1. Developmental Biology
  2. Neuroscience

Stereotyped terminal axon branching of leg motor neurons mediated by IgSF proteins DIP-α and Dpr10

  1. Lalanti Venkatasubramanian
  2. Zhenhao Guo
  3. Shuwa Xu
  4. Liming Tan
  5. Qi Xiao
  6. Sonal Nagarkar-Jaiswal
  7. Richard S Mann  Is a corresponding author
  1. Columbia University, United States
  2. Mortimer B. Zuckerman Mind Brain Behavior Institute, United States
  3. University of California, Los Angeles, United States
  4. Baylor College of Medicine, United States
https://doi.org/10.7554/eLife.42692
Share this article
Cite this article
  1. Lalanti Venkatasubramanian
  2. Zhenhao Guo
  3. Shuwa Xu
  4. Liming Tan
  5. Qi Xiao
  6. Sonal Nagarkar-Jaiswal
  7. Richard S Mann
(2019)
Stereotyped terminal axon branching of leg motor neurons mediated by IgSF proteins DIP-α and Dpr10
eLife 8:e42692.
https://doi.org/10.7554/eLife.42692
  2. Download BibTeX
  3. Download .RIS
7 figures, 5 videos, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Download asset Open asset
Sequential defasciculation and branching of developing Drosophila adult leg motor neurons.

(A) Development of Drosophila adult leg motor neurons across six distinct time points during pupariation – Late L3 (96 hr AEL/0 hr APF), Early Pupa (10 hr APF), Mid Pupa (20 hr and 25 hr APF), Late …

https://doi.org/10.7554/eLife.42692.002
Figure 1—figure supplement 1
Download asset Open asset
Sequential defasciculation and branching of developing Drosophila adult leg motor neurons.

(A, A’) Maximum projection confocal images of a T1 leg disc denoting the terminal position (white arrowhead) of LinA/15 leg MNs expressing myr::GFP (green; generated by lineage tracing) and all MNs …

https://doi.org/10.7554/eLife.42692.003
Figure 2 with 1 supplement
Download asset Open asset
Expression patterns of DIPs and dprs in Drosophila T1 adult leg neuro-musculature.

(A–B) dpr (A) and DIP (B) expression patterns in the Drosophila T1 adult leg for a subset of heterophilic binding partners identified by DIP-Dpr ‘interactome’ studies (Carrillo et al., 2015; Özkan …

https://doi.org/10.7554/eLife.42692.006
Figure 2—figure supplement 1
Download asset Open asset
Expression patterns of additional dprs in the Drosophila T1 adult leg.

(A) dpr expression patterns in the T1 adult leg. Bottom row – dpr6 and dpr10 expression was restricted to glutamatergic MNs in the T1 leg using a genetic intersectional approach (see Materials and …

https://doi.org/10.7554/eLife.42692.007
Figure 3 with 2 supplements
Download asset Open asset
Effects of mutating DIP-α on the terminal branching of α-leg MNs.

(A) Left Column: Proximal-Distal (P–D) oriented Fe and Ti T1 adult leg segments depicting axon muscle-targeting of three DIP-α expressing leg MNs labeled by DIP-α-A8-Gal4(86Fa)>20XUAS-6XGFP (green) …

https://doi.org/10.7554/eLife.42692.008
Figure 3—figure supplement 1
Download asset Open asset
Characterization of DIP-α and other DIP mutants.

(A) Genomic locus of DIP-α showing introns (black line), non-coding exons (grey rectangles) and coding exons (orange rectangles). The location of the DIP-α-A8 enhancer is denoted by a green bar (see …

https://doi.org/10.7554/eLife.42692.009
Figure 3—figure supplement 2
Download asset Open asset
Co-expression and phenotypic analysis of DIP-α and DIP-β in adult leg MNs.

(A) Expression of DIP-β in the α-ltm-leg MNs is confirmed by co-expression of DIP-β-T2A-QF > 10XQUAS-6XmCherry (red) along with DIP-α-A8-Gal4 > 20XUAS-6XGFP (green). (A) In the Fe (left), DIP-β is …

https://doi.org/10.7554/eLife.42692.010
Figure 4 with 2 supplements
Download asset Open asset
Spatial and temporal characterization of DIP-α expression.

