Glia relay differentiation cues to coordinate neuronal development in Drosophila

Abstract

Neuronal birth and specification must be coordinated across the developing brain to generate the neurons that constitute neural circuits. We used the Drosophila visual system to investigate how development is coordinated to establish retinotopy, a feature of all visual systems. Photoreceptors achieve retinotopy by inducing their target field in the optic lobe, the lamina neurons, with a secreted differentiation cue, epidermal growth factor (EGF). We find that communication between photoreceptors and lamina cells requires a signaling relay through glia. In response to photoreceptor-EGF, glia produce insulin-like peptides, which induce lamina neuronal differentiation. Our study identifies a role for glia in coordinating neuronal development across distinct brain regions, thus reconciling the timing of column assembly with that of delayed differentiation, as well as the spatiotemporal pattern of lamina neuron differentiation.

Fig. 1. Photoreceptors do not communicate directly with lamina precursors through EGF

(A) Schematic of lamina development in the optic lobes, which is coupled to photoreceptor development in the eye disc. Hh from photoreceptors drives lamina precursor (purple) birth and assembly into columns. Photoreceptor-EGF is required for precursor differentiation into neurons (yellow). Columns consist of 6–7 precursors, which differentiate in an invariant spatiotemporal pattern (yellow). (B) A horizontal view of an early pupal (P10–15hrs APF) eye disc and optic lobe showing photoreceptor axons marked by HRP (cyan). In the optic lobe, lamina precursors express Dac (magenta) and differentiated photoreceptors and neurons express Elav (yellow). Lamina cell bodies (magenta) are organized into columns that associate with photoreceptor axons. Wrapping glia, marked by membrane-targeted GFP (white) driven by a wrapping glia-specific Gal4, extended processes through the optic stalk and into the lamina, where they encapsulate lamina cells and photoreceptors progressively (inset in B”; arrowheads mark location of photoreceptors between glial processes and lamina cells). (C) Expressing Htl^DN in wrapping glia disrupted glial process infiltration into the lamina. Only cells immediately below glial processes differentiated (arrowhead in D”). (D) Lamina-specific Gal4 driving GFP showed normal lamina neuron differentiation. (E) Lamina-specific EGFR^DN and P35 co-expression did not affect neuronal differentiation. (F) Lamina-specific Aop^ACT and P35 co-expression led to loss of differentiated neurons (dashed bracket). (G) Lamina-specific MAPK^ACT expression led to premature Elav expression in columns. (H) Quantification of (E–F) as a percentage of differentiated cells in the 6 youngest lamina columns. Asterisks indicate significance with Mann-Whitney U-test p<0.01; #optic lobes examined indicated in brackets. (Scale bar = 10µm).

Fig. 2. L1–L4 differentiation requires photoreceptor-induced EGFR signaling in wrapping glia

Eye discs with wrapping glia marked by the pan-glial nuclear marker Repo (Magenta) and dpMAPK (yellow) in (A) rho3^−/+ and (B) rho3^−/− animals, quantified in (C) p<0.001; Mann-Whitney U-test; #discs indicated in brackets. (D–G) Optic lobes stained for Elav (yellow), Dac (magenta), HRP (cyan) and GFP (white) (D) A control wr. glia>GFP lamina. (E) When wrapping glia express EGFR^DN, only presumptive L5s differentiated (arrow head). (F) In a rho3^−/− animal, there was only a late differentiating presumptive L5 (See also Fig. S1C–G). (G) When wrapping glia express EGFR^ACT and GFP in a rho3^−/− background, the L1–L4 front of differentiation is restored (bracket). (H,I) Developmentally expressed subtype-specific markers used in combination to identify neuronal subtypes (16): Sloppy paired 2 (Slp2) alone marks L2 and L3; Slp2 and Seven up (Svp) together mark L1, Brain-specific homeobox (Bsh) alone marks L4, and Slp2 and Bsh together mark L5 (dashed line indicates lamina plexus). (H) In a control rho3^−/+ brain and (I) when wrapping glia drive EGFR^ACT (and GFP; not shown) in a rho3^−/− background, all cell types were recovered. (J) Quantification of (D–G) as a percentage of differentiated cells in the 6 youngest lamina columns. Asterisks indicate significance with Mann-Whitney U-test p<0.01; #optic lobes examined indicated in brackets. (Scale bar = 10µm).

Fig. 3. Wrapping glial Insulin-like peptides induce lamina neuronal differentiation

(A) Normal lamina neuronal differentiation in a control. (B) A chico^−/− brain lacked L1–L4 differentiation (dashed bracket). (C) Ilp6-Gal4 and (D) Ilp7-Gal4 drove expression of GFP (membrane or cytoplasmic, respectively) in wrapping glia and their extensions into the optic stalk (yellow arrows). (E) A rho3^−/− lamina. (F) A rho3^−/− animal with wrapping glia expressing Ilp6 showed L1–L4 differentiation (bracket). (G) A rho3^−/− animal with the lamina expressing InR^ACT showed neuronal differentiation (bracket). Elav (yellow), Dac (magenta), HRP (cyan) and GFP (white). (H) Quantification of (F,G) as a percentage of differentiated cells in the 6 youngest lamina columns. Asterisks indicate significance with Mann-Whitney U-test p<0.01; #optic lobes examined indicated in brackets. (Scale bar = 10µm).

Fig. 4. The signaling relay may serve to delay differentiation to ensure consistent column assembly

(A–D) Early pupal (stages indicated) eye-optic lobe complexes stained for Elav (yellow), Dac (magenta), HRP (cyan) and (C, D) GFP (white). Cyan dashed line marks the youngest photoreceptors. (A) Control. (B) rho3^−/−. (C) An early-onset pan-photoreceptor Gal4 driving GFP and Ilp6 in a rho3^−/− background. Differentiation was widespread and initiated in the youngest column (arrowhead), which contained ~4 lamina precursors. (D–E) A late-onset pan-photoreceptor Gal4 driving GFP and Ilp6 in a rho3^−/− background. (D) At ~10hrs APF, differentiation initiated only in old columns (arrowheads), but columns assembled 6–7lamina precursors/column. (E) At ~15hrs APF, GFP and Ilp6 were expressed in all photoreceptors. Differentiation was widespread but variable as some columns contained more differentiated neurons than their older neighbors (arrowhead). (Scale bar = 10µm).

Fig. 5. A signaling relay from photoreceptors to glia to lamina precursors instructs lamina differentiation

Model: Photoreceptors secrete EGF and FGF, which activate EGFR and FGFR respectively in wrapping glia. EGFR activation is required for glial expression of Ilps, which activate InR and MAPK in lamina precursors leading to L1–L4 differentiation. FGFR signaling regulates glia morphogenesis and process extension into the brain (3) and therefore indirectly regulates the timing and patterning of L1–L4 differentiation.