Metamorphosis of memory circuits in Drosophila reveals a strategy for evolving a larval brain
- Janelia Research Campus, United States
- Department of Biology, Friday Harbor Laboratories, University of Washington, United States
- Life Sciences Institute, University of Michigan, United States
Figures
Figure 1
Comparison of the effects of direct development versus metamorphosis on neurogenesis and the establishment of neuronal phenotypes.
In a direct developing insect like a cricket, embryogenesis produces a hatchling with a miniature cricket body plan. Its neuroblasts (NBs) generate their entire lineages during embryogenesis so that at hatching the CNS has its full complement of neurons and they already possess their mature phenotypes. In the metamorphic development of Drosophila, by contrast, a shortened embryonic phase redirects development to produce a simplified, larval body plan. Their neuroblasts produce only their early-born neuron types and neuronal phenotypes are modified for larval morphology and behavior. During larval growth, the arrested NBs reactivate to produce the rest of their neuronal lineages, but their young neurons arrest development soon after their birth. The species-typical body plan of the fly finally arises at metamorphosis and, in the CNS, remodeling larval neurons and maturing postembryonic-born neurons combine to make the mature nervous system of the fly.
Figure 2
The organization and development of the larval and adult mushroom bodies (MBs).
(A) The larval MB has a core of γ Kenyon neurons whose dendrites project to the calyx (CA) neuropil and whose axons extend through the peduncle and bifurcate into a vertical and medial lobe. Projections from three clusters of aminergic neurons, the PAL, PPL1, and PAM clusters divide the axon array into 10 computational compartments: IP and LP: intermediate and lower peduncle; LA: lateral appendix; UVL, IVL, LVL: upper, intermediate, and lower vertical lobe; SHA: shaft; UT, IT, LT: upper, intermediate, and lower toe. PPL1 input largely indicates punishment, PAM input indicates reward, and PAL is unknown. The diagrams below highlight in gray (left) the compartments that lose contact with γ neuron axons during pruning and (right) the compartments that are not incorporated into the adult MB. (B) The adult MB has 16 compartments. It contains regrown γ neurons (gray) that lack the larval-specific vertical branch along with late developing α′β′ (green) and αβ (orange) Kenyon cells. These together form the medial (β′,β) and vertical (α′, α) lobe systems of the adult. Compartment designations are numbered and based on the Kenyon cell axons that they contain. (C) Projection of a multicolor flip-out (MCFO) image from a larval brain showing two MB input neurons that project bilaterally to the upper (UVL) and intermediate (IVL) compartments of the vertical lobes. Blue: neuroglian staining. (D) Schematic of the microcircuitry characteristic of larval and adult compartments. (E) Developmental timeline of the production of the three major classes of Kenyon cells that make up the mature MB.
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Figure 2—source data 1
Examples of the adult anatomies of larval neurons MBIN-b1 and -b2 obtained by flip-switch-mediated immortalization of expression of line SS21716 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data1-v2.pptx
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Figure 2—source data 2
Examples of the adult anatomies of larval neurons DAN-c1 and DAN-d1 obtained by flip-switch-mediated immortalization of expression of lines MB586B and MB328B, respectively, late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data2-v2.pptx
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Figure 2—source data 3
Examples of the adult anatomy of larval neuron OAN-e1 obtained by flip-switch-mediated immortalization of expression of lines SS21716 and SS01958 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data3-v2.pptx
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Figure 2—source data 4
Examples of the adult anatomies of larval neurons MBIN-l1 and DAN-f1 obtained by flip-switch-mediated immortalization of expression of stable spilt lines late in larval life.
The anatomy of the adult form of MBIN-l1 was revealed using lines SS04484 and SS01624; that of DAN-f1 using lines MB065B and MB145B.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data4-v2.pptx
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Figure 2—source data 5
Examples of the adult anatomies of larval neurons DAN-g1 and OAN-g1 obtained by flip-switch-mediated immortalization of expression of stable spilt lines late in larval life.
The anatomy of the adult form of DAN-g1 was revealed using lines SS017164 and SS01755; that of OAN-g1 using lines SS20844 and SS4268.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data5-v2.pptx
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Figure 2—source data 6
Table showing the success rate for maintaining expression of the various larval neurons through metamorphosis.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig2-data6-v2.xlsx
Figure 3 with 2 supplements
The metamorphic fates of the larval mushroom body input neurons (MBINs).
(A) The fates of the larval MBINs that innervate the calyx and the 10 compartments of the larval MB. For larval MBINs that remain with the MB, the arrows show the relationship of their larval compartment to the one that they innervate in the mature, adult MB. The remaining MBINs die (skull), trans-differentiate (T) to supply non-MB circuits in the adult, or their fate is unknown (?). For the MBINs whose transmitter is unknown, they express tyrosine hydroxylase but their final secreted transmitter has not been determined. Compartment designations as in Figure 2. (B) Images comparing the larval and adult forms of the MBINs that persist through metamorphosis. The images of larval cells from Saumweber et al., 2018 Nature Comm. 9: 1104. Adult names based on Aso et al., 2014, Li et al., 2020, or this study.
