=quartile; white circle=median; gray dashed line=ss^OFF control median; black dashed line=ss^ON control median. * denotes p<0.05; ** denotes p<0.005; **** denotes p<0.0001. For D, F-J) Ctrl cells were compared to A, U, P, D, or R7 cells. For E, Ss^ON R7s were compared to Ss^OFF R7s. For D-J) n>70 cells for each region. D) WT. E) Compaction in Ss^ON R7s and Ss^OFF R7s. F) PΔ. G) EEΔ. H) sin. I) PRE12Δ. J) LEΔ.

Figure 7.. Proposed mechanism for stochastic R7 subtype specification.

Gray box=inactive enhancer; green box=active enhancer.