(A) T1 LinA/15 leg MN MARCM clones using OK371-Gal4 > 20XUAS-6XmCherry (magenta) and DIP-α-T2A-QF > 10XQUAS-6XGFP (green) to label leg MN cell bodies in the VNC at multiple developmental time …

https://doi.org/10.7554/eLife.42692.011
Figure 4—figure supplement 1
Download asset Open asset
Spatial and temporal characterisation of DIP-α expression.

(A) T1 LinA/15 leg MN cis2MARCM clones (Enriquez et al., 2018) using OK371-Gal4 > 20XUAS-6XmCherry (magenta) and DIP-α-T2A-QF > 10XQUAS-6XGFP (green) to label leg MN axons in the T1 adult Fe and Ti …

https://doi.org/10.7554/eLife.42692.012
Figure 4—figure supplement 2
Download asset Open asset
Temporal rescue of DIP-α in a mutant background.

(A) Temporal rescue at 75 hr APF of axon terminal branching of αFe-ltm (left column) and αTi-ltm (right column) in the proximal Fe of T1 adult legs in samples mutant for DIP-α using DIP-α-T2A-Gal4 > …

https://doi.org/10.7554/eLife.42692.013
Figure 5 with 1 supplement
Download asset Open asset
dpr10 Expression in muscles is necessary for terminal branching of the α-leg MNs.

(A) Terminal branching of the T1 α-leg MNs labeled by DIP-α-T2A-QF > 10XQUAS-6XGFP in dpr6 and dpr10 double and single mutants. Left – T1 legs; Right – Fe and Ti leg segments (axons, green; cuticle, …

https://doi.org/10.7554/eLife.42692.014
Figure 5—figure supplement 1
Download asset Open asset
dpr10 expression in muscles is necessary for terminal branching of the α-Leg MNs.

(A) Terminal branching of the T1 α-leg MNs in Fe and Ti segments labeled by DIP-α-T2A-QF > 10XQUAS-6XGFP (axons, green; cuticle, grey). Top – Muscle-specific RNAi knockdown of dpr10 using Mef2-Gal4 >…

https://doi.org/10.7554/eLife.42692.015
Figure 6 with 1 supplement
Download asset Open asset
DIP-α is required for terminal axon lengthening and branching 30 to 45 hr APF.

(A) Terminal axon branching of control (left) and DIP-α mutant (right) αTi-ltm and αTi-tadm leg MNs at 30 hr (top), 40 hr (middle) and 50 hr (bottom) APF using DIP-α-T2A-Gal4 > UAS-DIP-α and DIP-α-T2…

https://doi.org/10.7554/eLife.42692.016
Figure 6—figure supplement 1
Download asset Open asset
DIP-α is Required for terminal axon lengthening and branching 30 to 45 hr APF.

(A) Magnified inset from Figure 6A showing decreased αTi-ltm terminal axon branching in a DIP-α mutant (right) compared to the control (left) at 30 hr APF. Axons are labeled using DIP-α-T2A-Gal4 > 20…

https://doi.org/10.7554/eLife.42692.017
Figure 7 with 1 supplement
Download asset Open asset
Dpr10 Expression is gradually restricted to distal fibers of the Ti-ltm 30 to 45 hr APF.

(A–B) Dpr10 protein expression (grey) in the developing Ti-ltm and proximal tadm, labeled by Mef2 (magenta) along with αTi-ltm and αTi-tadm axons labeled by DIP-α-T2AQF > 10XQUAS-6XGFP (green) at 25 …

https://doi.org/10.7554/eLife.42692.021
Figure 7—figure supplement 1
Download asset Open asset
Dpr10 expression in the developing Ti-ltm in DIP-α mutant and overexpression contexts.