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Figure 3—source data 1
Examples of the adult anatomy of larval neuron MBON-a1 obtained by flip-switch-mediated immortalization of expression of lines SS01417 and SS00867 late in larval life.
The first line also revealed an occasional adult form of MBON-a2.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data1-v2.pptx
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Figure 3—source data 2
Examples of the adult anatomies of larval neurons MBON-a2 and MBON-b1,-b2 obtained by flip-switch-mediated immortalization of expression of stable spilt lines late in larval life.
The anatomy of the adult form of MBON-a2 was revealed using lines SS00872 and SS02006; that of MBON-b1,-b2 using lines SS01708 and SS01959.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data2-v2.pptx
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Figure 3—source data 3
Examples of the adult anatomies of larval neurons MBON-d1, MBON-e2, and MBON-f2 obtained by flip-switch-mediated immortalization of expression of stable spilt lines late in larval life.
The anatomy of the adult form of the three neurons was revealed using lines SS01705, SS04172, and SS04328, respectively.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data3-v2.pptx
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Figure 3—source data 4
Examples of the adult anatomies of larval neurons MBON-g1 and -g2 obtained by flip-switch-mediated immortalization of expression of lines SS02130 and SS02121 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data4-v2.pptx
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Figure 3—source data 5
Examples of the adult anatomies of larval neurons MBON-h1 and -h2 obtained by flip-switch-mediated immortalization of expression of line SS01725 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data5-v2.pptx
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Figure 3—source data 6
Examples of the adult anatomies of larval neurons MBON-j1 and MBON-j2 obtained by flip-switch-mediated immortalization of expression of lines SS01973 and SS00860 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data6-v2.pptx
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Figure 3—source data 7
Examples of the adult anatomies of larval neurons MBON-i1 and MBON-k1 obtained by flip-switch-mediated immortalization of expression of lines SS01962 and SS04236 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig3-data7-v2.pptx
Figure 3—figure supplement 1
Confocal projections showing the terminal, adult identity of larval mushroom body input neurons (MBINs) that undergo trans-differentiation at metamorphosis.
(A, B) Female and male versions of larval MBIN-b1 and -b2. Images to the right are a horizontal section at the level of the tick marks showing that in males the cell innervates the medulla (med) but in the female the medulla projection is reduced but it has extensive branching in the lobula (lob). Ot: optic tubercle. (C) Frontal projection showing the terminal adult morphology of larval cell OAN-e1. Image to right is a lateral section at level of the tick marks showing that the arbor is outside of the bundles of Kenyon cell axons. (C’) and (C”) are frontal slices at levels to relationship of arbor the γ, β', and β lobes of the medial loves (C’) and the α and α' lobes of the vertical lobes (C”). (D) Terminal adult anatomy of larval DAN-f1. (E) Terminal adult identity of larval MBIN-l1. Green: pseudo color representation of RFP; magenta: nc82. bi: bilateral, CRP: crepine, LAL: lateral accessory lobe, lob: lobula, med: medulla, OL: optic lobe, ot: optic tubercule, PAL: protocerebral anterior lateral, PPL: protocerebral posterior lateral, SMP: superior medial protocerebrum.
Figure 3—figure supplement 2
Confocal projections showing the terminal, adult structure of the larval neuron OAN-g1.
The adult cell is called OA-VPM3. FB: fan-shaped body, SP: superior protocerebrum. Green: pseudocolor representation of RFP; magenta: nc82.
Figure 4 with 1 supplement
The metamorphic fates of the larval mushroom body output neurons (MBONs).
(A) The fates of the larval MBONs that innervate the calyx and the 10 compartments of the larval MB. For MBONs that remain within the MB after metamorphosis, the arrows show the relationship of their larval compartment to the one that they innervate in the adult MB. The remaining MBONs trans-differentiate (T) to supply non-MB circuits in the adult, or their fate is unknown (?). Compartment designations as in Figure 2. Transmitters: green: acetylcholine; blue: glutamate; red: GABA; gray: unknown; checkered versions are presumed transmitters based on Li et al., 2020. (B) Images comparing the larval and adult forms of the MBONs that persist through metamorphosis. The images of larval cells from Saumweber et al., 2018 Nature Comm. 9: 1104. Adult names based on Aso et al., 2014, Li et al., 2020, or this study.
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Figure 4—source data 1
Examples of the adult anatomies of larval neuron APL obtained by flip-switch-mediated immortalization of expression of line SS01671 late in larval life.
- https://cdn.elifesciences.org/articles/80594/elife-80594-fig4-data1-v2.pptx
Figure 4—figure supplement 1
Confocal projections showing the terminal, adult identity of larval mushroom body output neurons (MBONs) that undergo trans-differentiation at metamorphosis.
Frontal views of the adult brain showing the terminal identities of (A) MBON-d2, (B) MBON-b1 and -b2, and (C) MBON-g1 and g2. (C') A magnified image of the boxed region of 'C' showing the terminals of the neuron in the intermediate section of the nodulus. Green: pseudocolor representation of RFP; magenta: nc82.
Figure 5 with 2 supplements
The metamorphic transformation of selected mushroom body input neurons (MBINs) during early metamorphosis and their mature phenotype in the adult.
