1. Cell Biology
  2. Developmental Biology
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Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling

  1. Arul Subramanian
  2. Lauren Fallon Kanzaki
  3. Jenna Lauren Galloway
  4. Thomas Friedrich Schilling  Is a corresponding author
  1. University of California, Irvine, United States
  2. Massachusetts General Hospital, Harvard Stem Cell Institute, United States
Research Article
Cite this article as: eLife 2018;7:e38069 doi: 10.7554/eLife.38069
7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 4 supplements
Axial tenocyte morphogenesis.

(A–C) Lateral views of live Tg(scx:mCherry) embryos showing developing tenocytes (A - 24 hpf, B - 36 hpf, C - 48 hpf). (A’–C’) Transverse views from 3D projections showing the positions of developing tenocytes in relation to the notochord (NC) and neural tube (NT) along the horizontal (HMS) and vertical myosepta (VMS) (arrows). Tenocytes form projections at 36–48 hpf (B’ and C’). (D–F) Diagrams of lateral views showing the morphology of tenocytes in the developing somites. (D’–F’) Diagrams of transverse views from 3D projections of live Tg(scx:mCherry) embryos show the development of tenocyte projections (E’ and F’). (G–I) Lateral views of co-immunostained Tg(scx:mCherry) embryos showing developing tenocytes (anti-mCherry - white) and muscle fibers (anti-MHC - green) (G – 24 hpf, H – 36 hpf, I – 48 hpf). (G’–I’) Transverse views from 3D projections of live Tg(scx:mCherry) embryos showing the positions of developing tenocytes (arrowheads in G’ and H’) in relation to the myotome. Scale bars = 20 microns.

https://doi.org/10.7554/eLife.38069.003
Figure 1—figure supplement 1
Axial tenocytes form polarized projections orthogonal to muscle fibers.

(A) Lateral and (B) transverse views of live 60 hpf Tg(scx:mCherry) embryos showing tenocytes, which are pseudocolored to highlight the depth of the 3D reconstructed image. Transgene expression is also observed in neuronal cell bodies in the neural tube. Scale bars = 10 microns.

https://doi.org/10.7554/eLife.38069.004
Figure 1—figure supplement 2
Cranial tenocyte morphogenesis correlates with onset of muscle contraction.

Co-immunostained Tg(scx:mCherry) embryos (anti-mCherry – red; anti-MHC – green) showing temporal changes in tenocyte morphogenesis in relation to developing muscles. (A, D, G – 48 hpf; B, E, H – 62 hpf; C, F, I, – 72 hpf). Ventral views of Tg(scx:mCherry) embryonic heads. Abbreviations: IMA – intermandibularis anterior, IMP – intermandibularis posterior, IH – interhyal, AM – adductor mandibulae, HH – hyohyal, SH – sternohyoideus, mc – Meckels cartilage, bh – basihyal, ch – ceratohyal. Scale bar = 20 microns.

https://doi.org/10.7554/eLife.38069.005
Figure 1–video 1
Axial tenocyte progenitors align along HMS and VMS following muscle fiber differentiation.

Time-lapse video of a developing Tg(scx:mCherry) embryo between 20 and 36 hpf at 15 min intervals. Tenocyte progenitors express scx:mCherry at 24 hpf, when muscle fibers have already formed initial attachments at the VMS. A transverse view shows migration of tenocyte progenitors to a medial position (HMS) around the notochord (NC) and along the VMS.

https://doi.org/10.7554/eLife.38069.006
Figure 1–video 2
Tenocyte projections are dynamic.

A time-lapse video of a developing Tg(scx:mCherry) embryo between 48 and 60 hpf at 15 min intervals shows activity of tenocyte projections along VMS and HMS.

https://doi.org/10.7554/eLife.38069.007
Figure 2 with 3 supplements
Tenocyte projection length and branching density is regulated by mechanical force.

