1. Cell Biology

Prickle isoforms determine handedness of helical morphogenesis

  1. Bomsoo Cho
  2. Song Song
  3. Jeffrey D Axelrod  Is a corresponding author
  1. Stanford University School of Medicine, United States
https://doi.org/10.7554/eLife.51456
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Cite this article
  1. Bomsoo Cho
  2. Song Song
  3. Jeffrey D Axelrod
(2020)
Prickle isoforms determine handedness of helical morphogenesis
eLife 9:e51456.
https://doi.org/10.7554/eLife.51456
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8 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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Morphology of wildtype dorsal mechanosensory bristles.

(A) Dorsal and ventral views showing adult dorsal mechanosensory bristles (red arrowheads), ventral chemo- and mechanosensory bristles (blue arrowhead) and dorsal chemosensory bristles (black …

Figure 1—figure supplement 1
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Schematic of the AWM bristles, and expansive views of adult and pupal control (w1118) and pkpk30 bristles.

(A) Schematic drawing showing the organization of the AWM bristles on a conceptually unfolded wing. Dorsal mechanosensory bristles (red and white), ventral mechanosensory bristles (blue) and …

Figure 1—video 1
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Morphology of 36 hr wildtype dorsal mechanosensory bristles displayed in 3D A reconstruction of confocal stacks displaying AWMs stained with Su(H) (green) to mark socket cells and RFP marking two shaft-socket cell pairs.

Note that the software displays the edges of shapes cut by the bounds of the displayed section as bright areas.

Figure 2 with 1 supplement
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Morphology of pkpk mutant dorsal mechanosensory bristles.

(A–A’) pkpk30 adult wing and equivalent region of a pkpk30 36 hr pupal wing stained for Su(H) and actin. (B–B’) 3D views of RFP clones in a pkpk30 36 hr pupal wing revealing reversed orientation of …

Figure 2—figure supplement 1
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Rose plots for control and pkpk30 socket cell orientations and apical rearrangement of anterior wing margin cells.

(A) Complete set of rose plots for control and pkpk30 socket cell orientations at times sampled (control - 115 sockets from six wings (24 hr), 105 sockets from seven wings (28 hr), 103 sockets from …

Figure 3 with 4 supplements
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3D images of shaft-socket pairs at different times reveal a clockwise helical growth in control and counterclockwise growth in pkpk30 mutant bristles.

(A) Reconstructed 3D images from varying angles of 24 hr, 28 hr, 32 hr and 36 hr shaft-socket clones marked with RFP and stained with Su(H). (B) Cartoon interpretation of images from panel A), …

Figure 3—figure supplement 1
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Bristle polarity in adult and socket cell rotation in pupal control (w1118), pkpk30, pksple1, pkpk-sple13, and, MS1096-Gal4 UAS-pksple (>>sple) wings at 24 hr and 32 hr apf.

(A–D) For pkpk30 (panel B) and >>sple (panel E), socket cell images (Su(H)) are from regions corresponding to those demarcated by the arrowheads. (C’, D’, E’) Quantification of socket cell rotation …

Figure 3—video 1
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Morphology of 24 hr wildtype dorsal mechanosensory bristles displayed in 3D A reconstruction of confocal stacks displaying AWMs stained with Su(H) (green) to mark socket cells and RFP marking shaft-socket cell pairs.
Figure 3—video 2
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Morphology of 28 hr wildtype dorsal mechanosensory bristles displayed in 3D A reconstruction of confocal stacks displaying AWMs stained with Su(H) (green) to mark socket cells and RFP marking shaft-socket cell pairs.
Figure 3—video 3
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Morphology of 32 hr wildtype dorsal mechanosensory bristles displayed in 3D A reconstruction of confocal stacks displaying AWMs stained with Su(H) (green) to mark socket cells and RFP marking shaft-socket cell pairs.
Figure 4 with 2 supplements
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Expression of Pkpk and Pksple isoforms in pupal wings.

(A–B’’) V5::Pk (A–A’’) and V5::Sple (B–B’’) in pupal wings of ages indicated. Red arrowheads mark AWM and yellow arrowheads mark veins L3 and L4. (C–D’) Surface views of V5::Pk at 24 hr (C–C’) or …

Figure 4—figure supplement 1
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V5::Pk and V5::Sple expression throughout wing development.