Confocal images track the GFP expression through the first 48 hr after pupariation (P); the adult images show flip-switch induced expression of red fluorescent protein. Arrowheads: growth cones; P+#: # hours after pupariation. Background staining for the developmental series is for Fasciclin II (magenta); for the adult, it is Bruchpilot (nc82) (blue). Lines used for developmental timelines: DAN-c1: JRC-SS03066, DAN-d1: JRC-MB328B, DAN-g1: JRC-SS01716, MBIN-l1: JRC-SS04484, MBIN-b1,-b2: JRC-SS21716.
Figure 5—figure supplement 1
Confocal images following the degeneration of GFP-labeled larval PAM neuron early in metamorphosis.
The cells are reduced to GFP-labeled fragments by 24 hr after pupariation. Inset is an enlarged version of the boxed region for line SS01949 showing the discrete axonal tufts in the shaft (SHA), upper toe (UT), and intermediate toe (IT) compartments. P+#: # hours after pupariation. Background staining for the developmental series is Fasciclin II (magenta); lines used for developmental timelines: DAN-h1, -i1, and -j1: JRC-SS01949; DAN-k1: JRC-SS01757.
Figure 5—figure supplement 2
Confocal images showing the early metamorphic changes in line JRC-SS01702 driving GFP expression in two mushroom body input neurons (MBINs): OAN-e1 and DAN-c1.
The subsequent images track the axonal tufts of the two neurons. That of DAN-c1 persists as a cohesive tuft associated with the mushroom bodies while OAN-e1 tufts retract from the mushroom bodies form a diffuse arbor over the superior medial protocerebrum. The adult image shows an example of flip-switch-induced expression of red fluorescent protein (RFP) in the adult. Two adult neurons were found in flip-out samples of this line: the above cell interpreted as the adult version of OAN-e1 and the adult version of DAN-c1 as confirmed by a second, clean DAN-c1 line (Figure 5). P+#: # hours after pupariation. Background staining for the developmental series is for Fasciclin II (magenta); for the adult it is nc82 (blue).
Figure 6 with 3 supplements
The metamorphic transformation of selected mushroom body output neurons (MBONs) during early metamorphosis and their mature phenotype in the adult.
Confocal images track the GFP expression through the first 48 hr after pupariation (P); the adult images show flip-switch-induced expression of red fluorescent protein. *Expression due to nontarget neurons in some driver lines; filled arrowheads: axonal growth cones; open arrowheads: dendritic growth cones; P+#: # hours after pupariation. Magenta: Fasciclin II; blue: Bruchpilot (nc82). Lines used for developmental timelines: MBON-a1: JRC-SS00867, MBON-a2: JRC-SS02006, MBON-g: JRC-SS02130, MBON-h: JRC-SS01725, MBON-j1: JRC-SS01973.
Figure 6—figure supplement 1
The early metamorphic transformation in selected larval mushroom body output neurons (MBONs) that assume similar phenotypes in the adult system.
Confocal images track the GFP expression through the first 48 hr after pupariation; the adult images show flip-switch-induced expression of red fluorescent protein (RFP) in the adult. Open triangles: dendritic growth cones; filled triangles: axonal growth cones: growth cones; P+#: # hours after pupariation. Background staining for the developmental series is for Fasciclin II (magenta); for the adult it is nc82 (blue). Lines used for developmental timelines: MBON d1: JRC- SS01705, MBON j1: JRC- SS01973, MBON-i1: JRC- SS04244, MBON-k1: JRC- SS01980, APL: JRC- SS01671.
Figure 6—figure supplement 2
Pruning and outgrowth of MBON-j2 as it transforms into its adult form named MBON 05.
At pupariation (P 0h), MBON-j2 has a dendritic arbor in the ipsilateral medial toe (MT) compartment and a contralateral axon arbor (AA). By P+8 hr, the dendritic arbor is gone and the axonal arbor has severely reduced. At P+18 hr, the cell has formed contralateral outgrowth areas for new dendritic (filled arrowhead) and axonal arbors (open arrowhead). P+24 h: dendritic growth invades the γ4 compartment (filled arrowhead) while the axonal region splits into multiple growth cones, one of which invades the γ2 compartment (open arrowhead); By P+48 hr, a dendritic tuft fills the γ4 compartment and axonal arbor is in γ2, but the cell shows the delayed invasion of γ1. Adult version of the cell is a red fluorescent protein version obtained by flip-switch treatment of MBON-j2 in the larva. Blue: nc82; green: green fluorescent protein; magenta: fasciclin II.
Figure 6—figure supplement 3
The early metamorphic transformation of larval mushroom body output neurons (MBONs) that show major redirections to adult mushroom body compartments.
