(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.

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 (1^o – primary, 2^o – secondary, 3^o – tertiary, 4^o – 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.

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.

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.

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.

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.