(A, A’) DIP-α mutant αTi-ltm axon labeled by DIP-α-T2A-Gal4 > 20XUAS-6XGFP (green) retains its ability to target regions of high Dpr10 expression (grey) in the developing Ti-ltm (labeled by Mef2, …

https://doi.org/10.7554/eLife.42692.022

Videos

Video 1
Download asset
Live imaging of developing LinA/15 leg MNs between ~25 - 37 hr APF.

WT Time lapse in vivo live imaging of developing T1 LinA/15 leg MNs expressing myr::GFP (yellow) between ~25 hr APF (00:00) to ~37 hr APF (12:20) (10 min interval, five fps). In the first frame the …

https://doi.org/10.7554/eLife.42692.004
Video 2
Download asset
Live imaging of developing LinA/15 Ti-ltm targeting leg MNs between ~40 - 50 hr APF.

WT Time lapse in vivo live imaging of developing T1 LinA/15 Ti-ltm targeting leg MNs expressing myr::GFP (yellow) between ~40 hr APF (00:00) to ~50 hr APF (12:20) (10 min interval, five fps). In the …

https://doi.org/10.7554/eLife.42692.005
Video 3
Download asset
Comparison of WT and DIP-α mutant Ti-ltm targeting leg MNs using live-imaging.

Time lapse in vivo live imaging of Ti-ltm targeting leg MNs, including αTi-ltm, in a DIP-α mutant animal between ~30 hr APF (00:00) to ~38 hr APF (08:30) (10 min interval, five fps). Leg MNs are …

https://doi.org/10.7554/eLife.42692.018
Video 4
Download asset
Live imaging of WT αTi-ltm leg MN between ~35 hr APF to ~45 hr APF.

Time lapse in vivo live imaging of αTi-ltm leg MNs in control (Video 4) and DIP-α mutant animal (Video 5), using DIP-α-T2A-Gal4 > UAS-DIP-α and DIP-α-T2A-Gal4/DIP-α– respectively, between ~35 hr APF …

https://doi.org/10.7554/eLife.42692.019
Video 5
Download asset
Live imaging of DIP-α mutant αTi-ltm leg MN between ~35 hr APF to ~45 hr APF.
https://doi.org/10.7554/eLife.42692.020