Lateral views of live Tg(scx:mCherry) embryos (48 hpf) showing tenocyte projections. Images are pseudocolored by depth from medial (red) to lateral (blue). Control embryos were imaged without stimulation (A) and after stimulation (B), and the length of tenocyte projections was compared with embryos injected with αBtx and imaged without (C) and with stimulation (D). Dot plot shows individual data points of tenocyte projection length under different conditions (E). The data points from each embryo are connected by a vertical line. NS – Not Stimulated, S – Stimulated. (n > 50 data points/embryo in three embryos/sample, p value was determined through ANOVA 1-way analysis ***<0.00001, **<0.0001). Histogram shows quantification of branch points along tenocyte projections per tenocyte in 36 hpf control and αBtx injected embryos for every level of branching (1o – primary, 2o – secondary, 3o – tertiary, 4o – quaternary). (n = 4, p value was determined through ttest *<0.01, ***<0.00001). The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 2—source data 1 and Figure 2—source data 2.

https://doi.org/10.7554/eLife.38069.008
Figure 2—source data 1

Measurements of tenocyte projection length along VMS.

https://doi.org/10.7554/eLife.38069.014
Figure 2—source data 2

Measurement of tenocyte projection branching complexity along VMS.

https://doi.org/10.7554/eLife.38069.015
Figure 2—figure supplement 1
Density of tenocyte projections is regulated by mechanical force.

Dot plot shows individual data points of tenocyte projection density at the ventral VMS in embryos injected with αBtx and imaged without and with stimulation. The data points from three VMSs in each embryo are connected by a vertical line. NS – Not Stimulated, S – Stimulated. (n ~ 10 embryos/sample, p value was determined through ANOVA 1-way analysis and Tukey test ***<0.00001). The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 2—figure supplement 1—source data 1.

https://doi.org/10.7554/eLife.38069.009
Figure 2—figure supplement 1—source data 1

Measurements of projection density along VMS.

https://doi.org/10.7554/eLife.38069.010
Figure 2—figure supplement 2
cacnb1 mutants show reduced length and branching of tenocyte projections.

Lateral views of immunostained (A) sibling and (B) cacnb1 mutant embryos carrying the Tg(scx:mCherry) transgene showing tenocyte projections in a pseuodocolored, depth-coded pattern. (C) Dot plot shows individual data points of tenocyte projection length in sibling and cacnb1 mutant embryos (n = 50 data points/embryo in eight embryos/sample, p-value was determined by Wilcoxon Rank Sum Test - < 0.0001). The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 2—figure supplement 2—source data 1.

https://doi.org/10.7554/eLife.38069.011
Figure 2—figure supplement 2—source data 1

Measurements of Tsp4b localization area.

https://doi.org/10.7554/eLife.38069.012
Figure 2—figure supplement 3
Cranial tenocyte patterning and morphogenesis is disrupted in pet mutants.

Ventral views of 98 hpf wild-type (A–C) and cacnb1; scx:mCherry (D–F) embryonic heads showing muscle fibers (green) and corresponding tenocytes (red). (G–J) Higher magnification views of control Tg(scx:mCherry) embryonic heads (panel A insets - color coded boxes) and (K–N) higher magnification views of cacnb1; scx:mCherry embryonic heads (panel D insets - color-coded boxes) showing tenocyte projections in different tendons (arrows). Scale bars = 20 microns.

https://doi.org/10.7554/eLife.38069.013
Figure 3 with 3 supplements
Tsp4b localization to VMS and tenocyte projections requires mechanical force.

