Extremes of lineage plasticity in the Drosophila brain

Abstract

An often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.

Figure 1. Mushroom body γ neuron population size is highly plastic

(A) Protein starvation of newly hatched larvae results in extra MB neurons (OK107+, p<0.01), which are of the γ class (EcRB1+, p<1×10⁻⁴ and 201Y+, p<1×10⁻¹⁰), by P0. No significant difference in other (EcRB1−) classes was observed. Control: Animals of genotype OK107-GAL4 or 201Y-GAL4; UAS-CD8::GFP raised on standard food. Sucrose: Animals of genotype OK107-GAL4 or 201Y-GAL4; UAS-CD8::GFP protein-starved in 20% sucrose solution for 48 hrs ALH then transferred to standard food. Standard error bars are shown, with Student’s t-test used to establish significance. (B) Percentage of clones induced at different developmental times (48, 72, 96, 120, and 144 hrs ALH) for each type of MB neuron. The three bars represent non-starved larvae (green triangles) and larvae starved from 48 hrs (orange triangles) or 60 hrs (blue triangles) ALH. The colored areas in each bar represent the percentage of clones for γ (blue), α′/β′ (magenta), pioneer α/β (yellow), and α/β (green) neurons, while the number indicates how many clones were analyzed for each treatment. Open bars with dashed outlines: not assayed. (C) Number of 201Y-GAL4-positive (γ) neurons at wandering third instar for control larvae and larvae protein-starved from 48 hrs ALH for six days before being transferred back to food.

Figure 2. Mushroom body α/β neuron population size is highly plastic

(A–F) The MBs of a wild-type fly (A,C,E) and a fly in which the PTTH neurons had been ablated by overexpressing the death gene grim using PTTH-GAL4 (B,D,F). The MBs were co-labeled with anti-Trio (magenta, γ and α′/β′ neurons) (A,B,E,F) and anti-FasII (green, γ and α/β neurons) (C,D,E,F). Scale bars: 20 μm.

Figure 3. MB temporal transitions are regulated by overall growth, not an internal counting mechanism

(A–F) The γ to α′/β′ transition is delayed in insulin receptor mutant animals. The MBs of wild-type (A,B) and InR^E19 homozygous (D,E) animals at specific days after larval hatching (ALH). The α′/β′ neurons were labeled by c305a-GAL4 (green), and the γ neurons were immunostained with anti-FasII Ab (magenta). Scale bars: 20 μm. The MBs of wild-type (C) and InR^E19 homozygous (F) animals at 24 hrs after puparium formation (APF). The partially pruned γ neurons (arrowheads) and the nascent α/β neurons (arrows) were immunostained with anti-FasII Ab (gray). Scale bars: 20 μm. (G–J) InR^E19 homozygous mushroom body clones induced in newly hatched larvae are smaller than wild-type clones but still feature all neuron classes. A wild-type (G) and InR^E19 homozygous (H) MB clone labeled by OK107-GAL4 in adult. The α′/β′ neurons labeled by c305a-GAL4 in a wild-type (I) and InR^E19 (J) mushroom body clone at 4 days ALH. Scale bars: 20 μm.

Figure 4. Protein starvation delays temporal identity transitions without altering final neuronal composition of the antennal lobe lineages

(A) Comparison of adult antennal lobes from fed control larvae to those of protein-starved larvae. 2D starvation: larvae were protein-starved from 0–48 hrs ALH then replaced on normal food. 4D starvation: larvae were starved from 48 hrs ALH for 4 days then replaced on normal food. adPN NB clones were labeled by acj6-GAL4, and lAL NB clones were labeled by acj6-GAL4 and GH146-GAL4. (B,C) The uniglomerular PN classes labeled by heat shock at different developmental timepoints (48, 72, 96, 120, 144 and 168 hrs ALH). The two bars at each timepoint represent fed control larvae (green triangles) and larvae starved from 48 hrs ALH (blue triangles). The colored areas in each bar represent the percentage of clones that belonged to particular temporal classes: DC2-DC3 (blue), VA1d-VM2 (magenta), DM6-VM5d (yellow) and VM5v-DL2d (green) neurons in adPN (B), and DM2-VA7m (blue), VL3/DA1 (magenta) and VM1-DM5 (yellow) neurons in lAL (C). Open bars with dashed outlines: not assayed. (D,E) Proliferation speeds of adPN (D) and lAL (E) NBs for fed controls (blue line) and animals protein-starved from 0–48 hrs ALH (magenta line). Brains were dissected at different developmental timepoints and incubated in S2 medium containing EdU (100 μg/ml) for 2 hrs at 25°C before fixation.