Lineage-guided Notch-dependent gliogenesis by Drosophila multi-potent progenitors

Abstract

Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that glial precursors with distinct developmental fates exist that govern region-specific gliogenesis. Here, we have mapped the glial progeny produced by the Drosophila type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis.

Keywords: Apoptosis; Astrocyte; Ensheathing glia; Gliogenesis; Notch; Proliferation.

Fig. 1.

CLIn reveals glial cells made by type II NBs. Representative images of anti-mCD8 immunostained (green) and nc82-counterstained (blue) adult fly brains. (A) Genetics for labeling all brain glia (repo-GAL4 driving UAS-mCD8::GFP expression). (B-C‴) Complete expression pattern of brain glia (n=5). (B) Maximum projection image of a z-stack. (C-C‴) Single confocal sections showing glia distribution from posterior (C) to anterior (C‴). MB calyx (CA), protocerebral bridge (PB), inferior bridge (IB), superior posterior slope (SPS), inferior posterior slope (IPS), lobula (LO), lobula plate (LOP), medulla (ME), superior medial procerebrum (SMP), superior intermediate protocerebrum (SIP), superior lateral protocerebrum (SLP), superior clamp (SCL), inferior clamp (ICL), pedunculus (PED), lateral horn (LH), fan-shaped body (FB), noduli (NO), gorget (GOR), posteriorlateral protocerebrum (PLP), vest (VES), wedge (WED), posterior ventrolateral protocerebrum (PVLP), anterior ventrolateral protocerebrum (AVLP), ellipsoid body (EB), MB medial and vertical lobes (ML and VL), spur (SPU), crepine (CRE), epaulette (EPA), lateral accessory lobe (LAL), anterior optic tubercle (AOTU), antennal lobe (AL), and gnathal ganglia (GNG). We follow the naming nomenclature of Ito et al. (2014). (D) CLIn technique marks only the repo-GAL4-positive glia within type II NB lineages targeted with stg14-KD. The∩represents an intersection. (E) Glia innervating only subsets of brain neuropils, particular the CX, are visible after intersection. n=7. (F-F‴) Single confocal sections showing the major neuropils outlined and covered by glia of type II lineages. (G) Independent labeling of type II glia and all brain glia via dual genetic systems (GAL4-UAS and LexAp65-lexAop). (H,H′) Glial cells (green) in PB and CA regions. Magenta, Repo staining. Repo∩T2 indicates the presence of type II lineage glia. (I,I′) Glial cell distributions after inducing apoptosis in all type II lineage glia (Repo∩T2; grim). (J) Quantification of glial cell numbers. n=14 and 10, respectively. Scale bars: 50 μm.

Fig. 2.

Glial cells originating from individual type II NBs. (A-H′) Composite confocal images of adult fly brains with the offspring of the type II NB labeled in magenta, in which the repo-GAL4-positive glia, if any, are further marked in green. The magenta/green and green-only views of the same brains, counterstained with nc82 mAb (blue), are shown side by side. Note absence of detectable glia in the DM1 (A′), DM4 (D′) and DL2 (H′) lineages, and presence of three distinct glial subsets, including outer chiasm glia (Xg^o), inner chiasm glia (Xgⁱ), lateral cell body rind glia (LCBRg) of the OL, in the DL1 clone (G′). DM1, n=17; DM2, n=10; DM3, n=8; DM4, n=3; DM5, n=15; DM6, n=12; DL1, n=7; DL2, n=8. The LCBRg, located between ME and PVLP, was present in 86% of DL1 samples. (I-M′) CLIn with dEaat-GAL4 marks the astrocyte-like glia (green) in gliogenic type II lineages (magenta). No astrocyte-like glia exist in the DL1 lineage (M′). DM2, n=4; DM3, n=8; DM5, n=10; DM6, n=10; DL1, n=2. (N-R′) CLIn with GAL4-MZ709 marks the ensheathing glia (green) in gliogenic type II lineages (magenta). Xg^o and Xgⁱ, but not LCBRg, express the ensheathing glial marker (compare R′ with G′). DM2, n=6; DM3, n=5; DM5, n=7; DM6, n=4; DL1, n=5. Scale bars: 50 μm. See also Fig. S2.

Fig. 3.

Diversity and location variability of DM5 INP1 glia. (A-C′) Composite confocal images of nc82-counterstained (blue) adult fly brains in which the DM5-INP1 sublineage is labeled in magenta (A-C) and the glial offspring positive for dEaat1-GAL4 (A-B′, n=3 samples) or for GAL4-MZ709 (C,C′, n=6 samples) are marked in green. Insets in A-C′ show single confocal sections of the corresponding dashed box areas in A-C. Magenta-only labels the neurons as well as additional types of glia. We can identify ensheathing glia morphology (arrowheads) in A-B′ and astrocyte-like glial morphology (arrow) in C,C′. The clone shown in B (five neurons and glia) is a sub-clone of DM5 INP1 (eight neurons and glia). Scale bars: 50 μm. See also Figs S2 and S3.

Fig. 4.

