Polarized transport of membrane and secreted proteins during lumen morphogenesis.

A ubiquitous feature of animal development is the formation of fluid-filled cavities or lumina, which transport gases and fluids across tissues and organs. Among different species, lumina vary drastically in size, scale, and complexity. However, all lumen formation processes share key morphogenetic principles that underly their development. Fundamentally, a lumen simply consists of epithelial cells that encapsulate a continuous internal space, and a common way of building a lumen is via opening and enlarging by filling it with fluid and/or macromolecules. Here, we discuss how polarized targeting of membrane and secreted proteins regulates lumen formation, mainly focusing on ion transporters in vertebrate model systems. We also discuss mechanistic differences observed among invertebrates and vertebrates and describe how the unique properties of the Na+/K+-ATPase and junctional proteins can promote polarization of immature epithelia to build lumina de novo in developing organs.

Knock-in tagging in zebrafish facilitated by insertion into non-coding regions.

Zebrafish provide an excellent model for in vivo cell biology studies because of their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers because of inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes that are crucial for epithelial biology and organ development, including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC and the ECM receptor Integrin ß1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

Zebrafish Erc1b mediates motor innervation and organization of craniofacial muscles in control of jaw movement.

BACKGROUND: Movement of the lower jaw, a common behavior observed among vertebrates, is required for eating and processing food. This movement is controlled by signals sent from the trigeminal motor nerve through neuromuscular junctions (NMJs) to the masticatory muscles. Dysfunctional jaw movements contribute to craniomandibular disorders, yet the pathophysiology of these disorders is not well understood, as limited studies have been conducted on the molecular mechanisms of jaw movement. RESULTS: Using erc1b/kimm533 genetic loss of function mutant, we evaluated lower jaw muscle organization and innervation by the cranial motor nerves in developing zebrafish. Using time-lapse confocal imaging of the erc1b mutant in a transgenic fluorescent reporter line, we found delayed trigeminal nerve growth and disrupted nerve branching architecture during muscle innervation. By automated 3D image analysis of NMJ distribution, we identified an increased number of small, disorganized NMJ clusters in erc1b mutant larvae compared to WT siblings. Using genetic replacement experiments, we determined the Rab GTPase binding domain of Erc1b is required for cranial motor nerve branching, but not NMJ organization or muscle attachment. CONCLUSIONS: We identified Erc1b/ERC1 as a novel component of a genetic pathway contributing to muscle organization, trigeminal nerve outgrowth, and NMJ spatial distribution during development that is required for jaw movement.

GRIK5 Genetically Regulated Expression Associated with Eye and Vascular Phenomes: Discovery through Iteration among Biobanks, Electronic Health Records, and Zebrafish.

Although the use of model systems for studying the mechanism of mutations that have a large effect is common, we highlight here the ways that zebrafish-model-system studies of a gene, GRIK5, that contributes to the polygenic liability to develop eye diseases have helped to illuminate a mechanism that implicates vascular biology in eye disease. A gene-expression prediction derived from a reference transcriptome panel applied to BioVU, a large electronic health record (EHR)-linked biobank at Vanderbilt University Medical Center, implicated reduced GRIK5 expression in diverse eye diseases. We tested the function of GRIK5 by depletion of its ortholog in zebrafish, and we observed reduced blood vessel numbers and integrity in the eye and increased vascular permeability. Analyses of EHRs in >2.6 million Vanderbilt subjects revealed significant comorbidity of eye and vascular diseases (relative risks 2-15); this comorbidity was confirmed in 150 million individuals from a large insurance claims dataset. Subsequent studies in >60,000 genotyped BioVU participants confirmed the association of reduced genetically predicted expression of GRIK5 with comorbid vascular and eye diseases. Our studies pioneer an approach that allows a rapid iteration of the discovery of gene-phenotype relationships to the primary genetic mechanism contributing to the pathophysiology of human disease. Our findings also add dimension to the understanding of the biology driven by glutamate receptors such as GRIK5 (also referred to as GLUK5 in protein form) and to mechanisms contributing to human eye diseases.

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts.

Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.

Rediscovering the Rete Ovarii: a secreting auxiliary structure to the ovary.

The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray's Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.

Notochord segmentation in zebrafish controlled by iterative mechanical signaling.

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning.

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2.

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aorta's (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces.

TNF Promoter Hypomethylation Is Associated With Mucosal Inflammation in IBD and Anti-TNF Response.

Background and Aims: Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the tumor necrosis factor (TNF) promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and nonresponders. Methods: We obtained mucosal biopsies from 200 participants (133 IBDs and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 nonresponders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated intestinal epithelial cells from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF nonresponders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed 2 missense variants in DNA methyltransferase 1, 1 of which had reduced function in vivo. Conclusion: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

TNF promoter hypomethylation is associated with mucosal inflammation in IBD and anti-TNF response.

Background and aims: Inflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. Methods: We obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. Conclusions: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

Self-organization of apical membrane protein sorting in epithelial cells.