Confocal images track the GFP expression through the first 48 hr after pupariation; the adult images show flip-switch-induced expression of red fluorescent protein (RFP) in the adult. Top shows early stages in the metamorphosis of MBON-c1. This example of MBON-c1 has an atypical ectopic branch (e) that leads to the larval calyx. Most larval cells lack this branch. Subsequent images show the progression of arbor loss and outgrowth through the 24 hr after pupariation. The yellow and white triangles show comparable junctions in the cell through time. The inset at P+24 hr is a sub-stack projection through the calyx (CA) neuropil showing that growth cones have invaded this neuropil. p: peduncle; green: green fluorescent protein; magenta: fasciculin II; blue: N-cadherin. Bottom follows the changes in MBON e2 and MBON f2 as they are redirected from the larval vertical lobe to the adult medial lobe. Triangles: growth cones; P+#: # hours after pupariation. Background staining for the developmental series is for fasciclin II (magenta); for the adult it is nc82 (blue). Lines used for developmental timelines: MBON c1: JRC-SS21789, MBON e2: JRC-SS04559, MBON-f2: JRC-SS04320.
Figure 7 with 1 supplement
The phenotypes of neurons made by thoracic neuroblast (NB) 4–2 during its embryonic and postembryonic phases of neurogenesis.
Neurons are generated pairwise during both phases to produce the 13A and 13B hemilineages. (A) Four examples of embryonic born, 13B interneurons that function in the larva. All are commissural interneurons having ipsilateral dendrites and contralateral output sites. Boxed image shows the neurons overlapping in segment T1. (B) Example of a postembryonic clone of NB4-2 showing the neurons of the two hemilineages at the end of larval life. (C) Confocal projection of the adult form of larval-born, 13B interneurons expressed in the SS04739 driver line. (D, E) The larval and adult phenotypes of one of the embryonic born 13B interneurons, T13t. (D) shows the three pairs of T13t neurons as revealed by the SS02006 driver line. (E) shows Flip-switch clones of the same cells showing their adult phenotypes.
Figure 7—figure supplement 1
Confocal images of dorsal (top) and transverse (bottom) views of the early metamorphosis of the T3 pair of T13t cells: the dendritic arbor is gone by 8 hr after pupariation (P+8h), contralateral growth cones are evident by P+24h, and the arbor is near its maximal extent by P+48h.
Through this period, the expanding neuropil pulls the cell bodies (*) to their adult position near the midline.
Figure 8
The postembryonic-born mushroom body input neurons (MBINs) and mushroom body output neurons (MBONs) of the adult mushroom bodies.
(A) Summary of the origins of the adult MBINs that arise during the postembryonic period. Numbers give the number of neurons in each of the PAM groups (from Aso et al., 2014). (B) Summary of the origins of the adult MBONs that arise during the postembryonic period. NB: neuroblast; ?: adult MBONs whose origins are unknown. (C) Results of twin-spot MARCM approach showing the sequential postembryonic birth of FLAa2 lineage neurons that innervate α' and α compartments. (C), (C’), and (C”) images are produced by successively later heatshocks in the larva; green cells are produced after the heatshock while the red cells (shown only in C) are the remainder of the FLAa2 lineage. (D) Twin spot MARCM results from the type II DL1 lineage. The leftmost panel shows the progeny of an intermediate neural progenitor (INP) in green and the remainder of the lineage in red. The remaining panels show GMC clones with an MBON neuron and its paired sister fan-shaped body (FSB) neuron, both in green. The arbors identifying the individual MBONs are marked. Background staining (blue) is for Bruchpilot (nc82).
Figure 9
Stability and changes in mushroom body (MB) compartments during metamorphosis.
(A) Developmental fates of the larval compartments through metamorphosis. Larval compartments that are lost at metamorphosis are uncolored. Those incorporated into the mature structure share the same color in the two stages. (B) Summary of transmitter output from the MB compartments in the larval and adult stages. The larval LP (=adult ped) and LVL (=adult γ 2) compartments switch transmitters through metamorphosis. Compartment designations as in Figure 2. (C) Summary of the roles of mushroom body output neuron (MBON) compartment shifting, MBON trans-differentiation, and MBON recruitment in producing the output configuration of the mature MB. Persisting neurons in the lobe compartments are DAN-c1/PPL1 01, DAN-d1/PPL1 03, and MBON-d1/MBON 11; the new adult-specific neuron is MBON 12; and MBON-c1/MBE-CA shifts to the calyx.
Figure 10
Fate of circuit connections in the mushroom body (MB) through metamorphosis.
(A) Matrix showing the overlap of mushroom body input neuron (MBIN) axon terminals with mushroom body output neuron (MBON) dendrites in the same compartment. MBIN-MBON overlap only in the larval stage (blue), only in the adult stage (magenta), or in both stages (yellow). Rows and columns that are grayed out for cells whose identity is unknown for either the larval or adult stage. Larval/adult names are provided for each cell, with the red names being the terminal identity of neurons that do not innervate the adult MB. (B) Matrix showing the cells that make MBON-MBON connections only in their larval configuration (blue), only in their adult configuration (magenta), or in both configurations (yellow).
Figure 11
The lineage relationships of the major neuron types of the larval and adult mushroom bodies (MBs).