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(D. melanogaster)		OK371-Gal4BDSC #26160RRID:BDSC_26160
Genetic reagent
(D. melanogaster)		Vglut-T2A-QF2BDSC #60315RRID:BDSC_60315
Genetic reagent
(D. melanogaster)		10 C12-Gal4BDSC #47841RRID:BDSC_47841
Genetic reagent
(D. melanogaster)		dpn > KDRT > Cre;
Act > LoxP > LexA,
LexA-myr::GFP; UAS-KD		PMID:24561995
Genetic reagent
(D. melanogaster)		Mef2-QF2BDSC #66469RRID:BDSC_66469
Genetic reagent
(D. melanogaster)		13XLexAop2-6XGFPBDSC #52265RRID:BDSC_52265
Genetic reagent
(D. melanogaster)		10XQUAS-6XGFPBDSC #52264;
this paper		RRID:BDSC_52264VK0027 insertion
generated for this paper.
Genetic reagent
(D. melanogaster)		10XQUAS-6XmCherryBDSC #52269;
BDSC #52270		RRID:BDSC_52269;
RRID:BDSC_52270
Genetic reagent
(D. melanogaster)		20XUAS-6X-GFPBDSC #52261;
BDSC #52262; This paper		RRID:BDSC_52261;
RRID:BDSC_52262		86Fa insertion
generated for this paper.
Genetic reagent
(D. melanogaster)		20XUAS-6X-mCherryBDSC #52268RRID:BDSC_52268
Genetic reagent
(D. melanogaster)		MiMIC-T2A-Gal4 linesBDSC #7838;
BDSC #76200; This paper		RRID:BDSC_78385;
RRID:BDSC_76200		Additional lines are listed
in Supplementary file 1.
generated by S.Nagarkar
Jaiswal, H.Bellen and
M.Courgeon, C. Desplan.
Genetic reagent
(D. melanogaster)		DIP-α-A8-Gal4This paperattp2 and 86Fa insertions
generated for this paper;
See Materials and methods
Genetic reagent
(D. melanogaster)		DIP-α-T2A-QF2This paperMiMIC Trojan Swaps
generated for this paper;
See Materials and methods
Genetic reagent
(D. melanogaster)		DIP-β-T2A-QF2This paperMiMIC Trojan Swaps
generated for this paper;
See Materials and methods
Genetic reagent
(D. melanogaster)		DIP-α1-7 also referred to as DIP-αnull2PMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		DIP-β1-95This paperGenerated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		DIP-γ1-67PMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		UAS-DIP-α-V5PMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		UAS-DIP-βVK0027 insertion
generated for this paper.
Genetic reagent
(D. melanogaster)		dpr6,10 - also referred to as dpr6-10LPMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		dpr101-29 also referred to as dpr10nullPMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		dpr61-116 also referred to as dpr6nullPMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		UAS-dpr10-V5PMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		UAS-dpr6-V5PMID: 30467079Generated by the Zipursky Lab
Genetic reagent
(D. melanogaster)		UAS-dpr10 RNAi VDRCVDRC# 103511
Genetic reagent
(D. melanogaster)		tub > FRT .Gal80>BDSC #38879;
BDSC #38880		RRID:BDSC_38879;
RRID:BDSC_38880
Genetic reagent
(D. melanogaster)		QUAS-DSCP-Flp0.2G (attp2)BDSC #30008RRID:BDSC_30008
Genetic reagent
(D. melanogaster)		tub-Gal80tsBDSC #7108RRID:BDSC_7108
Genetic reagent
(D. melanogaster)		DIP-α-GFSTFBDSC #60523RRID:BDSC_60523
Genetic reagent
(D. melanogaster)		dpr10-GFSTFBDSC #59807RRID:BDSC_59807
Genetic reagent
(D. melanogaster)		R13C09-Gal4BDSC #48555RRID:BDSC_48555
Genetic reagent
(D. melanogaster)		hkb-Gal4BDSC #62578RRID:BDSC_62578
Genetic reagent
(D. melanogaster)		DIP-ζ RNAi – TriP.HMS01671BDSC #38227RRID:BDSC_38227
Genetic reagent
(D. melanogaster)		FRT42DBDSC #1802RRID:BDSC_1802
Genetic reagent
(D. melanogaster)		FRT42D tubG80, tubQSPMID: 29395908
Genetic reagent
(D. melanogaster)		y,w,hs-Flp1.22, hs-Flp122.2(Chr 2)OtherGift from Gary Struhl
Genetic reagent
(D. melanogaster)		Mef2-Gal4 (Chr 2)OtherBellen Lab, provided
by R.Carrillo
AntibodyRabbit polyclonal
Anti-Mef2		PMID: 7839146RRID:AB_2568604Generated by
B.Paterson; (1:500)
AntibodySheep polyclonal
Anti-GFP		Bio-RadCat# 4745–1051(1:500)
AntibodyChicken polyclonal
Anti-GFP		AbcamCat# ab101863;
RRID:AB_10710875		(1:1000)
AntibodyMouse monoclonal
Anti-DIP-α		PMID: 30467079Generated by the
Zipursky Lab; (1:20)
AntibodyMouse monoclonal
Anti-Dpr10		PMID: 30467079Generated by the
Zipursky Lab; (1:500)
Recombinant
DNA reagent		T2A-QF2-Hsp70PMID:25732830RRID:Addgene_62944;
RRID:Addgene_62945
Recombinant
DNA reagent		pJFRC28-10XUAS-IVS-GFP-p10PMID:22493255RRID:Addgene_36431

Additional files

Supplementary file 1

DIP and dpr MiMIC-T2A-Gal4 lines .

https://doi.org/10.7554/eLife.42692.023
Supplementary file 2

Genotypes used for each figure.

https://doi.org/10.7554/eLife.42692.024
Transparent reporting form
https://doi.org/10.7554/eLife.42692.025

Download links