(A) Western blot from wing extracts from homozygous V5::Pk and V5::Sple eyes, and wings at various developmental stages, stained with anti-V5 and then anti-Pk[C] antibodies, validating the specific …

Figure 4—figure supplement 2
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Timing of expression and localization of Pkpk and/or Pksple correlates with handedness of helical rotation.

(A, B) V5::Pk and V5::Sple surface views at the margin of 24 hr, 28 hr and 32 hr pupal wings showing declining expression of Pk, first in socket cells and then in margin cells, and increasing …

Figure 5 with 1 supplement
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Core PCP components control shaft-socket rotation.

(A–D) Reversed bristle polarity in pkpk mutant compared to control is abrogated in pk fz and pk vang double mutants, demonstrating requirement for core PCP activity for polarity reversal in pkpk …

Figure 5—figure supplement 1
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Reversed Vang localization inpkpkmutant socket and margin cells.

Vang::EYFP expression clones were induced in pkpk mutant wings (32h). In the distal region of the AWM, where bristle polarity is reversed, Vang::EFYP is enriched on the distal side of both socket …

Figure 6 with 1 supplement
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Polarity propagates between bristle and margin cells.

3D reconstructed views of a Fz::EGFP wing (32 hr) with fz knockdown clones, stained for actin to mark wing hairs and bristle shafts, Su(H) to mark socket cells and RFP to indicate knockdown clones. …

Figure 6—figure supplement 1
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vangRNAi clone showing polarity propagation among bristles at the margin.

3D reconstructed views of a Vang::EYFP wing (32h) with vang knockdown clone, stained for Su(H) to mark socket cells and RFP to indicate knockdown clone. A clone involving 2 bristles (red arrowheads) …

Figure 7 with 1 supplement
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Pkpk and Pksple activity responds to Ds.

(A) A control (w1118) wing showing normally oriented bristles and rotated socket cells, and proximal V5::Sple. (B) A pkpk mutant wing with reversal of distal bristles, counter-rotated socket cells …

Figure 7—figure supplement 1
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A reversed ectopic Ds gradient re-orients core PCP domains.

(A–D) Quantification of socket cell rotation for the genotypes (32 hr) from Figure 7C–F: w118, MS1096 >dsRNAi ((A), 74 sockets from four wings), pkpk, MS1096 >dsRNAi ((B), 72 sockets from three …

Author response image 1
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pkpkand dll>ds;pkpkanterior wing margins.

Tables

Table 1
p values for comparison of rotation angles for control W1118 socket cells at different times apf.
W111824 hr
5.0°		28 hr
28.4°
24 hr
5.0°
28 hr
28.4°		<0.0001
32 hr
54.588°		<0.0001<0.0001
Table 2
Summary statistics for rotational angles.

CSD = Circular Standard Deviation.

GenotypeTimeGrand Mean Vector (GM)Length of Grand Mean Vector (r)Number of
means (wings)		Mean CSD
W111824 hr5.04°0.99664.14
2828.368°0.99574.075
3254.588°0.98162.940333
pkpk3024 hr0.63°0.98837.781333
32 hr348.482°0.946715.09
pkpk-sple1324 hr1.061°0.99733.687
32 hr47.59°0.99336.582
pksple124 hr17.8°0.97937.244667
32 hr59.288°0.9944.24425
MS1096 >> sple24 hr358.057°0.99435.796
32 hr333.296°0.964313.31267
fzR5224 hr3.351°0.99833.923333
32 hr20.471°0.905623.5668
dsh124 hr5.684°0.99625.023
32 hr20.144°0.944614.316
fmi RNAi24 hr1.15°0.99634.78
32 hr19.291°0.978411.741
vangstbm624 hr1.51°0.967311.52167
32 hr25.447°0.94614.21117
w1118 MS1096 > dsRNAi32 hr16.593°0.79433.10867
pkpk MS1096 > dsRNAi32 hr29.323°0.961314.37067
pksple MS1096 > dsRNAi32 hr6.596°0.97312.63867
pkpk-sple MS1096 > dsRNAi32 hr40.297°0.9936.219667

  1. Table 2—source datas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, containing measurement of rotation angles of socket cells in control and various PCP mutant wings, with associated statistics, are provided.