Lateral views of live control (A–C) and αBtx injected (D–F) Tg(scx:mCherry) embryos (48 hpf), injected with tsp4b-gfp mRNA showing localization of Tsp4b-GFP (green) (arrowheads) along the VMS and tenocyte projections (red). (I) Histogram shows the percentage of embryos with Tsp4b-GFP localized to VMS (n = 27, p value calculated by chi-squared test <0.05). (G–H) Lateral views of immunostained embryos showing Tsp4b protein localization detected immunohistochemically along VMS in control (G) and αBtx injected (H) embryos. (J) Dot plot shows individual data points of the fluorescent intensity of localized Tsp4b along the VMS in control and αBtx injected embryos. Three VMSs/embryo were sampled in control and αBtx-injected embryos. (n = 9, p value calculated by Wilcoxon Rank Sum Test - < 0.0001). Scale bars = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 3—source data 1 and Figure 3—source data 2.

https://doi.org/10.7554/eLife.38069.016
Figure 3—source data 1

Count of embryos showing localized or diffuse Tsp4b-GFP.

https://doi.org/10.7554/eLife.38069.023
Figure 3—source data 2

Measurements of Tsp4b fluorescence intensities along VMS.

https://doi.org/10.7554/eLife.38069.024
Figure 3—figure supplement 1
Early Lam and Fn organization do not depend on mechanical force.

Dot plot shows individual data points of the fluorescent intensity of localized Lam (A) and Fn (B) at MTJs along the VMS in control and αBtx-injected embryos at 48 hpf (A) and 24 hpf (B). Three VMSs/embryo were sampled in control and αBtx-injected embryos. (n = 9, p value calculated by ANOVA 1-way analysis and Tukey -*<0.002). Scale bars = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 3—figure supplement 1—source data 1 and Figure 3—figure supplement 1—source data 2.

https://doi.org/10.7554/eLife.38069.017
Figure 3—figure supplement 1—source data 1

Measurement of Laminin fluoresence intensity along VMS.

https://doi.org/10.7554/eLife.38069.018
Figure 3—figure supplement 1—source data 2

Measurement of Fibronectin fluoresence intensity along VMS.

https://doi.org/10.7554/eLife.38069.019
Figure 3—figure supplement 2
Tsp4b organization requires mechanical force.

Lateral view of immunostained 48 hpf embryos showing the localization of Tsp4b in control embryos without (A) and with stimulation (C), and αBtx-injected embryos without (B) and with stimulation (D). Histogram shows the mean area of Tsp4b localization in VMS (dotted region) (E). NS – Not Stimulated, S – Stimulated. Scale bar = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 3—figure supplement 2—source data 1.

https://doi.org/10.7554/eLife.38069.020
Figure 3—figure supplement 2—source data 1

Measurements of Tsp4b localization area.

https://doi.org/10.7554/eLife.38069.021
Figure 3—figure supplement 3
Mechanical force regulates expression of Tsp4b.

Histogram shows relative expression of rpl13a, scxa and tsp4b in 24 hpf and 48 hpf control and αBtx-injected embryos. (p value calculated by ANOVA 1-way analysis and Tukey test -**<0.001).

https://doi.org/10.7554/eLife.38069.022
Microtubule-rich tenocyte projections control tendon ECM localization.

Lateral views of live 48 hpf Tg(scx:mCherry) embryos injected with EGFP-alpha-Tubulin mRNA (A–C) showing localization of a-Tubulin along the length of projections colocalized with mCherry to mark in tenocytes. Transverse views of 3-D reconstructed live 60 hpf embryos showing tenocyte projections in DMSO-treated (D) and Nocodazole (Noco)-treated (E) embryos. Transverse view of 3-D reconstructed 60 hpf embryos immunostained for Tsp4b showing localization of Tsp4b in DMSO treated (F) and Noco treated (G) samples. Quantification of Tsp4b localization intensity in VMS (H) and distribution of Tsp4b aggregates in VMS (I) of DMSO-treated and Noco-treated embryos. (p value calculated by t-test for samples with unequal variance *<0.05, ***<0.0005). Scale bars = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 4—source data 1 and Figure 4—source data 2.

https://doi.org/10.7554/eLife.38069.025
Figure 4—source data 1

Mesurements of Tsp4b fluorescence intensities along VMS.

https://doi.org/10.7554/eLife.38069.026
Figure 4—source data 2

Count of Tsp4b aggregates along VMS.

https://doi.org/10.7554/eLife.38069.027
TGFβ signaling regulates tenocyte morphogenesis.

