Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships

  1. Julia L Meng
  2. Yupu Wang
  3. Robert A Carrillo
  4. Ellie S Heckscher  Is a corresponding author
  1. Department of Molecular Genetics and Cell Biology, University of Chicago, United States
  2. Program in Cell and Molecular Biology, University of Chicago, United States
  3. Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, United States
  4. Grossman Institute for Neuroscience, University of Chicago, United States
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Prolonged expression of Hunchback alters RP motor neuron synapses.

(A) Illustration of the late stage larvae (L3), body is organized into repeated left-right, mirror image hemisegments. Muscles (in gray) have a stereotyped pattern. (B) Illustration of muscles in a …

Figure 1—source data 1

Test for difference in the number of synaptic branches on ventral muscles in NB3-1>Hb compared to Control.

https://cdn.elifesciences.org/articles/56898/elife-56898-fig1-data1-v2.docx
Figure 1—figure supplement 1
NB3-1 generates RP motor neurons, with known features.

(A) Illustration of NB3-1 lineage progression. Each gray arrowhead represents cell division. Large circles are neuroblasts and smaller circles are neurons. Abbreviations: MN is motor neuron, IN is …

Figure 1—figure supplement 2
In embryos, prolonged expression of Hunchback generates more RP motor neurons with early born molecular identity.

(A–B) Illustrations of NB3-1 lineage progression. Each gray arrowhead represents cell division. Each gray arrowhead represents cell division. Large circles are neuroblasts, and smaller circles are …

Figure 1—figure supplement 2—source data 1

Test for difference in the number of embryonic RP MN molecular identities in NB3-1>Hbcompared to Control.

https://cdn.elifesciences.org/articles/56898/elife-56898-fig1-figsupp2-data1-v2.docx
Increased synaptic branches contain pre and postsynaptic markers necessary for function.

(A) Illustration of subcellular localization of neuromuscular synapse markers. Brunchpilot labels active zones, Discs large is a scaffolding protein strongly localized at post-synapse, and GluRIIA …

Altered synapses onto Ventral muscles are functional.

(A) Illustration of neuromuscular junction (NMJ) electrophysiology approach on third instar larvae (L3). Abbreviation NT is neurotransmitter. (B) Illustration highlighting (darkened) Muscle 30 whose …

Figure 3—source data 1

Test for the difference in EPSP, mEPSP, and Quantal Content for muscle 14 and 6 in NB3-1>Hb compared to Control.

https://cdn.elifesciences.org/articles/56898/elife-56898-fig3-data1-v2.docx
Figure 4 with 1 supplement
Precocious expression of Castor alters U motor neuron synapses.

(A–B) Illustrations of NB7-1 lineage progression. Each gray arrowhead represents cell division. Each gray arrowhead represents cell division. Large circles are neuroblasts, and smaller circles are …

Figure 4—source data 1

Test for difference in the number of synaptic branches on dorsal muscles in NB7-1>Cas compared to Control.

https://cdn.elifesciences.org/articles/56898/elife-56898-fig4-data1-v2.docx
Figure 4—figure supplement 1
In embryos, precocious expression of Castor generates fewer U motor neurons at the expense of later born neurons.

(A–B) Illustrations of NB7-1 lineage progression. Each gray arrowhead represents cell division. Each gray arrowhead represents cell division. Large circles are neuroblasts, and smaller circles are …

Figure 5 with 1 supplement
Precocious expression of Pdm alters U motor neuron synapses.

(A–C) Illustration of NB7-1 lineage progression. Each gray arrowhead represents cell division. Each gray arrowhead represents cell division. Large circles are neuroblasts, and smaller circles are …

Figure 5—source data 1

Test for difference in the number of synaptic branches on dorsal muscles in NB7-1>Pdm compared to Control.

https://cdn.elifesciences.org/articles/56898/elife-56898-fig5-data1-v2.docx
Figure 5—figure supplement 1
In embryos, precocious expression of Pdm generates either more or fewer U motor neurons depending on A/P positioning.

(A) Illustration of NB7-1 lineage progression. Each gray arrowhead represents cell division. Each gray arrowhead represents cell division. Large circles are neuroblasts, and smaller circles are …

Figure 6 with 1 supplement
Altered synapses onto Dorsal muscles are functional.