Blocking cell death in Repo-positive cells or entire lineages reveals lineage-characteristic phenotypes. (A) Genetics for blocking apoptosis in type II lineages. (B-K′) Composite confocal images of adult fly brains carrying various CLIn clones of type II lineages. All the offspring of the CLIn clones are labeled in magenta, and the offspring positive for repo-GAL4 are also marked in green for morphology and in blue for nuclei (lacZ). The clone identity and the transgene used to block apoptosis in Repo-positive offspring (UAS-p35) or entire progeny (lexAop-p35) are indicated. The dashed boxes in D-K are shown at higher magnification on the right (D′-K′). (B-D′) Increase of DM5 glia (B,B′) by either UAS-p35 (C,C′) or lexAop-p35 expression (D,D′). (E-G′) The DM6 glial cells (E,E′) are increased after lexAop-p35 (G,G′), but not UAS-p35, expression (F,F′). (H,H′) Glial cells from DM5-INP1 (right hemisphere). (I-K′) The DM6-INP1 do not contain glial cells (I,I′). Blocking apoptosis by lexAop-p35 rescued some glia (K,K′), while blocking apoptosis by UAS-p35 fails to rescue any glia (J,J′). In H,I, brains were contaminated with a DM5 NB clone and a type I lineage clone, respectively. Scale bars: 50 μm. See also Figs S4, S5 and S6.

Fig. 5.

Notch is required for gliogenic switch. (A) Illustration of twin-spot MARCM, which marks the homozygous mutant clone with mCD8::GFP and its paired wild-type sister clone with rCD2::RFP. (B-D″) Su(H) mutant clones lack glia. Twin-spot MARCM clones of the DM5 lineage induced shortly after larval hatching and examined in adult fly brains counterstained with anti-Repo Ab (blue). (B-D) Merged views of B′-D′ and B″-D″. The glia-like processes (arrow) are present on both green and magenta sides of the control DM5 twin-spot clones (B-B″, n=3). By contrast, the glia-like processes are not seen on the mutant green side of the twin-spot MARCM clones derived from Su(H) heterozygous precursors (C′,D″, n=3 and 2, respectively). (E-F″″) Su(H) mutant clones have excess neurons at the expense of glia. Composite confocal images of the late larval brain carrying twin-spot MARCM clones. Partial projection of the INP cell body region (small box in E,F) and the glia region (big box in E,F) are shown in E′,F′ and E″,F″, respectively. Single confocal sections of INP glia (green box in E″,F″) and NB glia (E″″,F″; red box in E″,F″) are shown in E‴,F‴ and E″″,F″″, respectively. (E-E″″) Wild-type DM5 and DM5-INP1 twin-spot clone. The INP1 example shown has seven neurons (E′) and two glial cells (E‴). On average, DM5-INP1 has 7.7±0.5 neurons and 1.1±0.4 glial cells (n=7). Arrowheads indicate glial cells. (F-F″″) Wild-type DM5 and mutant DM5-INP1 twin-spot clone. The INP1 example shown has 14 neurons (F′) and no glia (F‴). The cell in F‴ is negative for Repo (arrow). On average, mutant INP1 has 13±2 neurons and 0±0 glial cells (n=4). Scale bars: 50μm in B-D″; 20 μm in E-F″″. Data are mean±s.d.

Fig. 6.

Notch promotes glia expansion. Composite confocal images of adult fly brains with the type II glial population labeled in green. Blue indicates nc82 counterstaining. (A) Glial cell distribution in wild-type type II lineages (n=6). (B) Type II glia-specific RNAi knockdown of Delta, a Notch ligand, effectively reduces the glial coverage of neuropil regions (n=6). (C) Type II glia-specific overexpression of the Notch intracellular domain (NICD) greatly increases the glial coverage of neuropil regions (n=6). (D) Quantification of glial cell number by staining nuclear localized β-galactosidase after Notch manipulations driven by repo-GAL4. n=11, 7 and 10, respectively. (E-G) Quantifications of cell number, total volume and individual cell volume of astrocyte-like glia after Notch manipulations driven by dEaat1-GAL4. (E,F) n=10, 10 and 8, left to right columns. (G) n=11, 20 and 7, left to right columns. (H,I) Quantifications of cell number and total volume of ensheathing glia after Notch manipulations driven by R56F03-GAL4. n=9, 8 and 7, left to right columns. *P<0.05, **P<0.01, ***P<0.001; n.s., not significant. Scale bars: 50 μm. See also Fig. S7.

Fig. 7.

A working model of gliogenesis in Drosophila type II lineages. Type II NBs divide asymmetrically to self-renew and give rise to INPs. Each INP also undergoes multiple rounds of asymmetrical division to self-renew and generates ganglion mother cells (GMCs), which divide once to produce two neurons. Although the exact source of glial precursors remains to be determined, Notch signaling is required for gliogenic switch. The glial precursor might initially be positive for Gcm, which then turn on the downstream glial genes, including repo (Jones, 2005; Viktorin et al., 2011). The glial precursor generates both astrocyte-like and ensheathing glia. The expansion of both glial subtypes is Notch dependent. Lineage identity governs multiple stages of glial development, controlling which INPs produce glial precursors, which glial precursors undergo apoptosis before repo is expressed, the expanding potential of glial precursors, the diversity of glial progeny and their final distribution.