FOXD1 is required for 3D patterning of the kidney interstitial matrix.

BACKGROUND: The interstitial extracellular matrix (ECM) is comprised of proteins and glycosaminoglycans and provides structural and biochemical information during development. Our previous work revealed the presence of transient ECM-based structures in the interstitial matrix of developing kidneys. Stromal cells are the main contributors to interstitial ECM synthesis, and the transcription factor Forkhead Box D1 (Foxd1) is critical for stromal cell function. To investigate the role of Foxd1 in interstitial ECM patterning, we combined 3D imaging and proteomics to explore how the matrix changes in the murine developing kidney when Foxd1 is knocked out. RESULTS: We found that COL26A1, FBN2, EMILIN1, and TNC, interstitial ECM proteins that are transiently upregulated during development, had a similar distribution perinatally but then diverged in patterning in the adult. Abnormally clustered cortical vertical fibers and fused glomeruli were observed when Foxd1 was knocked out. The changes in the interstitial ECM of Foxd1 knockout kidneys corresponded to disrupted Foxd1+ cell patterning but did not precede branching dysmorphogenesis. CONCLUSIONS: The transient ECM networks affected by Foxd1 knockout may provide support for later-stage nephrogenic structures.

3D Mapping Reveals a Complex and Transient Interstitial Matrix During Murine Kidney Development.

BACKGROUND: The extracellular matrix (ECM) is a network of proteins and glycosaminoglycans that provides structural and biochemical cues to cells. In the kidney, the ECM is critical for nephrogenesis; however, the dynamics of ECM composition and how it relates to 3D structure during development is unknown. METHODS: Using embryonic day 14.5 (E14.5), E18.5, postnatal day 3 (P3), and adult kidneys, we fractionated proteins based on differential solubilities, performed liquid chromatography-tandem mass spectrometry, and identified changes in ECM protein content (matrisome). Decellularized kidneys were stained for ECM proteins and imaged in 3D using confocal microscopy. RESULTS: We observed an increase in interstitial ECM that connects the stromal mesenchyme to the basement membrane (TNXB, COL6A1, COL6A2, COL6A3) between the embryo and adult, and a transient elevation of interstitial matrix proteins (COL5A2, COL12A1, COL26A1, ELN, EMID1, FBN1, LTBP4, THSD4) at perinatal time points. Basement membrane proteins critical for metanephric induction (FRAS1, FREM2) were highest in abundance in the embryo, whereas proteins necessary for integrity of the glomerular basement membrane (COL4A3, COL4A4, COL4A5, LAMB2) were more abundant in the adult. 3D visualization revealed a complex interstitial matrix that dramatically changed over development, including the perinatal formation of fibrillar structures that appear to support the medullary rays. CONCLUSION: By correlating 3D ECM spatiotemporal organization with global protein abundance, we revealed novel changes in the interstitial matrix during kidney development. This new information regarding the ECM in developing kidneys offers the potential to inform the design of regenerative scaffolds that can guide nephrogenesis in vitro.

Comparative Analysis of the Extracellular Matrix Proteome across the Myotendinous Junction.

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.

Extracellular matrix protein composition dynamically changes during murine forelimb development.

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. Here, we quantified ECM protein dynamics in the murine forelimb during appendicular musculoskeletal morphogenesis (embryonic days 11.5-14.5) using tissue fractionation, bioorthogonal non-canonical amino acid tagging, and mass spectrometry. Our analyses indicated that ECM protein (matrisome) composition in the embryonic forelimb changed as a function of development and growth, was distinct from other developing organs (brain), and was altered in a model of disease (osteogenesis imperfecta murine). Additionally, the tissue distribution for select matrisome was assessed via immunohistochemistry in the wild-type embryonic and postnatal musculoskeletal system. This resource will guide future research investigating the role of the matrisome during complex tissue development.

Mechanical loading is required for initiation of extracellular matrix deposition at the developing murine myotendinous junction.

The myotendinous junction (MTJ) contributes to the generation of motion by connecting muscle to tendon. At the adult MTJ, a specialized extracellular matrix (ECM) is thought to contribute to the mechanical integrity of the muscle-tendon interface, but the factors that influence MTJ formation during mammalian development are unclear. Here, we combined 3D imaging and proteomics with murine models in which muscle contractility and patterning are disrupted to resolve morphological and compositional changes in the ECM during MTJ development. We found that MTJ-specific ECM deposition can be initiated via static loading due to growth; however, it required cyclic loading to develop a mature morphology. Furthermore, the MTJ can mature without the tendon terminating into cartilage. Based on these results, we describe a model wherein MTJ development depends on mechanical loading but not insertion into an enthesis.

An Integrative Biology Approach to Quantify the Biodistribution of Azidohomoalanine In Vivo.

Background: Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. We have previously demonstrated that labeling the in vivo murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion. Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Results: To address these gaps, we created a deterministic, compartmental model of the kinetic transport and incorporation of Aha in mice. Model results demonstrate the ability to predict Aha distribution and protein labeling in a variety of tissues and dosing paradigms. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing plasma and liver metabolomes following various Aha dosing regimens. We show that Aha administration induces minimal metabolic alterations in mice. Conclusions: Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We expect this model to be a useful tool to guide future experiments utilizing this technique to study proteomic responses to stimuli. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00760-4.

Metabolic labeling of secreted matrix to investigate cell-material interactions in tissue engineering and mechanobiology.

Re-creating features of the native extracellular matrix (ECM) with engineered biomaterials has become a valuable tool to probe the influence of ECM properties on cellular functions (e.g., differentiation) and toward the engineering of tissues. However, characterization of newly secreted (nascent) matrix and turnover, which are important in the context of cells interacting with these biomaterials, has been limited by a lack of tools. We developed a protocol to visualize and quantify the spatiotemporal evolution of newly synthesized and deposited matrix by cells that are either cultured atop (2D) or embedded within (3D) biomaterial systems (e.g., hydrogels, fibrous matrices). This technique relies on the incorporation of a noncanonical amino acid (azidohomoalanine) into proteins as they are synthesized. Deposited nascent ECM components are then visualized with fluorescent cyclooctynes via copper-free cycloaddition for spatiotemporal analysis or modified with cleavable biotin probes for identification. Here we describe the preparation of hyaluronic acid hydrogels through ultraviolet or visible light induced cross-linking for 2D and 3D cell culture, as well as the fluorescent labeling of nascent ECM deposited by cells during culture. We also provide protocols for secondary immunofluorescence of specific ECM components and ImageJ-based ECM quantification methods. Hyaluronic acid polymer synthesis takes 2 weeks to complete, and hydrogel formation for 2D or 3D cell culture is performed in 2-3 h. Lastly, we detail the identification of nascent proteins, including enrichment, preparation and analysis with mass spectrometry, which can be completed in 10 d.

Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse.

INTRODUCTION: Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative in vivo characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development. METHODS: Pregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC-MS/MS. For turnover studies, embryos were harvested 0-48 h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed via western blotting. RESULTS: We developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3 h and peak labeling was around 6 h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction. CONCLUSIONS: The methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.