The four Kenyon cell neuroblasts (MBp) divide continuously from mid-embryogenesis until just before adult emergence. Three classes of Kenyon cells (different colors) are made in succession. The remaining neuroblasts have discrete embryonic and postembryonic phases of neurogenesis; neurons made during the first phase make the larval CNS. At metamorphosis, they are combined with neurons from the postembryonic phase to make the adult CNS. Origins and fates of MB neurons: (red) neurons that function in both the larval and adult MB; (blue) neurons that function in the larval MB but switch to non-MB circuits in the adult; (purple) postembryonic-born cells that function only in the adult MB: (white) neurons that function outside of the MBs. The name of the larval neuroblast and its embryonic lineage is paired with the most likely postembryonic lineage. Most postembryonic lineages are type I, in which progeny arise pairwise fashion from division of successive ganglion mother cells (shown as paired lines of cells). DL1 and DL2 show a type II pattern of division that increases the number of neurons produced because each neuroblast division produces an intermediate precursor cell that then divides to produce a small number of ganglion mother cells (shown as more disorganized clusters). See text for details.
Figure 12
The origin of spatial-temporal information used to determine neuronal phenotypes.
(A) The early neuronal phenotypes within a lineage are determined by birth order of the ganglion mother cells (GMC). Birth order is encoded by a temporal program of transcription factor expression in the parent neuroblast (NB) as it divides. Transcription factor expression at the time of division is inherited by the GMC and its daughter neurons. Differences between the daughters is established by Notch (N) signaling with one sibling expressing the Notch-on (‘A’) fate and the other the Notch-off (‘B’) fate. This information, along with lineage identity factors, acts through a battery of terminal selector genes to establish neuronal phenotypes. (B) A hypothetical scheme to explain the metamorphic pattern of mushroom body input neuron (MBIN) recruitment and loss in the PPL1 cluster of dopamine neurons. It proposes that the Castor (cas) expressing neurons that are born in the DL1 lineage just before and after the embryonic neurogenic arrest are fated to become MBINs. The earlier born Pdm expressing neurons are also dopaminergic but their adult function is outside of the MB. Larval specification factors, though, modify how they interact with the terminal selector genes thereby transforming them into MBINs while the larval stage is maintained. (C) A hypothetical scheme using neurons of the DAL-V2/3 lineage to illustrate how sibling fates might be temporarily altered to recruit larval MBONs. In this scheme, two successive GMCs divide to produce one daughter that is an MBON and one that innervates the central complex, a dichotomy established by Notch signaling. With the evolution of the larva, Notch signaling is suppressed in the daughters during embryogenesis, allowing both to assume a similar fate – that of a larval MBON. With the reestablishment of normal Notch signaling at metamorphosis, the transformed daughter loses her MBON features and becomes a central complex neuron.
Figure 13
Summary of the relationship of the function of embryonic-born adult neurons to their temporary functions in the larval mushroom body (MB).
Neurons that function in the MB of both larva and adults typically have similar positions in both structures, although some of the adult medial lobe mushroom body output neurons (MBONs) shift to the larval vertical lobes. Other neurons innervating the larval vertical lobes and the larval-specific intermediate peduncle compartment are fated for adult functions outside of the MB. CC: central complex; LAL: lateral accessory lobe; LH: lateral horn; OL: optic lobes; RIP: dead neurons; SMP: superior medial protocerebrum.
Tables
Table 1
Metamorphic fates of larval mushroom body extrinsic neurons.
| Larval name | Compartment | Lineage * | Adult identity | Ref for adult identity |
|---|---|---|---|---|
| MBINs | ||||
| OAN-a1,a2 | CX | VUM† | OA-VUM2a | Busch et al., 2009 |
| MBIN-b1,b2 | IP | DPLd | PAL-OL | Mao and Davis, 2009; this study |
| DAN-c1 | LP | CPd2/3 | PPL1 01 (γ1pedc) | Aso et al., 2014; Li et al., 2020 |
| MBIN-c1 | LP | CPd2/3 | PPL1 01 (γ1pedc) | Aso et al., 2014; Li et al., 2020 |
| DAN-d1 | LA | CPd2/3 | PPL1 03 (γ2α'1) | Aso et al., 2014; Li et al., 2020 |
| MBIN-l1 | LA | BLV a3/4 | LAL>bi-CRP | This study |
| OAN-e1 | UVL | CPd2/3 | PPL1-SMP | Mao and Davis, 2009; this study |
| MBIN-e2 | UVL | CPd2/3 | Unknown | |
| DAN-f1 | IVL | CPd2/3 | PPL1-bi-SMP | Mao and Davis, 2009; this study |
| DAN-g1 | LVL | CPd2/3 | PPL1 02 (γ1) | Aso et al., 2014; Li et al., 2020 |
