1. Developmental Biology
  2. Cell Biology

Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal

  1. Ariel M Pani  Is a corresponding author
  2. Bob Goldstein
  1. University of North Carolina at Chapel Hill, United States
https://doi.org/10.7554/eLife.38325
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Cite this article
  1. Ariel M Pani
  2. Bob Goldstein
(2018)
Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal
eLife 7:e38325.
https://doi.org/10.7554/eLife.38325
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6 figures, 2 videos, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
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Tagged Wnt/EGL-20 is biologically functional and forms a long-range, anteroposterior gradient in vivo.

(a) transmitted light images of adult C. elegans with wild-type egl-20, the egl-20 loss-of-function mutant egl-20(n585), mNG-tagged egl-20, or YPET-tagged egl-20 showing normal external anatomy in mN…

https://doi.org/10.7554/eLife.38325.003
Figure 1—source data 1

Positions of Q neuroblast descendants in wild type, egl-20(n585), EGL-20::mNG, and EGL-20::YPET strains.

Source data corresponding to Figure 1b. Positions of Q neuroblast progeny after migration were quantified by using the non-motile URX neuron in the head and PLM neurons in the tail as fiducial markers. Relative positions of Q neuroblast progeny AQR, AVM, PVM, and PQR were calculated as a percentage of the distance between URX and PLM.

https://doi.org/10.7554/eLife.38325.007
Figure 1—source data 2

Fluorescence intensity values for EGL-20::YPET, EGL-20::mNG, and Pegl-20 > 2 x mKate2::PH.

Source data corresponding to Figure 1f,h. Fluorescence intensity values were obtained in FIJI (Schindelin et al., 2012) by drawing a line the width of the worm from head to tail and using the ‘plot profile’ function. Off-worm background in a nearby region was then subtracted from the raw pixel intensities.

https://doi.org/10.7554/eLife.38325.008
Figure 1—figure supplement 1
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Tissue-specific Wnt/EGL-20::mNG localization.

(a) Maximum intensity projection of surface planes in an L1 larva highlighting Wnt/EGL-20::mNG (green) localization to body wall muscles marked by Pmyo-3>mCherry::PH (magenta). (b) Maximum intensity …

https://doi.org/10.7554/eLife.38325.004
Figure 1—figure supplement 2
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Wnt/EGL-20 localization and plasma membrane architectures of source cells.

(a) Maximum intensity projection of surface optical sections in the posterior of an L2 larva showing tagged Wnt (green) and plasma membranes of source cells marked by Pegl-20>2x mKate2::PH …

https://doi.org/10.7554/eLife.38325.005
Figure 1—figure supplement 3
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Visualization of the native egl-20-expressing cell lineage in vivo.

(a) Design of an endogenously engineered, bicistronic flp::F2A::egl-20::mNG allele to drive FLP-based recombination in cells that natively express egl-20. (b) Design of a cell-lineage reporter based …

https://doi.org/10.7554/eLife.38325.006
Figure 2 with 1 supplement
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Endogenously tagged Wnt/EGL-20 localizes to responding QL neuroblasts that do not directly contact Wnt source cells.

(a–c) Maximum intensity projections showing Wnt/EGL-20::mNG protein (green), plasma membranes of EGL-20 source cells marked by Pegl-20>2x mKate2::PH (red), and responding Q neuroblast and seam cell …

https://doi.org/10.7554/eLife.38325.009
Figure 2—figure supplement 1
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Wnt/EGL-20::mNG localization in QR neuroblast descendants and seam cells.

(a–b) Maximum intensity projections of sub-surface optical sections on the right side of an L1 stage larva showing bright Wnt/EGL-20::mNG punctae (green) that localize to anteriorly migrating QR …

https://doi.org/10.7554/eLife.38325.010
Figure 3 with 3 supplements
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Endogenously tagged Wnt/EGL-20 and receptors co-localize on QL neuroblast protrusions.

(a, b) Endogenously tagged Wnt/EGL-20::mNG (green) and Frizzled/MIG-1::mKate2 (red) (a) or Frizzled/LIN-17::mScarlet-I (red) (b) punctae overlap and localize to protrusions from QL neuroblasts …

https://doi.org/10.7554/eLife.38325.011
Figure 3—figure supplement 1
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Endogenously tagged Dishevelled/MIG-5 localizes to QL neuroblast protrusions.

