Extracellular matrix protein composition dynamically changes during murine forelimb development

Abstract

21 22 33 investigating the role of the matrisome during complex tissue development. 34 35 Keywords: mouse; extracellular matrix; matrisome; limb development; murine morphogenesis and growth; 36 mass spectrometry; bioorthogonal non-canonical amino acid tagging; osteogenesis imperfecta; brain 37 38 102 These methods can be easily extended to investigating the role of the matrisome in other systems during 103 morphogenesis and growth. 104 105 6 Results and Discussion 106 Fractionation of whole embryonic tissue increased ECM protein identification and enabled temporal 107 analysis of the matrisome 108We previously demonstrated that fractionation of E15.5 murine embryos facilitated the analysis of the 109 matrisome via LC-MS/MS (Saleh, et al., 2019a). To confirm that fractionation provides the same benefits 110 for earlier embryonic timepoints wherein the ECM may be less cross-linked, E12.5 wild-type (WT) 111 embryos were homogenized then fractionated using buffers designed to selectively extract cytosolic (C), 112 nuclear (N), membrane (M) and cytoskeletal (CS) proteins, leaving behind an ECM-rich insoluble (IN) 113 pellet (Naba, et al., 2015) (Figure S1A). All samples were processed for LC-MS/MS, raw protein intensities 114 were determined by MaxQuant (Cox and Mann, 2008) and proteins were categorized into cellular 115 compartments (Saleh, et al., 2019a). Within the homogenate, 20 ECM proteins were identified; however, 116 these proteins only represented 0.8% ± 0.3% of the total raw intensity (Figure S1B, Table S1). In contrast, 117 the percentage of matrisome in the IN pellet was 91.5% ± 3.5%, indicating tissue fractionation significantly 118 increased the identification of ECM proteins in E12.5 embryos ( Table S1). The amount of ECM protein in 119 the C, N and M fractions contributed to less than 1.0% ± 0.9% of the total raw intensity. In comparison, 120 ECM proteins contributed 2.3% ± 1.1% of the total raw intensity of the CS fractions. For subsequent studies, 121 the CS and IN fractions were analyzed via LC-MS/MS separately, while the C, N and M fractions were 122 combined into one 'CNM' fraction. 123To investigate whether fractionation was amenable for temporal analysis of the embryonic matrisome, 124 the CNM, CS and IN fractions of E11.5-E14.5 WT embryos were analyzed by LC-MS/MS (Figure 1A). 125Across all fractions and timepoints, 3227 proteins were identified, 122 of which were part of the matrisome 126 (Table S2). Less than 1% and 5% of the raw intensity in CNM and CS fractions, respectively, were 127 attributed to ECM proteins, consistent with our previous study (Saleh, et al., 2019a) (Figures 1B, S1). 128Importantly, the matrisome was enriched in the IN fraction, where at least 30% of the raw intensity was 129 from ECM proteins. 130 7 GO analysis was conducted on the 50 most abundant proteins in each fraction for E14.5 embryos. 131"Cellular Component" terms indicated proteins extracted in the C, N and M buffers were from intracellular 132 compartments, and the most significant "Biological Process" ter...

Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

“…To visualize the distribution of myogenic progenitors in the context of the ECM, Pax3 Cre /ZsGreen1 + (Pax3GFP + ) limbs were cleared using SeeDB. These tissues were also counterstained for ECM highly expressed in the developing limb: tenascin-C (TNC), fibrillin-2 (FBN2), and type V collagen (COL5) (Jacobson et al, 2020b). Proximally, Pax3GFP + cells were loosely aggregated (Figure 1F-H''; *), whereas they were oriented along the proximal-distal (pd) axis of the limb towards the distal tip of the limb (arrowhead).…”

3D mapping reveals a complex and transient interstitial matrix during murine renal development

“…[91][92][93] These proteins were potentially extracted in the less stringent buffers at earlier timepoints as the ECM was still being incorporated into a cohesive network. 31 This is supported by the observation that some enzymes involved in ECM crosslinking, 4,94 including LOXL1, LOXL2, PXDN, TGM1, and TGM2, increased in abundance in the postnatal and adult kidney ( Figure S3; Table S1).…”

“…To assess if the kidney matrisome is specialized by E14.5, ECM protein abundance was compared to whole embryos ( Figure 2A; Table S2). 31 There was a significantly greater amount of basement membrane proteins in the kidney ( Figure 2B-C). Gene ontology (GO) analysis for proteins exclusive or significantly elevated in the kidney generated terms associated with basement membrane ( Figure 2D).…”

“…[28][29][30] E14.5 kidneys proteins were compared to E14.5 whole embryo data from (Jacobson et al, submitted). 31 LC-MS/MS analysis: Protein groups were filtered using Microsoft Excel (2019) to exclude proteins identified by less than two unique and razor peptides, reverse hits, or identified as potential contaminants (with the exception of keratins, THBS1, LUM, TPM2, PFN1, and COMP).…”

Microgravity Effects on the Matrisome

“…The proteomic analysis of the muscle and skeletal morphogenesis of a mouse embryo forelimb detected 122 matrisomal proteins, with their ratio varying significantly depending on the embryogenesis stage. Most of the proteins were represented by collagens and glycoproteins [ 47 ].…”