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Programmed Cell Death in Type II Neuroblast Lineages Is Required for Central Complex Development in the Drosophila Brain

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Programmed Cell Death in Type II Neuroblast Lineages Is Required for Central Complex Development in the Drosophila Brain

Yanrui Jiang et al. Neural Dev.

Abstract

Background: The number of neurons generated by neural stem cells is dependent upon the regulation of cell proliferation and by programmed cell death. Recently, novel neural stem cells that amplify neural proliferation through intermediate neural progenitors, called type II neuroblasts, have been discovered, which are active during brain development in Drosophila. We investigated programmed cell death in the dorsomedial (DM) amplifying type II lineages that contribute neurons to the development of the central complex in Drosophila, using clonal mosaic analysis with a repressible cell marker (MARCM) and lineage-tracing techniques.

Results: A significant number of the adult-specific neurons generated in these DM lineages were eliminated by programmed cell death. Programmed cell death occurred during both larval and pupal stages. During larval development, approximately one-quarter of the neuronal (but not glial) cells in the lineages were eliminated by apoptosis before the formation of synaptic connectivity during pupal stages. Lineage-tracing experiments documented the extensive contribution of intermediate neural progenitor-containing DM lineages to all of the major modular substructures of the adult central complex. Moreover, blockage of apoptotic cell death specifically in these lineages led to prominent innervation defects of DM-derived neural progeny in the major neuropile substructures of the adult central complex.

Conclusions: Our findings indicate that significant neural overproliferation occurs normally in type II DM lineage development, and that elimination of excess neurons in these lineages through programmed cell death is required for the formation of correct neuropile innervation in the developing central complex. Thus, amplification of neuronal proliferation through intermediate progenitors and reduction of neuronal number through programmed cell death operate in concert in type II neural stem-cell lineages during brain development.