| OAN-g1 | LVL | Unknown | OA-VPM3 | Busch et al., 2009 |
| DAN-h1 | SHA | DAL CM-1/2 | Dead | This study |
| DAN-i1 | UT | DAL CM-1/2 | Dead | This study |
| DAN-j1 | IT | DAL CM-1/2 | Dead | This study |
| DAN-k1 | LT | DAL CM-1/2 | Dead | This study |
| MBONs: | ||||
| MBON-a1 | CX | CPv2/3 | MBON 29 (γ4γ5) | This study |
| MBON-a2 | CX | CPv2/3 | MBON 22 (calyx) | Aso et al., 2014; Li et al., 2020 |
| MBON-b1,-b2 | IP | BLVa3/4 | LH-LN | Dolan et al., 2019 |
| MBON-b3 | IP | CPv2/3 | Unknown | |
| MBON-c1 | LP | BLDc | MBE-CA | This study |
| MBON-d1 | LA | DAL CM-1/2 | MBON 11 (γ1pedc>α/β) | Aso et al., 2014; Li et al., 2020 |
| MBON-d2 | LA | BAmd2 | SMP>IB | This study |
| MBON-e1 | UVL | CPd2/3 | Unknown | |
| MBON-e2 | UVL, IVL, LVL | DAM-d1 | MBON 03 (β'2mp) | Aso et al., 2014; Li et al., 2020 |
| MBON-f2 | IVL | DAL cl2 | MBON 30 (γ1,γ2,γ3) | Li et al., 2020 |
| MBON-f1 | IVL | CPd | Unknown | |
| MBON-g1,g2 | LVL | DAL-V2/3 | LAL.s-NO2i.b | Wolff and Rubin, 2018 |
| MBON-h1 | SHA | DAL-V2/3 | MBON 09 (γ3β'1) | Aso et al., 2014; Li et al., 2020 |
| MBON-h2 | SHA | DAL-V2/3 | MBON 08 (g3) | Aso et al., 2014; Li et al., 2020 |
| MBON-i1 | UT | DAM-d1 | MBON 04 (β'2-bilat) | Aso et al., 2014; Li et al., 2020 |
| MBON-j1 | IT | DAM-d1 | MBON 02 (β2β'2a) | Aso et al., 2014; Li et al., 2020 |
| MBON-j2 | IT | DAL CM-1/2 | MBON 05 (γ4>γ1,γ2) | Aso et al., 2014; Li et al., 2020 |
| MBON-k1 | LT | DAM-d1 | MBON 01 (γ5β'2a) | Aso et al., 2014; Li et al., 2020 |
| APL | UT,LT,LA,VL,CX | BLV a3/4 | APL | Aso et al., 2014; Li et al., 2020 |
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CX, calyx; IP: intermediate peduncle; LP: lower peduncle; UVL: upper vertical lobe; IB: inferior bridge; IVL: intermediate vertical lobe; LVL: lower vertical love; LA: lateral appendix; OT: optic tubercle; SHA: shaft; SMP: superior medial protocerebrum; UT: upper toe; IT: intermediate toe; LT: lower toe.
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*
Lineage designations from Saumweber et al., 2018.
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†
Lineage assumed to be from the ventral unpaired neuroblast because of position and nature of neurons.
Table 2
Developmental origins of adult mushroom body input neurons (MBINs) and mushroom body output neurons (MBONs) that do not come from remodeled larval, extrinsic mushroom body neurons.
| Neuron | # | Origin | Lineage | Reference |
|---|---|---|---|---|
| MBINs | ||||
| PPL1_04 (α'3) | 1 | Postembryonic | DL2 | Ren et al., 2016 |
| PPL1-05 (α'2α2) | 1 | Postembryonic | DL2 | Ren et al., 2016 |
| PPL1-06 (α3) | 1 | Postembryonic | DL2 | Ren et al., 2016 |
| PAM 01 (γ5) | 19 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 02 (β'2a) | 8 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 03 (β2β'2a) | 4 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 04 (β2) | 16 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 05 (β'2p) | 10 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 06 (β'2m) | 15 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 07 (γ4<γ1γ2) | 5 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 08 (γ4) | 26 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 09 (β1ped) | 6 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 10 (β1) | 6 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 11 (α1) | 7 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 12 (γ3) | 11 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 13 (β'1ap) | 7 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 14 (β'1) | 8 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM 15 (γ5β'2a) | 3 | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| PAM γ4/5 | ? | Postembryonic | CREa1A, CREa2A | Lee et al., 2020 |
| MBONs | ||||
| MBON 06 (β1>α] | 1 | Unknown | ||
| MBON 07 (α1) | 2 | Unknown | ||
| MBON 10 (β'1) | 8 | Unknown | ||
| MBON 12 (γ2α'1) | 2 | Postembryonic | FLAa2 | This study |
| MBON 13 (α'2) | 2 | Postembryonic | FLAa2 | This study |
| MBON 14 (α3) | 2 | Postembryonic | FLAa2 | This study |
| MBON 15 (α'1) | 2 | Postembryonic | DL1 | This study |
| MBON 16 (α'3ap) | 1 | Postembryonic | DL1 | This study |
| MBON 17 (α'3m) | 2 | Postembryonic | DL1 | This study |
| MBON 18 (α2sc) | 1 | postembryonic | DL1 | This study |
| MBON 19 (α2p3p) | 2 | Postembryonic | DL1 | This study |
| MBON 21 (γ4,γ5) | 1 | Unknown | ||
| MBON 23 (α2sp) | 1 | Postembryonic | DL1 | This study |
| MB-DPM | 1 | Postembryonic | Unknown | Mayseless et al., 2018 |
-
Adult names according to Aso et al., 2014 and Li et al., 2020, except PAM γ4/5, which is based on Lee et al., 2020.
Table 3
Comparison of transmitter expression in larval and adult forms of mushroom body output neurons (MBONs) and mushroom body input neurons (MBINs).
-
TH, tyrosine hydroxylase.
