Differing Strategies Despite Shared Lineages of Motor Neurons and Glia to Achieve Robust Development of an Adult Neuropil in Drosophila

Abstract

In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages is not understood. We show that two stem cells that produce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. We suggest that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution.

Keywords: astrocyte; cell lineage; ensheathing glia; motor neurons; neuroblast; neuropil; neuropil glia; plasticity; robustness; stem cell.

Figure 1. Cellular Organization of the Adult Thoracic NG

(A–C) Adult VNCs immunostained with anti-BRP (neuropil marker, blue) (A1–C) and anti-Dll (NG marker, green) (B) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1 and A2), H2B::RFP (nuclear marker, red) (B) or the multicolor system FB1.1 (single-cell marker, red, yellow, and green) (C) under the control of alrm-Gal4. (B) Astrocyte (H2B::RFP⁺, Dll⁺): arrow, EG (Dll⁺): arrowhead. (C) Different astrocytes with processes occupying different neuropil territories: arrows. See also Figure S1 and Movie S1. (D–F) Adult VNCs immunostained with anti-BRP (neuropil marker, blue) (D1–F) and anti-Dll (NG marker, green) (E) with EG-expressing mCD4::GFP (membrane marker, green) (D1 and D2), H2B::RFP (nuclear marker, red) (E) or the multicolor system FB1.1 (single-cell marker, red, yellow, and green; Hadjieconomou et al., 2011) (F) under the control of R56F03-Gal4. (E) Astrocyte: arrow (Dll⁺), EG (H2B::RFP⁺, Dll⁺): arrowhead. (D) arrow: in more than half of the samples analyzed (n = 10) a cell body of an EG (GFP ⁺, Dll⁺) was observed inside the neuropil, next to axon bundles. (F) Different EG with processes occupying different neuropil territories: arrows. See also Figure S1 and Movie S1. (G and H) Prothoracic neuromeres with EG-expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (H). (G) Asterisk: Elav⁺ neuron wrapped by an EG. Enlargements of the boxed regions are to the right of each panel. See also Movie S2. (I and J) Prothoracic neuromeres with EG-expressing mCD4::GFP (I) and GFP::mCD8::HRP (J) immunostained with anti-BRP (blue) (I) or labeled with DAB (brown) (J). (K) Low-magnification electron microscope image of the boxed region in (J). (L–O) Enlargement of the boxed regions in (K). (L2) Enlargement of the boxed region in (L1). Note: (L1) and (L2) show the organization of the EG processes around the neuropil while (M)–(O) show the EG processes inside the neuropil. Pink lines outline axons. PG, perineurial glia; SPG, subperineurial glia; NG, neuropil glia; C, cortex; Np, neuropil; NL, neural lamella. (P) Bottom, schematic of adult CNS (blue: neuropils, gray: cortex); top, single astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using MARCM. Axes: green (posterior), blue (dorsal), red (medial). (Q) Average number of NG in the adult VNC, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4, respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate SD. ProNm, Prothoracic neuromere; AMesoNm, Accessory mesothoracic neuromere; MesoNm, Mesothoracic neuromere; MetaNm, Metathoracic neuromere; ANm, Abdominal neuromeres; FS, frontal cross-section; TS, transverse cross-section; 3D, 3-dimensional reconstruction of confocal image stack; pMP, partial maximum projection.

Figure 2. Developmental Origin of the Adult NG

(A) 3D rendering of the thoracic segments of a L3 VNC (A1) labeled with anti-BRP (mature neuropil marker, blue) and anti-NCad (mature and immature neuropil marker, red). The glia surrounding the six immature leg neuropils express mCD8::GFP (green, left image only) under the control of Dll-Gal4. (T1, T2, and T3 indicate the three thoracic segments.) (A2) Following metamorphosis, 3D rendering of adult VNCs with astrocytes and EG-expressing GFP under the control of alrm-Gal4 (right) and R56F03-Gal4 (left) and costained with anti-BRP (red). (B–D) L3 CNSs in which glia surrounding the six immature leg neuropils express mCD8::GFP under the control of Dll-Gal4 and co-stained with anti-BRP (blue), anti-NCad (red) (B1–B3), anti-Repo (blue), anti-Dll (red) (C1–C3), or anti-Repo (blue), anti-PH3 (red) (D1–D3). (D2 and D3) Arrows point to glia expressing PH3. (E and F) Adult VNCs with NG expressing nuclear GFP (green) (E1–E5) or the multicolor Flybow system FB1.1 (yellow, red, and green) (see STAR Methods for details) and immunostained with anti-Repo (blue), anti-Dll (red) (E1–E5), or anti-Pros (blue) (F1–F5). (E1–E5) Arrow points to adult NG-expressing GFP. (F1–F5) Arrow: astrocyte (mCherry⁺, Pros⁺), arrowhead: EG (mCherry⁺, Pros⁻). See also Figure S2. (G) Left: schematic of an L3 CNS. The immature leg neuropils and proliferating NG are indicated. Right: schematic of an adult VNC with the adult leg NG. N, average number of proliferating NG in each L3 thoracic hemisegment.