Table 2—source data 1

Source data for w1118.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data1-v2.xlsx
Table 2—source data 2

Source data for pk30.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data2-v2.xlsx
Table 2—source data 3

Source data for pkpk-sple13.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data3-v2.xlsx
Table 2—source data 4

Source data for pksple1.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data4-v2.xlsx
Table 2—source data 5

Source data for MS1096-GAL4; UAS-pksple.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data5-v2.xlsx
Table 2—source data 6

Source data for fzR52.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data6-v2.xlsx
Table 2—source data 7

Source data for dsh1.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data7-v2.xlsx
Table 2—source data 8

Source data for fmiRNAi.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data8-v2.xlsx
Table 2—source data 9

Source data for vangstbm6.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data9-v2.xlsx
Table 2—source data 10

Source data for w1118; MS1096-GAL4; UAS-dsRNAi.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data10-v2.xlsx
Table 2—source data 11

Source data for pkpk; MS1096-GAL4; UAS-dsRNAi.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data11-v2.xlsx
Table 2—source data 12

Source data for pksple; MS1096-GAL4; UAS-dsRNAi.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data12-v2.xlsx
Table 2—source data 13

Source data for pkpk-sple; MS1096-GAL4; UAS-dsRNAi.

https://cdn.elifesciences.org/articles/51456/elife-51456-table2-data13-v2.xlsx
Table 3
p values for comparison of rotation angles for control and pk-related genotypes at 32 hr apf. ns = not significant.
32 hr anglesW1118
54.6°		pksple1
59.3°		pkpk-sple13
47.6°		pkpk30
348.5°
W1118
54.6°
pksple1
59.3°		(ns) 0.0702
pkpk-sple13
47.6°		0.01430.0343
pkpk30
348.5°		<0.0001<0.00010.0004
>>Pksple
333.2°		0.0012<0.00010.001(ns) 0.1826
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Drosophila melanogaster)pkpk-sple13Gubb et al., 1999, PMID: 10485852BDSC:41790;
FLYB:FBal0060943;
RRID:BDSC_41790		FlyBase symbol: pkpk-sple-13
Genetic reagent (Drosophila melanogaster)pkpk-sple14Gubb et al., 1999, PMID: 10485852FLYB:FBal0035401FlyBase symbol: pkpk-sple-14
Genetic reagent (Drosophila melanogaster)pkpk30Gubb et al., 1999, PMID: 10485852BDSC:44229;
FLYB:FBal0101223;
RRID:BDSC_44229		FlyBase symbol: pk30
Genetic reagent (Drosophila melanogaster)pksple1Gubb et al., 1999, PMID: 10485852BDSC:422;
FLYB:FBal0016024;
RRID:BDSC_422		FlyBase symbol: pksple-1
Genetic reagent (Drosophila melanogaster)vangA3Taylor et al., 1998, PMID: 9725839FLYB:FBal0093183FlyBase symbol:
VangA3
Genetic reagent (Drosophila melanogaster)vangstbm6Wolff and Rubin, 1998, PMID: 9463361BDSC:6918;
FLYB:FBal0062424;
RRID:BDSC_6918		FlyBase symbol:
Vangstbm-6
Genetic reagent (Drosophila melanogaster)fzR52Krasnow and Adler, 1994, PMID: 7924994FLYB:FBal0004939FlyBase symbol: fz23
Genetic reagent (Drosophila melanogaster)dsh1Bloomington Drosophila Stock CenterBDSC:5298;
FLYB:FBal0003138;
RRID:BDSC_5298		FlyBase symbol: dsh1
Genetic reagent (Drosophila melanogaster)UAS-pkspleBloomington Drosophila Stock CenterBDSC:41780;
FLYB:FBti0148928;
RRID:BDSC_41780		FlyBase symbol:
P{UAS-sple+}3
Genetic reagent (Drosophila melanogaster)UAS-pkRNAiVienna Drosophila Resource CenterVDRC:v101480; FLYB:FBst0473353; RRID:FlyBase_FBst0473353FlyBase symbol:
P{KK109294}VIE-260B
Genetic reagent (Drosophila melanogaster)UAS-fmiRNAiBloomington Drosophila Stock CenterBDSC:26022;