Lateral views of immunostained Tg(scx:mCherry) control (A–D) and SB431542-treated (E–H) embryos showing nuclei (DAPI), tenocytes (anti-mCherry) and pSMAD3 (anti-pSMAD3). (I) Localization of pSMAD3 was quantified as fluorescent intensity of nuclear pSMAD3 signal (marked by yellow dotted ROI) and plotted as a dot plot showing data points (n = 9, p value was calculated by t test ***<0.000005). (D, H) Pseudocolored 3D projections show tenocyte cell projections in control (D) and SB 431542 treated embryos (H). (J) Dot plot shows individual data points representing tenocyte projection lengths (n = 50 data points/embryo in nine embryos/sample, p value was calculated by Wilcoxon Rank Sum test ***<0.00005). Representative muscle nuclei are marked by a blue continuous ROI. Scale bars = 10 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 5—source data 1 and Figure 5—source data 2.

https://doi.org/10.7554/eLife.38069.028
Figure 5—source data 1

Measurements of pSMAD3 fluorescence intensities in tenocyte nuclei along VMS.

https://doi.org/10.7554/eLife.38069.029
Figure 5—source data 2

Measurements of tenocyte projection length along VMS.

https://doi.org/10.7554/eLife.38069.030
Figure 6 with 3 supplements
TGFβ signaling in tenocytes requires mechanical force.

Lateral views of 48 hpf immunostained Tg(scx:mCherry) control (A–C) and αBtx injected (D–F) embryos showing nuclei (DAPI), tenocytes (anti-mCherry) and pSMAD3 (anti-pSMAD3) (marked by yellow-dotted ROI). (G) Localization of pSMAD3 was quantified as fluorescent intensity of nuclear pSMAD3 signal and plotted as a dot plot (n = 4, p value was calculated by t-test **<0.005). (H) Dot plot shows individual tenocyte projection lengths (p value was calculated by t-test **<0.00005). Representative muscle nuclei are marked by a blue continuous ROI. Scale bar = 10 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 6—source data 1 and Figure 6—source data 2.

https://doi.org/10.7554/eLife.38069.031
Figure 6—source data 1

Measurements of Tenocyte projection length along VMS.

https://doi.org/10.7554/eLife.38069.036
Figure 6—source data 2

Measurements of tenocyte nuclei pSMAD3 fluorescence intensity along VMS.

https://doi.org/10.7554/eLife.38069.037
Figure 6—figure supplement 1
TGFβ signaling is elevated in response to mechanical force.

(A–F) Single plane images showing lateral views of paralyzed (αBtx) 48 hpf embryos without stimulation (A–C) and after stimulation (D–F), immunostained to show nuclei (DAPI), tenocytes (anti-mCherry) and pSMAD3 (anti-pSMAD3) (marked by yellow dotted ROI). Cell bodies are outlined by dotted lines. (G) pSMAD3 localization was quantified as fluorescent intensity of nuclear pSMAD3 signal and plotted as a dot plot (n = 3, p value was calculated by Wilcoxon Rank Sum test **<0.0005). NS – Not Stimulated, S – Stimulated. Representative muscle nuclei are marked by a blue continuous ROB. Scale bar = 10 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 6—figure supplement 1—source data 1.

https://doi.org/10.7554/eLife.38069.032
Figure 6—figure supplement 1—source data 1

Measurements of tenocyte nuclei pSMAD3 fluorescence intensity along VMS.

https://doi.org/10.7554/eLife.38069.033
Figure 6—figure supplement 2
Mechanical force regulates expression of genes involved in tendon development.