(A) Illustration of subcellular localization of neuromuscular synapse markers. Brunchpilot labels active zones, Discs large is a scaffolding protein strongly localized at post-synapse, GluRIIA is a …

Figure 6—figure supplement 1
Calcium imaging protocol, analysis, and examples.

(A–D) Illustration of calcium imaging protocol and analysis are shown. See Materials and methods for details. (E–H) Images of calcium signals on L3 Muscle 4. Control is NB7-1-GAL4/+; …

Summary of results in this study.

(A) Illustration of WT (wildtype) neuroblast lineage progression. Different patterns in the neuroblast represent temporal transcription factor expression. Outgrowth projecting from neuron represent …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)DIP-alpha-GAL4Robert Carrillo
(UChicago)
Genetic reagent (D. melanogaster)ac:VP16, gsb:v8v (aka NB7-1-GAL4)Minoree Kohwi (Columbia)
Genetic reagent (D. melanogaster)MHC-CD8-GCaMP6f-ShBloomington stock center [BL] 67739BDSC Cat# 67739, RRID:BDSC_67739
Genetic reagent (D. melanogaster)UAS-Hb; UAS-Hb/TM2Bloomington stock center [BL] 32198BDSC Cat# 32198, RRID:BDSC_32198
Genetic reagent (D. melanogaster)5172 J-GAL4 (aka NB3-1-GAL4)Haluk Lacin (Washington University)
Genetic reagent (D. melanogaster)w1118Bloomington stock center [BL] 36005BDSC Cat# 36005, RRID:BDSC_36005
Genetic reagent (D. melanogaster)UAS-CasChris Doe (University of Oregon)
Genetic reagent (D. melanogaster)UAS-HA-pdm2/CyO; UAS-HA-pdm2/TM3Chris Doe (University of Oregon)
Genetic reagent (D. melanogaster)UAS-myr-GFPBloomington stock center [BL] 32198BDSC Cat# 32198, RRID:BDSC_32198
Antibodyrabbit anti-Eve (polyclonal)Heckscher Lab1:1000
Antibodyrabbit anti-Castor (polyclonal)Chris Doe (Oregon)1:1000
Antibodyguinea pig anti-Hb (polyclonal)John Rientz (UChicago)1:1000
Antibodyguinea pig anti-Kruppel (polyclonal)John Rientz (UChicago)1:1000
AntibodyMouse anti-Eve (monoclonal)3C10DSHB Cat# 3C10
Anti-even skipped
RRID:AB_528229
1:50
Antibodychicken anti-GFP (polyclonal)Aves #GFP-1020Aves Labs Cat# GFP-1020, RRID:AB_100002401:1000
Antibodyrat anti-Worniu (polyclonal)Abcam #ab1963621:250
Antibodymouse anti-En
(monoclonal)
4D9DSHB Cat# 4D9 anti-engrailed/invected, RRID:AB_5282241:5
Antibodyguinea pig anti-Runt
(polyclonal)
John Rientz (UChicago)1:500
Antibodyguinea pig anti-HB9 (polyclonal)Heather Broiher (Case Western)1:1000
Antibodymouse anti-Cut (monoclonal)2B10DSHB Cat# 2B10
anti-Cut homeobox
RRID:AB_528186
1:50
Antibodyrat anti-Zfh2
(polyclonal)
Chris Doe (Oregon)1:800
Antibodyrabbit anti-Smad3 (pMad) (polyclonal)Abcam #52903Abcam Cat# ab52903, RRID:AB_8825961:300
Antibodymouse anti-Brp (monoclonal)NC82Creative Diagnostics Cat# DMAB9116MD, RRID:AB_23926641:50
Antibodymouse anti-DLG
(monoclonal)
4F3DSHB Cat# 4F3 anti-discs large, RRID:AB_5282031:500
Antibodymouse anti-GluRIIA
(monoclonal)
8B4D2DSHB Cat# 8B4D2 (MH2B), RRID:AB_528261:25
AntibodyCy3-HRP
(polyclonal)
Jackson ImmunoResearch 123-025-021Jackson ImmunoResearch Labs Cat# 123-025-021, RRID:AB_23389541:400
Antibody647-Phallion
(polyclonal)
Thermofisher A22287Thermo Fisher Scientific Cat# A22287, RRID:AB_26201551:600

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