Figure 3. Coordinated Development of NG and Adult Neuropil

(A–E) Thoracic segments of larval VNCs at different stages (A, early L2; B, early L3; C, mid L3; D, late L3, E, 5 hr after pupal formation) with proliferating NG-expressing mCD8::GFP under the control of Dll-Gal4 and labeled with anti-BRP (blue), anti-NCad (red). Asterisks indicate axons of incoming leg sensory neurons. See also Figure S3 and Movies 3 and 4. (E1–E3) At this stage, the expression of BRP is almost undetectable, probably a consequence of the remodeling and/or degradation of the larval neuropils. (F–K) ProNm of an pupal VNC with astrocytes (F–H) or EG (I–K) expressing a membrane-tagged GFP under the control of alrm-Gal4 and R56F03-Gal4, respectively, and co-stained with anti-Repo (blue) and anti-NCad (red) (F, G, I, and J), anti-BRP (blue), and anti-Dll (red) (H and K). (F and I) Processes of astrocytes (arrow) and EG (arrowhead) are first observed invading the neuropil. (L) Average number of NG at different stages (number of samples analyzed at each stage = 4) with schematics of transverse sections of T1 or ProNm. NG (green), larval neuropils (blue), and leg neuropils (red) are indicated. Error bars indicate SD. ATS, anterior transverse section; PTS, posterior transverse section; APF, after pupa formation.

Figure 4. NGBs Produce Leg Motor Neurons and All Thoracic NG

(A and B) Thoracic segments of an L3 VNC labeled with anti-BRP (blue) and anti-Dll (red) containing MARCM clones expressing mCD8::GFP under the control of both VGlut-Gal4 and repo-Gal4. Lin A, Lin ?, and Lin D clones produce motor neurons (arrowhead) and glia (arrow). (A1–A5) Sample with Lin A, Lin B and Lin ? clones; note that Lin B does not produce glia. (B1–B5) Sample with a Lin D clone. Asterisks mark motor neuron axons exiting the VNC. (C–E) Lin A (C1–C3), Lin ? (D1–D3) and Lin D (E1–E3) cis² MARCM clones in a ProN labeled with anti-BRP (blue) (C1–C2, D1–D2, E1–E2) and in a T1 Leg (C3, D3, E3) expressing mCD8::RFP in NG (red) and mCD8::GFP (green) under the control of repo-Gal4 and VGlut-QF, respectively.

Figure 5. Lin A Initially Generates Postmitotic Motor Neurons and Proliferating Glia and Then Only Motor Neurons

(A–C) 3D reconstruction of all six (A1), three (B1), or a single (C) Lin A clones labeled with myr::GFP (green) generated by a Lin A tracing system in the thoracic segments of a late L3 VNC, and immunostained with anti-Dpn (red), anti-Elav (cyan), and anti-Repo (blue); A1 and C1 are ventral views, B1 and C2 are lateral views. C3 shows a series of frontal cross-sections (FS) of Lin A from ventral (1) to dorsal (4). NB: Lin A NB/NGB, red arrowhead, GMC: Lin A GMC, black arrowhead, Ns: Lin A neurons, cyan arrowhead. Axes in A2, B2: green (posterior, Post), blue (dorsal, D), red (medial, M). A2 and B2 show plots of Lin A progeny as seen in A1 and B1, respectively, color coded according to cell type. Blue, Repo⁺; red, Dpn⁺; cyan, Elav⁺, orange: (GMC/IMC, ganglion mother cell/intermediate mother cell, Dpn⁻, Elav⁻, Repo⁻); note: some of these GMC/IMC could be immature neurons or glia. (D–J) Ventral view of a 3D reconstruction of single Lin A clones in a T2 segment at different time points during L3 (D1–J1) and graphs of each Lin A cell from two perspectives (D2–J2 and D3–J3). Axes: green (posterior, Post), blue (dorsal, D), red (medial, M). Shown are: (D) 75–78 hr AEL, (E) 96–99 hr AEL, (F) 99–102 hr AEL, (G) 102–105 hr AEL, (H) 120–123 hr AEL, (I) 123–126 hr AEL, and (J) 126–129 hr AEL. (K) Average number of Lin A cell types at different L3 stages. Number of samples analyzed at each stage: (75-to 78-hr AEL): n = 16; (78-to 81-hr AEL): n = 16; (96-to 99-hr AEL): n = 8; (99-to 102-hr AEL): n = 10; (102-to 105-hr AEL): n = 7; (120-to 123-hr AEL): n = 9; (123-to 126-hr AEL): n = 8; (126-to 129-hr AEL: n = 7. Error bars indicate SD. AEL, after egg laying. “Lin A” refers to all cells labeled by GFP. (L and M) Lin A QMARCM/MARCM clone in a ProNm generated before the first post-embryonic division and labeled with Repo (blue). Motor neurons produced by one daughter cell are labeled with mCD8::GFP under the control of VGlut-QF, and all the cells produced by the sister daughter cell are labeled with RFP under the control of tubulin-Gal4 (L1, L2). RFP labels a single motor neuron targeting a body wall muscle (M) and NG when the clones are induced at early L3 or earlier. Number of samples analyzed: n > 30. (N) Inferred pattern of Lin A divisions. Lin A.1 produces at least one glioblast (GB) and one motor neuron targeting the body wall; Lin A.2 also produces at least one glioblast and the remaining Lin A motor neurons. We refer to the mother of a glioblast and motor neuron as an intermediate mother cell (IMC) and the progeny of the Glioblast as proliferating glia (pG) Later cell divisions of Lin A.2 generate a GMC, which produce one motor neuron and a sister cell that undergoes apoptosis (blue star; Truman et al., 2010).