(A) qRT-PCR analysis shows relative expression of scxa, tsp4b, tgfbip and ctgfa2 genes in control (wild-type) embryos and paralyzed embryos (αBtx-inj) (without and with stimulation) (p value was calculated by ANOVA 1-way analysis and Tukey test **<.005). (B) ddPCR analysis shows absolute expression level of scxa, tsp4b, tgfbip and ctgfa2 genes in control (wild-type) embryos and paralyzed embryos (αBtx-injected) (without and with stimulation) in whole embryos.

https://doi.org/10.7554/eLife.38069.034
Figure 6—figure supplement 3
TGFβ signaling and tenocyte projection integrity affect tendon gene expression.

Real time PCR analysis shows relative expression of scxa, tsp4b, tgfbip and ctgfa2 genes in control (DMSO treated) embryos, SB431542-treated embryos (A) and Nocodazole-treated embryos (B).

https://doi.org/10.7554/eLife.38069.035
TGFβ-mediated mechanotransduction is essential for tenocyte differentiation and morphogenesis.

(A) In the presence of tensile force from muscle contraction (1) changes in ECM organization and other factors lead to release of active Tgfβ ligand (2). Tgfβ ligand binds to receptors on tenocytes to increase pSMAD3 signaling (3), secretion of ECM components (4) and growth/branching of microtubule rich projections (5). Cartoon depiction of tenocyte morphogenesis in the presence of mechanical force (during onset of muscle contraction in embryonic development or through electrical stimulation of paralyzed embryos). (B) In the absence of mechanical force (before onset of muscle contraction or in paralyzed embryos) there is reduced active Tgfβ ligand, pSMAD3 signaling, expression of ECM proteins and growth/branching of projections. Depiction of tenocyte morphogenesis in the absence of mechanical force.

