Temporal progression of Drosophila medulla neuroblasts generates the transcription factor combination to control T1 neuron morphogenesis

Abstract

Proper neural function depends on the correct specification of individual neural fates, controlled by combinations of neuronal transcription factors. Different neural types are sequentially generated by neural progenitors in a defined order, and this temporal patterning process can be controlled by Temporal Transcription Factors (TTFs) that form temporal cascades in neural progenitors. The Drosophila medulla, part of the visual processing center of the brain, contains more than 70 neural types generated by medulla neuroblasts which sequentially express several TTFs, including Homothorax (Hth), eyeless (Ey), Sloppy paired 1 and 2 (Slp), Dichaete (D) and Tailless (Tll). However, it is not clear how such a small number of TTFs could give rise to diverse combinations of neuronal transcription factors that specify a large number of medulla neuron types. Here we report how temporal patterning specifies one neural type, the T1 neuron. We show that the T1 neuron is the only medulla neuron type that expresses the combination of three transcription factors Ocelliless (Oc or Otd), Sox102F and Ets65A. Using CRISPR-Cas9 system, we show that each transcription factor is required for the correct morphogenesis of T1 neurons. Interestingly, Oc, Sox102F and Ets65A initiate expression in neurons beginning at different temporal stages and last in a few subsequent temporal stages. Oc expressing neurons are generated in the Ey, Slp and D stages; Sox102F expressing neurons are produced in the Slp and D stages; while Ets65A is expressed in subsets of medulla neurons born in the D and later stages. The TTF Ey, Slp or D is required to initiate the expression of Oc, Sox102F or Ets65A in neurons, respectively. Thus, the neurons expressing all three transcription factors are born in the D stage and become T1 neurons. In neurons where the three transcription factors do not overlap, each of the three transcription factors can act in combination with other neuronal transcription factors to specify different neural fates. We show that this way of expression regulation of neuronal transcription factors by temporal patterning can generate more possible combinations of transcription factors in neural progeny to diversify neural fates.

Keywords: Neuroblasts; Neuronal differentiation; Neuronal morphogenesis; Temporal patterning; Temporal transcription factors.

Figure 1.. Oc, Ets65A and Sox102F are expressed in T1 neurons.

(A) A schematic drawing of the normal T1 morphology in red. LA: lamina. ME: Medulla. (B-B’) The expression of Oc (green) in the adult medulla carrying T1LexA>LexopRFP (red). (C,D) The expression of Ets65A::GFP (green in C) or Sox102F::GFP (green in D) partially overlaps with that of Oc (blue) in the 3^rd instar larval medulla. The early-born (deeper layer) neurons are to the left, and later born (more superficial layers) neurons are to the right. Neurons expressing both appear cyan in the overlay. White arrow indicates one example. (E,E’) Neurons that express both Sox102F (red) and Oc (blue) also express Ets65A::GFP (green). (F,F’) Sox102F::GFP (green) is expressed in the same neurons as Sox102F immunostaining (red) in the 3^rd instar larval medulla. (G,G’) In the 3^rd instar larval medulla, the expression of Ets65A::GFP (green) is in the nucleus of the same cells that express Ets65A mRNA as shown by in situ hybridization against all isoforms (purple). (H–H”) T1 neurons (red) in the adult medulla express both Ets65A::GFP (green) and Oc (blue). (I,I’) T1 neurons (red) in the adult medulla express both Sox102F (green) and Oc (blue).

Figure 2.. Knocking down of Oc, Sox102F and Ets65A using CRISPR-Cas9 system.