Maximum intensity projections of surface optical sections showing mNG::MIG-5 punctae (green) (a, c) localize to protrusions (white arrow) from QL neuroblasts that respond to Wnt signaling, marked by …

https://doi.org/10.7554/eLife.38325.012
Figure 3—figure supplement 2
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Extensive overlap between Wnt punctae and two Frizzled homologs in an early L1 animal.

Maximum intensity projection of surface optical sections showing the posterior right side of an early L1 animal including the QR neuroblast and seam cells V4-6. (a) Wnt/EGL-20::mNG; (b) …

https://doi.org/10.7554/eLife.38325.013
Figure 3—figure supplement 3
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Extensive overlap between Wnt punctae and two Frizzled homologs in a late L1 animal.

Maximum intensity projection of midline optical sections showing the posterior of a late L1 animal. (a) Wnt/EGL-20::mNG; (b) Frizzled/MIG-1::2x mKate2; (c) Frizzled/LIN-17::mTurq2; (d) merged image …

https://doi.org/10.7554/eLife.38325.014
Figure 4 with 1 supplement
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Distribution of endogenously tagged Wnt/EGL-20 secreted from different cell types suggests a common pool of extracellular ligand.

(a) design of an endogenous egl-20 tagged with: :FRTmKate2::STOPFRTmNG cassette to visibly distinguish Wnt/EGL-20 molecules produced by different cell types. By default, this allele expresses egl-20:…

https://doi.org/10.7554/eLife.38325.015
Figure 4—figure supplement 1
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Ectopically expressed Wnt/EGL-20 spreads extracellularly and localizes to native receiving cells.

(a) Design of a transgene to ectopically express Wnt/EGL-20::mNG (green) and a 2x mTurq2::PH plasma membrane marker (blue) in posterior tail epithelial cells using a promoter fragment from the Wnt …

https://doi.org/10.7554/eLife.38325.016
Figure 5 with 1 supplement
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FRAP shows rapid Wnt recovery consistent with free extracellular spreading in vivo.

(a, b) Images of Wnt/EGL-20::YPET fluorescence recovery at selected time points during the first 5:30 after photobleaching in a mid-body region. The bleached region of interest included a responding …

https://doi.org/10.7554/eLife.38325.017
Figure 5—figure supplement 1
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Tagged Wnt recovery after photobleaching over 90 min.

(a) Images of Wnt/EGL-20::YPET fluorescence recovery during the first 90 min after photobleaching in a mid-body region with images acquired every 3 min. Arrows indicate the anterior boundary of the …

https://doi.org/10.7554/eLife.38325.018
Figure 6 with 1 supplement
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Extracellular spreading shapes long-range Wnt dispersal.

(a) Normal Wnt/EGL-20::YPET fluorescence in the posterior of a late L1 larva; (b) Schematic diagram of the Morphotrap system and Wnt/EGL-20::YPET distribution in Pmyo-3 >Morphotrap and control …

https://doi.org/10.7554/eLife.38325.021
Figure 6—source data 1

Fluorescence intensity values for EGL-20::YPET in control and Morphotrap animals.

Source data corresponding to Figure 6f. Fluorescence intensity values were obtained in FIJI (Schindelin et al., 2012) by drawing a line the width of the worm from head to tail and using the ‘plot profile’ function. Off-worm background in a nearby region was then subtracted from the raw pixel intensities.

https://doi.org/10.7554/eLife.38325.023
Figure 6—source data 2

Non-muscle fluorescence intensity values for EGL-20::YPET in control and Morphotrap animals.

Source data corresponding to Figure 6g. Wnt/EGL-20::YPET levels outside of body wall muscle were calculated by measuring the mean pixel intensity of a region of interest anterior to the egl-20-expressing cells that did not include body wall muscles marked by a Pmyo-3-driven transgene. Control and Morphotrap worms were imaged with identical settings on the same slides, and off-worm background in a nearby region was subtracted from the raw pixel intensities.

https://doi.org/10.7554/eLife.38325.024
Figure 6—figure supplement 1
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Intracellular Wnt-Morphotrap aggregations caused by ubiquitous Morphotrap expression and abnormal morphologies in cells with multi-copy extrachromosomal arrays.