Figure 6. Lin A.1 and Lin A.2 Produce Astrocytes and EG, While Lin D Produces Only Astrocytes

(A–C) Schematic representation of the cis² MARCM experiments labeling Lin A.2 (A), Lin A.1 (B), and Lin D (C). (D–H) Lin A.2 (D and G), Lin A.1 (E and H), and Lin D (F) cis² MARCM clones in a ProNm stained with anti-BRP (blue), with motor neurons expressing mCD8::GFP, astrocytes expressing mCherry, and EG-expressing mCD8::RFP, under the control of VGlut-QF, and alrm-Gal4 or R56F03-Gal4, respectively. Number of VNCs analyzed: Lin A astrocyte: n = 10, Lin A EG: n = 4, Lin D astrocyte: n = 4, Lin Z astrocytes: n = 13. See also Figure S4 and Movie S5. (I) Summary of progeny produced by Lin A and Lin D. (J–L) Schematic representation of tub-Gal4 MARCM experiments in (M)–(O) labeling Lin A.2 (J), Lin A.1 (K), and Lin D (L). (M–O) Lin A.2 (M), Lin A.1 (N), Lin D (O) MARCM clones labeled with mCD8::GFP and nGFP under the control of tub-Gal4 in a thoracic segment of an L3 VNC and immunostained with anti-Dpn (red), anti-Elav (cyan), and anti-Repo (blue). (M2, N2, O2) are successive frontal cross-sections (FS) from ventral (V) to dorsal (D). pG, proliferating glia (Repo⁺).

Figure 7. Gliogenesis Is Not Stereotyped and Depends on Inter-lineage Competition

(A–R) Three examples each of Lin A.2 (A–C, G–I, M–O) and Lin A.1 (D–F, J–L, P–R) cis² MARCM clones expressing alrm > mCherry (A–F) or R56F03 > mCD8:RFP (G–L) and VGlut > mCD8::GFP (M–R). Astrocyte and EG clone morphology is variable from animal to animal, while motor neuron clones have a uniform, lineage-specific morphology. (A–G) Arrows point to NG processes and arrowheads point to NG cell bodies that extend into or are located in a different neuromere than the ProNm in which the clone originated. (S) Schematic summarizing the Lin A and Lin D NGBs and their progeny. Green indicates a Lin A.2 MARCM clone induced in the NGB; red indicates all proliferating glia. Bottom right: schematic of a thoracic neuromere, showing all (red) or just Lin A.2 (green outline) astrocytes with their processes entering the neuropil. (T) Same as (S) showing three possible outcomes for a Dll⁻ Lin A.2 MARCM clone: no compensation, an increase in astrocyte processes, or an increase in astrocyte number. (U and V) WT (U) and Dll⁻ (V) Lin A.2 MARCM clones in the right ProNm, labeled with mCD8::GFP (green) under the control of VGlut-Gal4 and repo-Gal4, and co-stained with anti-BRP (blue). All astrocytes are labeled with mCherry under the control alrm-QF. Frontal (FS) (V3) and transverse (TS) (V4) sections showing that both thoracic neuropils appear to be equally invaded by astrocyte processes even if the neuropil contains a Dll⁻ Lin A.2 clone. (W) Average number of repo > H2B:RFP⁺ NG, alrm > H2B:RFP⁺ astrocytes and R56F03 > H2B:RFP⁺ EG in WT and Dll⁻ cis² MARCM Lin A.2 clones. Error bars indicate SD. See also Figure S6. Number of samples analyzed = 4 (X) Average number of astrocytes in WT and Dll⁻ MARCM Lin A.2 clones (GFP⁺, mCherry⁺), compared to the total number of astrocytes (GFP⁻, mCherry⁺). Error bars represent SD. Number of samples analyzed for each type of neuromere = 4.