Temporal patterning of Drosophila medulla neuroblasts controls neural fates

Abstract

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. Here we describe how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors-Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless-are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. We provide evidence that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, our work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts.

Figure 1. The developing medulla

a. Model of a larval brain showing that the NE (blue) gives rise to the lamina on the lateral (L) side and to the medulla on the medial (M) side. A wave of neurogenesis (light red) converts NE cells (blue) into NBs (red). VNC: ventral nerve cord. b. Surface view showing NE (Phalloidin, blue), medulla NBs (Dpn, red), and lamina neurons (Elav, purple). c. Cross-section model showing NBs (red), GMCs (green), and neurons (purple). A single NB clone is shown by grey thick outlines. d. Cross-section view showing the NE (DE-Cadherin, blue), medulla NBs (Dpn, red), medulla GMCs (Pros, green), medulla and lamina neurons (elav-Gal4>UASCD8-GFP, purple). a-d. Small Red arrow: the wave of neurogenesis.

Figure 2. A temporal sequence of TFs in medulla NBs

a,b. Surface views showing that NBs sequentially express: a. Hth (red), Ey (blue), Slp1 (green) and D (red). b. D (red) and Tll (cyan). c-i. Cross-section views showing the expression of the five TFs in NBs and their progeny. c,c’. Hth (red), Ey (blue), Dpn(green). d,d’. Ey (blue), Slp1 (green). e,e’. D (red). The dashed line separates the two populations of D⁺ neurons (see text). f,f’. In a NB clone (βGal, green in f, dashed circles in f’), the NB is Ey⁺ (blue, small arrow), while its progeny are Ey⁺ or Hth⁺ (red, open arrows). g,g’. In a NB clone (βGal, white in inset), the NB is D⁺ (red, small arrow). It has generated Slp⁺ (green) GMCs (arrowhead), and Ey⁺ (blue) neurons (open arrows). h,h’. The oldest NBs (small arrows) express Tll (cyan in h), Dpn (red) and nuclear Pros (blue in h’). i. Tll⁺ NB progeny (small arrows) lose Tll (cyan), and turn on Repo (red) while migrating (along the dashed arrow) to become medulla neuropil glia (arrowhead). j. Schematic model. For simplicity, the overlap between TFs is not shown; only one NB/GMC is shown for each stage. D expression in the deeper neuron population is not shown. Empty cells indicate a subset of neurons born during the Ey, Slp or D windows do not maintain the NB TF. k. Model showing each NB sequentially expresses five TFs.

Figure 3. Cross-regulations between TFs in the gene cascade

a,a’. Surface view: in an ey^J5.71 mutant clone marked by lack of GFP (red) and of Ey (blue), Slp1 (green) is lost in NBs. b. Cross-section view: in ey^J5.71 mutants, Hth (red) is only in the youngest NBs (Dpn marking all NBs, blue). c. Summary model. d-d”. Surface view: in slp mutant MARCM clones (GFP, white in d, dashed line in d’,d’’), NBs continue to express Ey (blue) and do not turn on D (red). e. Summary model. f-f’’. Surface view: in D mutant clones marked by lack of GFP (white in f, dashed line in f-f’’), NBs continue to express Slp1 (green) and do not turn on Tll (cyan). g. Summary model. h. Model summarizing cross-regulations between the five TFs. (*): sufficient but not required.

Figure 4. Notch-dependent asymmetric division of medulla GMCs

All panels are cross-section views with Ap (green) and Ey (blue). a. A subset of Hth⁺ neurons (red) are Ap⁺ while Ey⁺ neurons are intermingled with Ap⁺ neurons. b. D⁺ neurons above the dashed line co-express Ap; D expression below the dashed line is in Ap⁻ neurons. c,c’. Two daughters of a GMC are labeled by GFP (red). One is Ap⁺ and the other is Ap⁻. d-d’’. Wild type tubGal4 MARCM clones marked by CD8-GFP (red) contain both Ap⁺ and Ey⁺ neurons. e-f’’. Su(H) mutant MARCM clones (CD8-GFP, red in e, blue in f). e-e’’. Ap is lost and Ey expanded. f-f’’’. D (red) in NBs is not affected (open arrow) but D and Ap are lost in D⁺ NB progeny (above the dashed line, white arrow); the deeper layer of D expression in Ap⁻ neurons (below the dashed line, star) is expanded. g, A simplified schematic model.

Figure 5. Hth and Ey are required for neuronal diversity

All images are cross-section views of larval medulla. a,a’. In wild type, Bsh (blue) is in neurons expressing both ap-LacZ (green), an enhancer trap that perfectly mimics Ap expression, and Hth (red). b,b’. Bsh (blue), but not Hth (red) is lost in Su(H) mutant clones (GFP, green). c. Bsh (blue) is lost in hth^P2 mutant clones (GFP, green). d. Bsh (blue) is ectopically expressed when UAS-GFP::Hth is driven by tubGal4 in a MARCM clone (GFP, red). e, In wild type, Dfr (red) is expressed in two-three rows of Ap⁺ (green) neurons. There are also Dfr⁺Dac⁺(blue) Ap⁻ neurons in a more superficial layer. f. The Ap⁺Dfr⁺ neurons (below the dashed line) are intermingled with Ey⁺ (blue) neurons. g,g’. Dfr expression (red) is lost in ey^J5.71 mutant clones marked by lack of GFP (green in g, dashed line in g’). h,h’. Dfr⁺ (red) neurons are expanded in slp mutant clones (GFP, green). In this region there are very few Dfr⁺ Dac⁺ (blue) neurons. The expanded Dfr⁺ neurons do not express Dac.

Figure 6. Slp is required for neuronal diversity

a-b’. Cross-section views of larval medulla, with Toy in red and Ap (or ap-lacZ) in blue. a. In wild type, Toy⁺ neurons in the deeper layer are Ap⁻. The superficial Toy⁺ neurons are Ap⁺ and are intermingled with Slp1⁺ neurons (green). b,b’. In slp mutant clones (GFP, green in b, dashed outline in b’), Toy⁺Ap⁺ neurons disappear. c,d. Adult medulla with OrtC1-Gal4>UAS-CD8GFP (green), ap-lacZ (blue) and Toy (red). c. Horizontal view. d. View through the medulla cortex. e,f. Flip-out clones in adults (OrtC1-Gal4, hsFLP, UAS-FRT-STOP-FRT-CD8GFP). Arrows point to neuron cell bodies. e. Tm20. Photoreceptor axons in blue. f. Tm5 and TmY10. g,h. OrtC1-Gal4 MARCM clones in adults. g. wt. h. slp mutant. i. Simplified model showing neuronal TF markers expressed in progeny of NBs of different stages. The lineage is approximate and does not take into account regional differences. The brackets for “D” indicate that D is not maintained in all N^ON progeny of D⁺ NBs.