Polarized epithelial cells are characterized by the asymmetric distribution of proteins between apical and basolateral domains of the plasma membrane. This asymmetry is highly conserved and is fundamental to epithelial cell physiology, development, and homeostasis. How proteins are segregated for apical or basolateral delivery, a process known as sorting, has been the subject of considerable investigation for decades. Despite these efforts, the rules guiding apical sorting are poorly understood and remain controversial. Here, we consider mechanisms of apical membrane protein sorting and argue that they are largely driven by self-organization and biophysical principles. The preponderance of data to date is consistent with the idea that apical sorting is not ruled by a dedicated protein-based sorting machinery and relies instead on the concerted effects of oligomerization, phase separation of lipids and proteins in membranes, and pH-dependent glycan interactions.

Distinct roles for luminal acidification in apical protein sorting and trafficking in zebrafish.

Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis.

Lysosome-Rich Enterocytes Mediate Protein Absorption in the Vertebrate Gut.

The guts of neonatal mammals and stomachless fish have a limited capacity for luminal protein digestion, which allows oral acquisition of antibodies and antigens. However, how dietary protein is absorbed during critical developmental stages when the gut is still immature is unknown. Here, we show that specialized intestinal cells, which we call lysosome-rich enterocytes (LREs), internalize dietary protein via receptor-mediated and fluid-phase endocytosis for intracellular digestion and trans-cellular transport. In LREs, we identify a conserved endocytic machinery, composed of the scavenger receptor complex Cubilin/Amnionless and Dab2, that is required for protein uptake by LREs and for growth and survival of larval zebrafish. Moreover, impairing LRE function in suckling mice, via conditional deletion of Dab2, leads to stunted growth and severe protein malnutrition reminiscent of kwashiorkor, a devastating human malnutrition syndrome. These findings identify digestive functions and conserved molecular mechanisms in LREs that are crucial for vertebrate growth and survival.

Sheath Cell Invasion and Trans-differentiation Repair Mechanical Damage Caused by Loss of Caveolae in the Zebrafish Notochord.

The notochord, a conserved axial structure required for embryonic axis elongation and spine development, consists of giant vacuolated cells surrounded by an epithelial sheath [1-3]. During morphogenesis, vacuolated cells maintain their structural integrity despite being under constant mechanical stress [4]. We hypothesized that the high density of caveolae present in vacuolated cells [5, 6] could buffer mechanical tension. Caveolae are 50- to 80-nm membrane invaginations lined by cage-like polygonal structures [7, 8] formed by caveolin 1 (Cav1) or Cav3 and one of the cavin proteins [6, 9-11]. Recent in vitro work has shown that plasma membrane caveolae constitute a membrane reservoir that can buffer mechanical stresses such as stretching or osmotic swelling [12]. Moreover, mechanical integrity of vascular and muscle cells is partly dependent on caveolae [13-15]. However, the in vivo mechano-protective roles of caveolae have only begun to be explored. Using zebrafish mutants for cav1, cav3, and cavin1b, we show that caveolae are essential for notochord integrity. Upon loss of caveola function, vacuolated cells collapse at discrete positions under the mechanical strain of locomotion. Then, sheath cells invade the inner notochord and differentiate into vacuolated cells, thereby restoring notochord function and allowing normal spine development. Our data further indicate that nucleotides released by dying vacuolated cells promote sheath cell vacuolization and trans-differentiation. This work reveals a novel structural role for caveolae in vertebrates and provides unique insights into the mechanisms that safeguard notochord and spine development.

Animal model of Sar1b deficiency presents lipid absorption deficits similar to Anderson disease.

Anderson disease (ANDD) or chylomicron retention disease (CMRD) is a rare, hereditary lipid malabsorption syndrome associated with mutations in the SAR1B gene that is characterized by failure to thrive and hypocholesterolemia. Although the SAR1B structure has been resolved and its role in formation of coat protein II (COPII)-coated carriers is well established, little is known about the requirement for SAR1B during embryogenesis. To address this question, we have developed a zebrafish model of Sar1b deficiency based on antisense oligonucleotide knockdown. We show that zebrafish sar1b is highly conserved among vertebrates; broadly expressed during development; and enriched in the digestive tract organs, brain, and craniofacial skeleton. Consistent with ANDD symptoms of chylomicron retention, we found that dietary lipids in Sar1b-deficient embryos accumulate in enterocytes. Transgenic expression analysis revealed that Sar1b is required for growth of exocrine pancreas and liver. Furthermore, we found abnormal differentiation and maturation of craniofacial cartilage associated with defects in procollagen II secretion and absence of select, neuroD-positive neurons of the midbrain and hindbrain. The model presented here will help to systematically dissect developmental roles of Sar1b and to discover molecular and cellular mechanisms leading to organ-specific ANDD pathology. Key messages: Sar1b depletion phenotype in zebrafish resembles Anderson disease deficits. Sar1b deficiency results in multi-organ developmental deficits. Sar1b is required for dietary cholesterol uptake into enterocytes.

Trafficking mechanisms of extracellular matrix macromolecules: insights from vertebrate development and human diseases.

Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates.

The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development.

ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)-mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1's oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization.

The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrix proteins in skeletal morphogenesis.

Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM), yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER) similar to those found in coat protein II complex (COPII)-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d) but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated ECM deposition, such as arthritis, fibrosis or osteoporosis.