FLYB:FBti0114752;
RRID:BDSC_26022		Flybase symbol:
P{TRiP.JF02047}attP2
Genetic reagent (Drosophila melanogaster)UAS-fzRNAiBloomington Drosophila Stock CenterBDSC:34321;
FLYB:FBti0140932;
RRID:BDSC_34321		Flybase symbol:
P{TRiP.HMS01308}attP2
Genetic reagent (Drosophila melanogaster)UAS-vangRNAiBloomington Drosophila Stock CenterBDSC:34354;
FLYB:FBti0140967;
RRID:BDSC_34354		Flybase symbol:
P{TRiP.HMS01343}attP2
Genetic reagent (Drosophila melanogaster)UAS-dsRNAiBloomington Drosophila Stock CenterBDSC:32964;
FLYB:FBti0140473;
RRID:BDSC_32964		Flybase symbol:
P{TRiP.HMS00759}attP2
Genetic reagent (Drosophila melanogaster)UAS-dsMatakatsu and Blair, 2004, PMID: 15240556FLYB:FBtp0019964Flybase symbol:
P{UAS-ds.T}
Genetic reagent (Drosophila melanogaster)dll-GAL4Bloomington Drosophila Stock CenterBDSC:3038;
FLYB:FBti0002783;
RRID:BDSC_3038		Flybase symbol:
P{GawB}Dllmd23
Genetic reagent (Drosophila melanogaster)MS1096-GAL4Bloomington Drosophila Stock CenterBDSC:8860;
FLYB:FBti0002374;
RRID:BDSC_8860		Flybase symbol:
P{GawB}BxMS1096
Genetic reagent (Drosophila melanogaster)armP-fz::EGFPStrutt, 2001, PMID:11239465FLYB:FBtp0014592Flybase symbol:
P{arm-fz.GFP}
Genetic reagent (Drosophila melanogaster)actP-vang::EYFPStrutt, 2002, PMID: 12137731FLYB:FBtp0015854Flybase symbol:
P{Act5C(-FRT)stbm-EYFP}
Genetic reagent (Drosophila melanogaster)actP > CD2>vang::EYFPStrutt, 2002, PMID: 12137731FLYB:FBtp0084387Flybase symbol:
P{Act5C(FRT.polyA)stbm-EYFP}
Genetic reagent (Drosophila melanogaster)ci-GAL4Croker et al., 2006, PMID: 16413529FLYB:FBtp0057188Flybase symbol:
P{ci-GAL4.U}
Genetic reagent (Drosophila melanogaster)UAS-mCherryBloomington Drosophila Stock CenterBDSC:38424;
FLYB:FBti0147460;
RRID:BDSC_38424		Flybase symbol:
P{UAS-mCherry.NLS}3
Genetic reagent (Drosophila melanogaster)actP > CD2>Gal4Bloomington Drosophila Stock CenterBDSC:30558;
FLYB:FBti0012408;
RRID:BDSC_30558		Flybase symbol:
P{GAL4-Act5C(FRT.CD2).P}S
Genetic reagent (Drosophila melanogaster)UAS-RFPBloomington Drosophila Stock CenterBDSC:30558;
FLYB:FBti0129814;
RRID:BDSC_30558		Flybase symbol:
P{UAS-RFP.W}3
Antibodygoat polyclonal anti-Su(H)Santa CruzSanta Cruz:sc-15183
RRID:AB_672840		1/200 (immunolabelling)
AntibodyMouse monoclonal anti-V5Thermo-FisherThermo_Fisher:R960-25, RRID:AB_25565641/200 (immunolabelling)
1/1000 (Western blotting)
AntibodyGuinea pig polyclonal anti-Pk[C]Olofsson et al., 2014,
PMID: 25005476		N/A1/800 (immunolabelling)
1/1000 (Western blotting)
AntibodyRat monoclonal anti-dEcadDSHBRRID:AB_5281201/200 (immunolabelling)
AntibodyMouse monoclonal anti-γ-TubulinSigma-AldrichSigma-Aldrich: T6557
RRID:AB_477584		1/1000 (Western blotting)
Recombinant DNA reagentpCFD4AddgeneRRID:Addgene_49411CRISPR gRNA backbone
Recombinant DNA reagentpDsRedattpAddgeneRRID:Addgene_51019Donor recombinant DNA backbone
Recombinant DNA reagentpCR-Blunt-II-TOPOThermo-FisherRRID:Addgene_29705Backbone for sub-cloning
Sequence-based reagentpkpk gRNA 1This papergRNA sequence in PCR primersATGGCTCAGGCCCGATCTAG
Sequence-based reagentpkpk gRNA 2This papergRNA sequence in PCR primersGTGGATCAACCCCTGGAAAC
Sequence-based reagentpksple gRNA 1This papergRNA sequence in PCR primersCTCGTAAATTTAGCTTCGAG
Sequence-based reagentpksple gRNA 2This papergRNA sequence in PCR primersAGATGCAATTTGGCCGCCCT

Additional files

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https://cdn.elifesciences.org/articles/51456/elife-51456-transrepform-v2.pdf

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