Table 4
Split GAL4 lines used to determine fates of larval mushroom body output neurons (MBONs) and mushroom body input neuron (MBINs).
| Cell name | Split line | Split line | Split line |
|---|---|---|---|
| MBIN-b1,b2 | SS21716 [FS, TL) | ||
| DAN-c1 | SS03066 (FS, TL) | MB586B (FS) | SS01702 (FS) |
| DAN-d1 | MB328B (FS, TL) | ||
| MBIN-l1 | SS04484 (FS, TL) | SS01624 (FS) | |
| OAN-e1 | SS36923 (FS) | SS01958 (FS) | |
| DAN-f1 (+DAN-c1) | MB065b (FS, TL) | MB145 (TL) | |
| DAN-g1 | SS01716 (FS, TL) | SS01755 (FS) | |
| OAN-g1 (sVPMmx) | SS25844 (FS) | SS04268 (FS) | |
| DAN-h1 | SS01949 (NC,TL) | SS01696 (NC) | MB440B (NC) |
| DAN-i1 | SS01949 (NC,TL) | MB196C (NC) | |
| DAN-j1 | SS01949 (NC,TL) | MB316B (NC) | MB340C (NC) |
| DAN-k1 | SS01757 (NC,TL) | MB198B (NC) | SS00616 (NC) |
| MBON-a1 | SS00867 (FS,TL) | SS01417 (FS) | |
| MBON-a2 | SS02006 (FS) | ||
| MBON-b1,b2 | SS01708 (FS, TL) | SS04112 (FS) | SS01959 (FS) |
| MBON-c1 | SS21789 (FS, TL) | ||
| MBON-d1 | SS01705 (FS,TL) | ||
| MBON-d2 | SS04231 (FS) | ||
| MBON-e2 | SS04559 (FS, TL) | ||
| MBON-f2 | SS04328 (FS, TL) | SS36248 (FS) | |
| MBON-g1,g2 | SS02130 (FS, TL) | SS02121 (FS) | |
| MBON-h1,h2 | SS01725 (FS, TL) | ||
| MBON-i1 | SS01771 | ||
| MBON-j1 | SS01973 (FS,TL) | SS01972 (FS) | |
| MBON-j2 | SS00860 (FS,TL) | ||
| MBON-k1 | SS01962 (FS) | SS01980 (TL) | |
| APL | SS01671 (FS, TL) |
-
Bold lines are the best lines for each cell.
-
FS, flip-switch immortalization; NC, no adult counterpart; TL, developmental timeline.
Table 5
Split GAL4 lines used in study.
| Split line | Target cell | AD | DBD |
|---|---|---|---|
| MB065b | DAN-f1 (+DAN-c1) | TH-p65ADZp in attP40 | R72B05-ZpGdbd in attP2 |
| MB145 | DAN-f1 (+DAN-c1) | R15B01-p65ADZp in attP40 | R72B05-ZpGdbd in attP2 |
| MB 196C | DAN-i1 | R58E02-p65ADZp in attP40 | R36B06-ZpGdbd in attP2 |
| MB198B | DAN-k1 | R58E02-p65ADZp in attP40 | R71D01-ZpGdbd in attP2 |
| MB316B | DAN-j1 | R58E02-p65ADZp in attP40 | R93G08-ZpGdbd in attP2 |
| MB328B | DAN-d1 | R82C10-p65ADZp in attP40 | R32F01-ZpGdbd in attP2 |
| MB340C | DAN-j1 | R93D10-p65ADZp in attP40 | R12G04-ZpGdbd in attP2 |
| MB440B | DAN-h1 | R30G08-p65ADZp in attP40 | R17D06-ZpGdbd in attP2 |
| MB586B | DAN-c1 | TH-p65ADZp in attP40 | R72G06-ZpGdbd in attP2 |
| SS00616 | DAN-k1 | 71D01-p65ADZp in VK00027 | 17D06-ZpGdbd in attP2 |
| SS00860 | MBON-j2 | w; R89G07-p65ADZ; MKRS/TM6B | R24E12-ZpGdbd in attP2 |
| SS00867 | MBON-a1 | w; R93G12-p65ADZ; MKRS/TM6B | R52E12-ZpGdbd in attP2 |
| SS01417 | MBON-a1 | w; R52E12-p65ADZp | R93G12-ZpGdbd in attP2 |
| SS01624 | MBIN-l1 | w; R84D07-p65ADZ | R37G09-ZpGdbd in attP2 |
| SS01671 | APL | R21D02-p65ADZp | R55D08-ZpGdbd in attP2 |
| SS01696 | DAN-h1 | 76F05-p65ADZp in attP40 | 95H02-ZpGdbd in attP2 |
| SS01702 | DAN-c1 | VT054895-p65ADZ in attP40 | R53C05-ZpGdbd in attP2 |
| SS01705 | MBON-d1 | R11E07-p65ADZp in attP40 | R52H01-ZpGdbd in attP2 |
| SS01708 | MBON-b1,b2 | R12G03-p65ADZp in attP40 | 21D02-ZpGdbd in attP2 |
| SS01716 | DAN-g1 | R14E06-p65ADZp in attP40 | R27G01-ZpGdbd in attP2 |
| SS01725 | MBON-h1,h2 | R20A02-p65ADZp in attP40; MKRS/TM6B | R28A10-ZpGdbd in attP2 |