(A) Oc expression (cyan) in control medulla (genotype T1LexA>LexopRFP/ UASCas9; eyGal4/+) with T1 labeled in red. (B) Oc expression (cyan) in ocgRNA medulla (genotype T1LexA>LexopRFP /UASCas9; eyGal4/ocgRNA) with T1 labeled in red. (C) Quantification of percentage of T1 neurons stained positive for Oc in control and ocgRNA brains, n=4 for control, n=8 for ocgRNA. Less than 100% ( ~93±1.8 %) of wild types T1s are stained positive for Oc, possibly due to focal planes or weak antibody signals, etc. Oc is lost in ~59±13 % of T1 neurons when Oc is knocked down with ocgRNA (t-test: p=6.24×10⁻⁶ ). (D) Sox102F expression (cyan) in control medulla (genotype T1LexA>LexopRFP/ UASCas9; eyGal4/+) with T1 labeled in red. (E) Sox102F expression (cyan) in Sox102FgRNA medulla (genotype T1LexA>LexopRFP/ UASCas9; eyGal4/Sox102FgRNA) with T1 labeled in red. (F) Quantification of percentage of T1 neurons stained positive for Sox102F in control and Sox102FgRNA brains. n=4 for control, n=9 for Sox102F-gRNA. Sox102F is lost in ~52±9% of T1 neurons in Sox102FgRNA brains. (t-test: p=8.39×10⁻⁸ ). (G–G”) In clones (marked in green) where Cas9 and Ets65AgRNA are expressed (genotype yw hs FLP; act>y+>Gal4 UAS GFP / UASCas9; UASEts65AgRNA/+), in situ hybridization of Ets65A (purple) in 3^rd instar larval medulla showed that Ets65A mRNA is partially knocked down in the clones. Note the clone margin perfectly correlates with that of higher Ets65A expression.

Figure 3.. Oc, Ets65A and Sox102F are each required for T1 neuron morphogenesis.

MCFO is used to visualize wild type or mutant T1 morphology with HA staining in green. In all panels, white arrowhead indicates the location of the cell body. (A–D) no gRNA wild type control (genotype: hsFLP/+; T1lexA /UASCas9; ey>Gal4^16F10, 13xLexAop2->dSTOP>-myr::smGFP-HA /+). (A) Densely labeled. (B) Sparsely labeled. (C–C”) Magnified view of the cell body marked with an arrowhead in (B) showing Sox102F (red) and Oc (blue) staining. (D) Drawing of the wild type T1 morphology. (E–H) oc is knocked down using ocgRNA (Genotype: hsFLP/+; T1lexA /UASCas9; ey>Gal4^16F10, 13xLexAop2->dSTOP>-myr::smGFP-HA / UAS-ocgRNA). (E) Densely labeled. (F) Sparsely labeled. (G-G”) Magnified view of the cell body marked with arrowhead in (F) showing Sox102F (red) and Oc (blue) staining. (H) Drawing of typical oc mutant T1 morphology. (I–L) Sox102F is knocked down using Sox102FgRNA (Genotype: hsFLP/+; T1lexA /UASCas9; ey>Gal4^16F10, 13xLexAop2->dSTOP>-myr::smGFP-HA / UAS-Sox102FgRNA). (I) Densely labeled. (J) Sparsely labeled. (K–K”) Magnified view of the cell body marked with arrowhead in (J) showing Sox102F (red) and Oc (blue) staining. (L) Drawing of typical Sox102F mutant T1 morphology. (M–P) Ets65A is knocked down using Ets65AgRNA (Genotype: hsFLP/+; T1lexA /UASCas9; ey>Gal4^16F10, 13xLexAop2->dSTOP>-myr::smGFP-HA / UAS-Ets65AgRNA). (M) Densely labeled. (N) Sparsely labeled. (O–O”) Magnified view of the cell body marked with arrowhead in (N) showing Sox102F (red) and Oc (blue) staining. (P) Drawing of typical Ets65A mutant T1 morphology.

Figure 4.. The three transcription factors initiate expression in neurons born at different temporal stages.