Figures

Figure 1
Figure 1
An increased number of neural cell somata is present in apoptosis-blocked dorsomedial (DM) lineages. (A, B) Neuropile labeled by nc82 and DM lineage-derived cells labeled with green fluorescent protein (GFP) in (A) wild-type flies and (B) DM lineage-specific apoptosis-blocked flies. Single confocal sections at the level of the fan-shaped body of the central complex are shown. (C, D) Neuropile labeled with nc82 and DM lineage-derived cell bodies labeled with GFP in (C) wild-type and (D) DM lineage-specific apoptosis-blocked flies. Single confocal sections at the level of the posterior dorsal midline are shown. Note the marked increase in the number of labeled DM lineage-derived cell somata in the apoptosis-blocked flies compared with wild-type. (A) and (C) are from the same preparation, as are (B) and (D), and both preparations are from pupae 48 hours after puparium formation. Scale bars = 20 μm. Genotypes: (A, C) UAS-flp; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP (B, D) UAS-flp; UAS-p35; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP.
Figure 2
Figure 2
Programmed cell death in dorsomedial (DM) lineages occurs during both larval and pupal development. (A-D) DM lineage cells expressing activated Caspase-3 (aCasp-3) at different times during post-embryonic development. Single confocal sections show activated Caspase-3 (red) and DM lineage cells labeled with mCD8::GFP (green) at (A) 48 and (B) 96 hours after larval hatching (ALH), and (C) 12 and (D) 24 hours after puparium formation (APF). Asterisks indicate cells undergoing apoptosis. (E) Average number of observed apoptotic cells per DM lineage at different stages during larval and pupal development (error bars represent the standard error). For each time point, more than 36 DM lineages were examined. Scale bars = 10 μm. Genotype: (A-D) UAS-mCD8::GFP; erm-Gal4R09D11.
Figure 3
Figure 3
Clonal analysis of apoptosis-blocked dorsomedial (DM) lineages during larval development. (A) Schematic representation of the six DM lineages located at the posterior dorsomedial edge of the brain hemisphere. Cell number was quantified for the DM1 lineage (marked in green). (B) Average number of cells in wild-type (light blue) and apoptosis-blocked (dark blue) DM1 lineage. (C) Average number of glial cells in wild-type (light blue) and apoptosis-blocked (dark blue) DM1 lineage. (D-F) A wild-type mosaic analysis with a repressible cell marker (MARCM) clone of a DM1 lineage; cells labeled with mCD8::GFP (green). Single confocal sections at the level of (D) the neuroblast (arrowhead), and (E) the secondary axon tract (arrow), and superimposition of all single optical sections containing (F) labeled cells are shown. (G-I) A MARCM clone of a cell death-blocked DM1 lineage; cells labeled with mCD8::GFP (green). Single confocal sections at the level of (G) the neuroblast (arrowhead), and (H) the secondary axon tract (arrow), with (I) superimposition of all single optical sections containing labeled cells are shown. (B, C) Error bars represent standard deviation. Scale bars = 20 μm. Genotypes: (D-F) hs-flp; tub-Gal4 UAS-mCD8::GFP; FRT2A tub-Gal80/FRT2A; (G-I) hs-flp; tub-Gal4 UAS-mCD8::GFP; FRT2A tub-Gal80/FRT2A H99.
Figure 4
Figure 4
Innervation of the central complex from wild-type dorsomedial (DM) lineages. Confocal sections show the neuropile (white, labeled with nc82) and neuronal innervation of the central complex from wild-type DM lineages (green, labeled with mCD8::GFP) in adult brains at the level of (A-D) the ellipsoid body, (E-H) the fan-shaped body, (I-L) the noduli, and (M-P) the protocerebral bridge of the central complex. (A, B, E, F, I, J, M, N) Overview of the neuropile and the central complex innervation from DM lineages. (C, D, G, H, K, L, O, P) Close-up view of the central complex neuropile and the neuronal innervation from DM lineages. Scale bars = 20 μm. Genotype: UAS-flp; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP.
Figure 5
Figure 5
Innervation of the central complex from apoptosis-blocked dorsomedial (DM) lineages. Confocal sections show the neuropile (white, labeled with nc82) and neuronal innervation of the central complex from apoptosis-blocked DM lineages (green, labeled with mCD8::GFP) in adult brains at the level of (A-D) the ellipsoid body, (E-H) the fan-shaped body, (I-L) the noduli, and (M-P) the protocerebral bridge of the central complex. (A, B, E, F, I, J, M, N) Overview of the neuropile and the central complex innervation from apoptosis-blocked DM lineages. (C, D, G, H, K, L, O, P) Close-up view of the central complex neuropile and the neuronal innervation from DM lineages. Arrows and arrowheads indicate misinnervation by labeled neuronal projections and misarrangement of neuropile in the central complex (see text for details). Scale bars = 20 μm. Genotype: UAS-flp; UAS-p35; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP.
Figure 6
Figure 6
Development of central complex innervation defects in apoptosis-blocked dorsomedial (DM) lineage neurons. (A-D) Confocal images of single sections at the level of (A, B) the ellipsoid body and (C, D) the fan-shaped body in wild-type pupal brains at 24 hours after puparium formation (APF). Neuropile labeled with (A, C) nc82 and (B, D) neuronal innervation from DM lineages labeled with mCD8::GFP. (E-H) Confocal images of single sections at the level of (E, F) the ellipsoid body and (G, H) the fan-shaped body in the pupal brains of DM-specific apoptosis-blocked flies at 24 hours APF. (E, G) Neuropile labeled with nc82 and (F, H) neuronal innervation labeled with mCD8::GFP. (F) Perturbations in the innervation of the developing EB are indicated by arrows. (I-L) Confocal images of single sections at the level of (I, J) the ellipsoid body and (K, L) the fan-shaped body and noduli in pupal brains of wild-type at 48 hours APF. Neuropile labeled with (I, K) nc82 and (J, L) neuronal innervation labeled with mCD8::GFP. (M-P) Confocal images of single sections at the level of (M, N) the ellipsoid body and (O, P) the fan-shaped body and noduli in pupal brains of DM-specific apoptosis-blocked flies at 48 hours APF. Neuropile labeled with (M, O) nc82 and (N, P) neuronal innervation labeled with mCD8::GFP. Misinnervation and misarrangement in the developing central complex are indicated by (N, P) arrows and arrowheads (see text for details). Scale bar = 20 μm Genotypes: (A-D, I-L) UAS-flp; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP (E-H, M-P) UAS-flp; UAS-p35; erm-Gal4R09D11 act>CD2>Gal4 UAS-mCD8::GFP.

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