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Thrombospondin-4 controls matrix assembly during development and repair of myotendinous junctions.

ABSTRACT: Tendons are extracellular matrix (ECM)-rich structures that mediate muscle attachments with the skeleton, but surprisingly little is known about molecular mechanisms of attachment. Individual myofibers and tenocytes in Drosophila interact through integrin (Itg) ligands such as Thrombospondin (Tsp), while vertebrate muscles attach to complex ECM fibrils embedded with tenocytes. We show for the first time that a vertebrate thrombospondin, Tsp4b, is essential for muscle attachment and ECM assembly at myotendinous junctions (MTJs). Tsp4b depletion in zebrafish causes muscle detachment upon contraction due to defects in laminin localization and reduced Itg signaling at MTJs. Mutation of its oligomerization domain renders Tsp4b unable to rescue these defects, demonstrating that pentamerization is required for ECM assembly. Furthermore, injected human TSP4 localizes to zebrafish MTJs and rescues muscle detachment and ECM assembly in Tsp4b-deficient embryos. Thus Tsp4 functions as an ECM scaffold at MTJs, with potential therapeutic uses in tendon strengthening and repair.

SUBMITTER: Subramanian A

PROVIDER: S-EPMC4096842 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Thrombospondin-4 controls matrix assembly during development and repair of myotendinous junctions.

Subramanian Arul A Schilling Thomas F TF

eLife 20140618

Tendons are extracellular matrix (ECM)-rich structures that mediate muscle attachments with the skeleton, but surprisingly little is known about molecular mechanisms of attachment. Individual myofibers and tenocytes in Drosophila interact through integrin (Itg) ligands such as Thrombospondin (Tsp), while vertebrate muscles attach to complex ECM fibrils embedded with tenocytes. We show for the first time that a vertebrate thrombospondin, Tsp4b, is essential for muscle attachment and ECM assembly ...[more]

PMID: 24941943

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Project description:PurposeTo determine thrombospondin-2 (TSP2) expression and its impact on postnatal retinal vascular development and retinal neovascularization.MethodsThe TSP2-deficient (TSP2(-/-)) mice and a line of TSP2 reporter mice were used to assess the expression of TSP2 during postnatal retinal vascular development and neovascularization. The postnatal retinal vascularization was evaluated using immunostaining of wholemount retinas prepared at different postnatal days by collagen IV staining and/or TSP2 promoter driven green fluorescent protein (GFP) expression. The organization of astrocytes was evaluated by glial fibrillary acidic protein (GFAP) staining. Retinal vascular densities were determined using trypsin digestion preparation of wholemount retinas at 3- and 6-weeks of age. Retinal neovascularization was assessed during the oxygen-induced ischemic retinopathy (OIR). Choroidal neovascularization (CNV) was assessed using laser-induced CNV.ResultsUsing the TSP2-GFP reporter mice, we observed significant expression of TSP2 mRNA in retinas of postnatal day 5 (P5) mice, which increased by P7 and remained high up to P42. Similar results were observed in retinal wholemount preparations, and western blotting for GFP with the highest level of GFP was observed at P21. In contrast to high level of mRNA at P42, the GFP fluorescence or protein level was dramatically downregulated. The primary retinal vasculature developed at a faster rate in TSP2(-/-) mice compared with TSP2(+/+) mice up to P5. However, the developing retinal vasculature in TSP2(+/+) mice caught up with that of TSP2(-/-) mice after P7. No significant differences in retinal vascular density were observed at 3- or 6-weeks of age. TSP2(-/-) mice also exhibited a similar sensitivity to the hyperoxia-mediated vessel obliteration and similar level of neovascularization during OIR as TSP2(+/+) mice. Lack of TSP2 expression minimally affected laser-induced CNV compared with TSP2(+/+) mice.ConclusionsLack of TSP2 expression was associated with enhanced retinal vascularization during early postnatal days but not at late postnatal times, and minimally affected retinal and CNV. However, the utility of TSP2 as a potential therapeutic target for inhibition of ocular neovascularization awaits further investigation.

Project description:The myotendinous junction is a highly interdigitated interface designed to transfer muscle-generated force to tendon. Understanding how this interface is formed and organized, as well as identifying tendon- and muscle-specific extracellular matrix (ECM), is critical for designing effective regenerative therapies to restore functionality to damaged muscle-tendon units. However, a comparative analysis of the ECM proteome across this interface has not been conducted. The goal of this study was to resolve the distribution of ECM proteins that are uniformly expressed as well as those specific to each of the muscle, tendon, and junction tissues. The soleus muscles from 5-month-old wild-type C57BL/6 mice were harvested and dissected into the central muscle (M) away from tendon, the junction between muscle and tendon (J) and the tendon (T). Tissues were processed by either homogenizing in guanidine hydrochloride or fractionating to isolate the ECM from more soluble intracellular components and then analyzed using liquid chromatography-tandem mass spectrometry. Overall, we found that both tissue processing methods generated similar ECM profiles. Many ECM were found across the muscle-tendon unit, including type I collagen and associated fibril-regulating proteins. The ECM identified exclusively in M were primarily related to the basal lamina, whereas those specific to T and J tissue included thrombospondins and other matricellular ECM. Type XXII collagen (COL22A1) was restricted to J, and we identified COL5A3 as a potential marker of the muscle-tendon interface. Immunohistochemical analysis of key proteins confirmed the restriction of some basal lamina proteins to M, tenascin-C to T, and COL22A1 to J. COL5A3, PRELP, and POSTN were visualized in the tissue surrounding the junction, suggesting that these proteins play a role in stabilizing the interface. This comparative map provides a guide for tissue-specific ECM that can facilitate the spatial visualization of M, J, and T tissues and inform musculoskeletal regenerative therapies.

Dataset Information

Thrombospondin-4 controls matrix assembly during development and repair of myotendinous junctions.

Publications

Thrombospondin-4 controls matrix assembly during development and repair of myotendinous junctions.

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