(a–c) Wnt/EGL-20::YPET (a), Peft-3 driven Morphotrap (b), and composite image (c) highlighting tagged Wnt-Morphotrap aggregrations (arrows) in Wnt-producing cells; (d–f) body wall muscle …

https://doi.org/10.7554/eLife.38325.022

Videos

Video 1
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FRAP experiment showing Wnt/EGL-20::YPET recovery over 5:30 in vivo.

Video of time lapse images showing Wnt/EGL-20::YPET fluorescence recovery every 30 s for the first 5:30 after photobleaching in a mid-body region including seam cells and a QL neuroblast. Top panel …

https://doi.org/10.7554/eLife.38325.019
Video 2
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FRAP experiment showing Wnt/EGL-20::YPET recovery over 81 min in vivo.

Video of time lapse images showing Wnt/EGL-20::YPET fluorescence recovery every 3 min for the first 81 min after photobleaching in a mid-body region. Wnt/EGL-20::YPET fluorescence was colored using …

https://doi.org/10.7554/eLife.38325.020

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(C. elegans)		cpIs89[Pwrt-2>2x mTurq2::PH::tbb-2
3'UTR loxN] I; cpIs85[Pegl-20 > 2 x
mKate2::PH::3xHA::tbb-2 3'UTR
loxN] II; egl-20(cp221[egl-20::m
NG^3xFlag]) IV		This paperLP515
Strain, strain
background
(C. elegans)		egl-20(cp221[egl-20::mNG^3xFlag])
IV; qyIs541[Pmyo-3>mCherry::P
H::tbb-2 3'UTR]		This paperLP673
Strain, strain
background
(C. elegans)		cpIs128[Pwrt2>2x mTurquoise2
::PH::3xHA::tbb-2 3'UTR loxN] I;
mig-1(cp360[mig-1::mKate2^
3xMyc]) I; egl-20(cp221[egl-20:
:mNG^3xFlag]) IV		This paperLP727
Strain, strain
background
(C. elegans)		cpIs89[Pwrt-2>2x mTurquoise2
::PH::tbb-2 3'UTR loxN] I; mig
-5(cp385[mNG-GLO^AID::mig-5]) II		Heppert et al., 2018LP728
Strain, strain
background
(C. elegans)		cpIs92 [Pmec-7>2x mTurq2::PH::
3xHA::tbb-2 3'UTR loxN] I;
cpIs129 [Pgcy-32 > 2 x mKate2::
PH::3xHA::tbb-2 3'UTR loxN] II;
egl-20(cp353[egl-20::mNG^3xFlag]) IV		This paperLP729
Strain, strain
background
(C. elegans)		cpIs92 [Pmec-7>2x mTurq2::PH:
:3xHA::tbb-2 3'UTR loxN] I;
cpIs129 [Pgcy-32 > 2 x mKate2::PH
::3xHA::tbb-2 3'UTR loxN] II		This paperLP730
Strain, strain
background
(C. elegans)		cpIs130[Pwrt-2>2x mKate2
::PH::3xHA::let-858 3'UTR::Ptag-168 >
HisCl1::tbb-2 3'UTR loxN] II; egl-20
(cp353[egl-20::mNG^3xFlag]) IV		This paperLP732
Strain, strain
background
(C. elegans)		cpIs130[Pwrt-2>2x mKate2::PH::
3xHA::let-858 3'UTR::Ptag-168 >
 HisCl1::tbb-2 3'UTR loxN] II;
egl-20(cp400[egl-20::YPET^3xFlag]) IV		This paperLP783
Strain, strain
background
(C. elegans)		cpIs156[Pwrt2>2x mTurquoise2::PH:
:3xHA::tbb-2 3'UTR SEC + loxN] I; lin-1
7(cp391[lin-17::mScarlet-C1^AID]) I;
egl-20(cp221[egl-20::mNG^3xFlag]) IV		This paperLP790
Strain, strain
background
(C. elegans)		mig-1(cp360[mig-1::mKate2^3xMyc])
I; lin-17(cp404[lin-17::mTurquoise2^AID])