| SS01755 | DAN-g1 | R46F09-p65ADZp | R14E06-ZpGdbd in attP2 |
| SS01757 | DAN-k1 | w; R48F09-p65ADZp; MKRS/ TM6B | R27A11-ZpGdbd in attP2 |
| SS01771 | MBON-i1 | w; 65A05-p65ADZ; MKRS/TM6B | 14C08-ZpGdbd in attP2 |
| SS01949 | DAN-h1, -i1, -j1 | VT026700-p65ADZp in attP40 | VT058464-ZpGDBD in attP2 |
| SS01958 | OAN-e1 | VT023826-p65ADZp in attP40 | R75F01-ZpGdbd in attP2 |
| SS01959 | MBON-b1,b2 | VT027952-p65ADZp in attP40 | R26A02-ZpGdbd in attP2 |
| SS01962 | MBON-k1 | VT033301-p65ADZp in attP40 | R27G01-ZpGdbd in attP2 |
| SS01972 | MBON-j1 | VT057469-p65ADZp in attP40 | 12C11-ZpGdbd/ TM3 in attP2 |
| SS01973 | MBON-j1 | VT057469-p65ADZp in attP40 | R18D09-ZpGdbd in attP2 |
| SS01980 | MBON-k1 | VT020613-p65ADZp in attP40 | VT033301-ZpGdbd in attP2 |
| SS02006 | MBON-a2 | w; 93G12-p65ADZ; MKRS/TM6B | 71E06-ZpGdbd in attP2 |
| SS02121 | MBON-g1,g2 | R21D06-p65ADZp in attP40 | R23B09-ZpGdbd in attP2 |
| SS02130 | MBON-g1,g2 | w; R23B09-p65ADZp; MKRS/ TM6B | R21D06-ZpGdbd in attP2 |
| SS03066 | DAN-c1 | VT054895-p65ADZ in attP40 | VT057278-ZpGdbd in attP2 |
| SS04112 | MBON-b1,b2 | VT027952-p65ADZp in attP40 | HAV5; CyO/Sco; 21D02-ZpGDBD in attP2 |
| SS04231 | MBON-d2 | VT032899-p65ADZp in attP40 | HAV5; CyO/Sp; 87G02-ZpGDBD in attP2 |
| SS04268 | OAN-g1 (sVPMmx) | VT012639-p65ADZp in attP40 | VT016127-ZpGdbd in attP2 |
| SS04328 | MBON-f2 | VT033301-p65ADZp in attP40 | VT029593-ZpGdbd in attP2 |
| SS04484 | MBIN-l1 | R37G09-p65ADZp in attP40 | VT007174-ZpGdbd in attP2 |
| SS21716 | MBIN-b1,b2 | VT048835-p65ADZp in attP40 | VT026664-ZpGdbd in attP2 |
| SS21789 | MBON-c1 | VT050247-p65ADZp in attP40 | VT050247-ZpGDBD in attP2 |
| SS25844 | OAN-g1 (sVPMmx) | VT040569-p65ADZp in attP40 | VT061921-ZpGdbd in attP2 |
| SS36248 | MBON-f2 | VT016795-p65ADZ in attP40 | VT029593-ZpGdbd in attP2 |
| SS36923 | OAN-e1 | VT054895-p65ADZ in attP40 | HAV5; CyO/Sco; 75F01-ZpGDBD in attP2 |
| SS04559 | MBON-e2 | w; 65A05-p65ADZ; MKRS/TM6B | VT045663-ZpGDBD in attP2 |
Table 6
Reagents used in this study.
| Reagent | Source | Catalog # |
|---|---|---|
| Mouse anti-bruchpilot | Developmental Studies Hybridoma Bank | Nc82-s |
| Rat anti-N cadherin | Developmental Studies Hybridoma Bank | DN-Ex #8 |
| Mouse anti-neuroglian | Developmental Studies Hybridoma Bank | BP 104 |
| Mouse anti-Fasciclin II | Developmental Studies Hybridoma Bank | 1D4 |
| Rabbit anti-DsRed | ClonTech | #632496 |
| Normal donkey serum | Jackson ImmunoResearch | #017-000-121 |
| AF488 donkey α-rabbit | Jackson ImmunoResearch | #711-545-152 |
| AF488 donkey α-mouse | Jackson ImmunoResearch | #711-585-151 |
| AF594 donkey α-rabbit | Jackson ImmunoResearch | #711-585-152 |
| AF594 donkey α-mouse | Jackson ImmunoResearch | #711-585-151 |
| AF649 donkey α-rat | Jackson ImmunoResearch | #711-605-153 |
| Mifepristone (RU-486) | Sigma-Aldrich | #M8046-100mg |
| S2 – Schneider’s Insect Medium | Sigma-Aldrich | #S01416 |
| DPX mountant | Electron Microscopy Sciences | #13512 |
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Metamorphosis of memory circuits in Drosophila reveals a strategy for evolving a larval brain
eLife 12:e80594.
https://doi.org/10.7554/eLife.80594