https://doi.org/10.7554/eLife.38069.038

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyRabbit anti Tsp4bSchilling labRRID: AB_27257931:500 dilution
AntibodyMouse anti Myosin
heavy chain
DSHBCat# A4.1025,
RRID: AB_528356
1:250 dilution
AntibodyChicken anti GFPAbcamCat# ab13970,
RRID: AB_300798
1:1000 dulution
AntibodyRat anti mCherryMolecular ProbesCat# M11217,
RRID: AB_2536611
1:500 dilution
AntibodyRabbit anti FibronectinAbcamCat# ab2413,
RRID: AB_2262874
1:200 dilution
AntibodyRabbit anti LamininAbcamCat# ab11575,
RRID: AB_298179
1:200 dilution
AntibodyRabbit anti pSMAD3Antibodies-onlineCat# ABIN1043888,
RRID: AB_2725792
1:500 dilution
AntibodyAlexa Fluor 488
conjugated Donkey
anti Chicken IgY
Jackson ImmunoresearchCat# 712-586-1531:1000 dulution
AntibodyDyLight 549 conjugated
Donkey anti Rabbit IgG
Jackson ImmunoresearchCat# 711-506-152,
RRID: AB_2616595
1:1000 dulution
AntibodyAlexa Fluor 488
conjugated Donkey
anti Rabbit IgG
Jackson ImmunoresearchCat# 711-545-152,
RRID: AB_2313584
1:1000 dulution
AntibodyCy5 conjugated
anti Mouse IgG
Jackson ImmunoresearchCat# 115-176-0711:1000 dulution
AntibodyAlexa Fluor 594
conjugated Donkey
anti Rat IgG
Jackson ImmunoresearchCat# 712-586-153,
RRID: AB_2340691
1:1000 dulution
AntibodyAlexa Fluor 488
conjugated anti Mouse
IgG
Jackson ImmunoresearchCat# 715-546-150;
RRID: AB_2340849
1:1000 dulution
AntibodyDiAmino PhyenylIndole
(DAPI)
InvitrogenCat# D1306,
RRID: AB_2629482
1:1000 dulution
Cell line (E. coli)Chemically competent
DH5alpha cells
Schilling Lab
Chemical compound, drugSB431542TocrisCat# 1614,
SID: 241182574
50 mM stock solution,
10 µM final concentration
Chemical compound, drugNocodazoleSigma-AldrichCat#1404,
SID: 24278535
33 mM stock solution,
0.33 mM final concentration
Chemical compound, drugTrizolInvitrogenCat# 15596018
Chemical compound, drug3-aminobenzoic acid
ethyl ester
methanesulfonate
Sigma-AldrichCat# A5040,
SID: 329770864
Commercial assay
or kit
mMessage mMachine
T7 ultra transcription
kit
AmbionCat# AM1345,
RRID: SCR_016222
Commercial assay
or kit
mMessage mMachine
T3 transcription kit
AmbionCat# AM1348,
RRID: SCR_016223
Commercial assay
or kit
mMessage mMachine
SP6 transcription kit
AmbionCat# AM1340,
RRID: SCR_016224
Commercial assay
or kit
Protoscript II first
strand cDNA synthesis
kit
New England BiolabsCat# E6560,
RRID: SCR_016225
Commercial assay
or kit
Luna universal
qPCR master mix
New England BiolabsCat# M3003,
RRID: SCR_016226
Commercial assay
or kit
Direct-zol RNA
Miniprep
Zymo ResearchCat# R2061,
RRID: SCR_016227
Commercial assay
or kit
QX200 EvaGreen
653 ddPCR Supermix
Bio-RadCat# 1864033
RRID: SCR_008426
Commercial assay
or kit
QX200 Droplet Generation
Oil for EvaGreen
Bio-RadCat# 1864005,
RRID: SCR_008426
Commercial assay
or kit
QX200 Droplet
Generator
Bio-RadCat# 1864002,
RRID: SCR_008426
Commercial assay or kitQX200 Droplet 657 ReaderBio-RadCat# 1864003,
RRID: SCR_008426
Commercial assay
or kit
Qubit SSDNA assay kitInvitrogenCat# Q10212,
SCR_008817
Commercial assay
or kit
Qubit 2.0 fluorometerInvitrogenCat# Q32866,
SCR_008817
Gene (Danio rerio)Tg(scx:mCherry)Galloway labN/A
Gene (Danio rerio)Cacnb1+/-Schilling labN/A
Sequence-based reagentPrimers for RT-PCR,
see Table S1
This paperN/A0.5 µM final concentration
Recombinant DNA
reagent
pmtb-t7-alpha-bungarotoxinAddgeneCat# 69542,
RRID: SCR_002037
Recombinant DNA
reagent
pIRESneo-EGFP-alpha
tubulin
AddgeneCat# 12298,
RRID: SCR_002037
Recombinant DNA
reagent
pmEGFP-Lifeact-7AddgeneCat# 54610,
RRID: SCR_002037
Software, algorithmSimple Neurite TracerFiji
Table 1
List of primer sequences used for RT-PCR.
https://doi.org/10.7554/eLife.38069.039
NameSequenceGene
rpl13a-fp-qpcrTCTGGAGGACTGTAAGAGGTATGCribosomal protein L13a
rpl13a-rp-qpcrAGACGCACAATCTTGAGAGCAG
rps13-fp-qpcrATAGGCGAAGTGTCCCCACAribosomal protein S13
rps13-fp-qpcrCAGTGACGAAACGCACCTGA
scxa-fp-qpcrGGAGAACTCGCAGCCCAAAscleraxis A
scxa-rp-qpcrAATCCCTTCACGTCGTGGTTT
tsp4b-fp-qpcrACAATCCACGAGACAACAGCthrombospondin 4b
tsp4b-rp-qpcrGCACTCATCCTGCCATCTCT
ctgfa-fp-qpcrCTTTACTGTGACTACGGCTCCconnective tissue growth factor a
ctgfa-rp-qpcrACAACTGCTCTGGAAAGACTC
tgfbip-fp-qpcrCCCCAATGTTTGTGCTATGCtgfβ induced peptide
tgfbip-rp-qpcrCTCCAATCACCTTCTCATATCCAG

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

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