I; egl-20(cp221[egl-20::mNG^3xFlag]) IV		This paperLP792
Strain, strain
background
(C. elegans)		cpIs92 [Pmec-7>2x mTurq2::
PH::3xHA::tbb-2 3'UTR loxN]
I; cpIs129 [Pgcy-32 > 2 x mKate
2::PH::3xHA::tbb-2 3'UTR loxN] II;
egl-20(n585) IV		This paper, CGCLP793egl-20(n585) crossed to LP730
Strain, strain
background
(C. elegans)		cpIs92 [Pmec-7>2x mTurq2::
PH::3xHA::tbb-2 3'UTR loxN]
I; cpIs129 [Pgcy-32 > 2 x mKate2
::PH::3xHA::tbb-2 3'UTR loxN] II;
egl-20(cp400[egl-20::
YPET^3xFlag]) IV		This paperLP795
Strain, strain
background
(C. elegans)		cpIs117[Peft-3::FRT > 2 x
mKate2::PH::let-858 3'UTR::FRT >
2 x mTurquoise2::PH::3x
HA::tbb-2 3'UTR + loxN] I;
egl-20(cp411[flp::F2A::egl-20]) IV;
egl-20(cp221[egl-20::m
NG^3xFlag]) IV;		This paperLP805
Strain, strain
background
(C. elegans)		cpIs158[Pmyo-3>pat-3sp::2x vhhGFP4::
CD8 tm::2x mTurquoise2::PH::tbb-2
3'UTR loxN] I; cpIs130[Pwrt-2>2x
mKate2::PH::3xHA::let-858 3'UTR
::Ptag-168 > HisCl1::tbb-2
3'UTR loxN] II; egl-20(cp400
[egl-20::YPET^3xFlag]) IV		This paperLP815
Strain, strain
background
(C. elegans)		cpIs159[egl-5(K enhancer)::pes-10
delta > flp::SL2::2x
mTurq2::PH::3xHA::tbb-2
3'UTR loxN] I;
egl-20(cp413[egl-20::FRT5T2::
mKate2::let-858 3'UTR::FRT5T2::
mNG^3xFlag]) IV		This paperLP817
Strain, strain
background
(C. elegans)		cpIs160 [Plin-44 > egl-20::mNG
::SL2::2x mTurq2::PH::3x
HA::tbb-2 3'UTR loxN] I;
cpIs130[Pwrt-2>2x
mKate2::PH::3xHA::let-858
3'UTR::Ptag-168 > HisCl1::
tbb-2 3'UTR loxN] II		This paperLP818
Recombinant
DNA reagent		Peft-3>Cas9+PU6>empty sgRNADickinson et al., 2013pDD162vector for Cas9 + sgRNA
cloning
Recombinant
DNA reagent		Peft-3>Cas9+ttTi5605 sgRNADickinson et al., 2013pDD122Cas9 + sgRNA targeting
genomic site near ttTi5605
Recombinant
DNA reagent		Peft-3>Cas9+ttTi4348 sgRNAThis paperpAP082Cas9 + sgRNA targeting
genomic site near ttTi4348.
Derived from pDD122.
Recombinant
DNA reagent		empty promoter > 2 x
mKate2::PH::3xHA::tbb-2
3'UTR loxN SEC loxN ttTi5605		This paperpAP087.2vector for plasma membrane
reporter insertions near
ttTi5605. Derived from pCFJ150.
Recombinant
DNA reagent		empty promoter > 2 x
mTurq2::PH::3xHA::tbb-2
3'UTR loxN SEC loxN ttTi4348		This paperpAP088vector for plasma membrane
reporter insertions near
ttTi4348. Derived from pCFJ352.
Recombinant
DNA reagent		mNG^SEC^3xFlagDickinson et al., 2015pDD268vector for cloning homologous
repair templates
Recombinant
DNA reagent		YPET^SEC^3xFlagDickinson et al., 2015pDD283vector for cloning homologous
repair templates
Recombinant
DNA reagent		mKate2^SEC^3xMycDickinson et al., 2015pDD287vector for cloning homologous
repair templates

Additional files

Supplementary file 1

PCR primers used to amplify transgene promoters.

https://doi.org/10.7554/eLife.38325.025
Transparent reporting form
https://doi.org/10.7554/eLife.38325.026

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