All images shown are of 3^rd instar larval medulla. (A–A’) UAS-NuLacZ reporter (red) driven by an ey> Gal4 initiates expression at the same time as endogenous Ey expression (blue). (B–B’) UAS-NuLacZ reporter (red) driven by a slp> Gal4 initiates expression at the same time as endogenous Slp expression (blue). (C–C’) UAS-NuLacZ reporter (red) driven by a D> Gal4 initiates expression at the same time as endogenous D expression (blue). (D–F) D>Gal4 is used to drive UAS-NuLacZ (red) in D stage (and all later stage) neuroblasts and progeny. (D) Almost all Ets65A::GFP (green) expressing cells express D>> LacZ. (E) Some Sox102F::GFP (green) expressing cells express D>> LacZ (yellow cells), while others do not (green cells). (F) Some Oc (blue) expressing cells express D>>LacZ (purple cells), while others do not (blue cells). (G-H) slp>Gal4 is used to drive UAS-NuLacZ (red) expression. (G) All neurons expressing Sox102F::GFP (green) also express slp>>LacZ. (H) All neurons expressing Oc (blue) also express slp>>LacZ. (I) ey>Gal4 is used to drive UAS-NuLacZ (red) in Ey stage (and all later stage) neuroblasts and progeny. All Oc (blue) expressing neurons also express LacZ(red).

Figure 5.. D, Slp and Ey are required for initiation of Ets65A, Sox102F and Oc expression in neurons, respectively.

All images shown are of 3^rd instar larval medulla. (A–A”) In situ hybridization of Ets65A mRNA (purple) in brains with D mutant clones marked by GFP (green). (B–B”) Immunostaining of Sox102F (red) and Oc (blue) in brains with D mutant clones marked by GFP (green). (C–C”) Immunostaining of Sox102F (red) in brains with slp mutant clones marked by GFP (green). (D) Immunostaining of Oc (blue) in brains with slp mutant clones marked by GFP (green). (E,E’) Immunostaining of Oc (blue) in brains with clones expressing eyRNAi driven by actin>Gal4 marked by GFP (green). (F) A model summarizing how temporal patterning generate different combinations of transcription factor expression in medulla neurons. Oc expression is started in late Ey stage, and is expressed in Notch-off progeny of Ey, Slp and D stages; Sox102F is expressed in Notch-on neurons born in the Slp and D temporal stages, and also in Notch-off neurons born in the D stage; Ets65A is expressed in Notch-off neurons born in the D stage, and continue to be expressed in progeny born at later stages. The Ap and Toy expression pattern are based on (Li et al., 2013).

Figure 6.. T1 neurons are derived from the Notch-off progeny and present in all spatial domains of the main medulla.

All images shown are of 3^rd instar larval medulla. (A–A”) The expression of Ets65A::GFP (green), Oc (blue) and ap>LacZ (red) in larval medulla. Cells expressing both Oc and Ets65A appear cyan in the overlay (A), and two examples are indicated by white arrows. (B–B”) The expression of Sox102F::GFP (green), Oc (blue) and ap>LacZ (red) in larval medulla. Cells expressing both Oc and Sox102F appear cyan in the overlay (B,B”), and two examples are indicated by cyan arrows. Cells expressing both Ap and Sox102F appear yellow in the overlay (B,B’) and two examples are indicated by yellow arrows. (C-C”) The expression of Sox102F (green), Toy (blue) and ap>LacZ (red) in larval medulla. Examples of cells expressing Sox102F but not Toy or ap>LacZ are indicated by green arrows; Examples of cells expressing all three are indicated by white arrows; there are also cells that express Toy and ap>Lac but not Sox102F, and examples are indicated by purple arrows. (D) A schematic drawing showing the different spatial domains of the main medulla (the center Vsx domain, Optix domains, Dpp domains) and the two tips that express Wg. Drawing not to scale. The red dashed line indicates the location of the focal planes for panels E and F, and the blue dashed line indicates the location of the focal plane for the panel G. (E) Cells expressing Oc (blue) and Sox102F (red) are present in the center Vsx domain and the two neighboring Optix (green) domains. Cells expressing both TFs appear purple in the overlay and examples are indicated by white arrows. (F) Cells expressing all three TFs: Oc (blue), Sox102F (red) and Ets65A::GFP (green) are present in the center Vsx domain and the two neighboring Optix domains. Cells expressing all three TFs appear white in the overlay and examples are indicated by white arrows. (G) Cells expressing all three TFs: Oc (blue), Sox102F (red) and Ets65A::GFP (green) are also present in the two Dpp domains (dorsal and ventral), but not present in the two tips (Wg domains).