RESUMEN
Dysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes. Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSALR) in comparison to wild type. Our integrative proteogenomics approach comprised gene- and splicing-level assessments for mRNAs and proteins. It recapitulated many known instances of aberrant mRNA splicing in DM1 and identified new ones. It enabled the design and targeting of splicing-specific peptides and confirmed the translation of known instances of aberrantly spliced disease-related genes (e.g., Atp2a1, Bin1, Ryr1), complemented by novel findings (Flnc and Ywhae). Comparative analysis of large-scale mRNA and protein expression data showed quantitative agreement of differentially expressed genes and splicing patterns between disease and wild type. We hence propose this work as a suitable blueprint for a robust and scalable integrative proteogenomic strategy geared toward advancing our understanding of splicing-based disorders. With such a strategy, splicing-based biomarker candidates emerge as an attractive and accessible option, as they can be efficiently asserted on the mRNA and protein level in coordinated fashion.
Asunto(s)
Distrofia Miotónica , Proteogenómica , Ratones , Animales , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología , Empalme Alternativo/genética , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.
Asunto(s)
Envejecimiento/metabolismo , Empalme Alternativo , Músculo Esquelético/metabolismo , Distrofia Miotónica/metabolismo , Receptores de Superficie Celular/biosíntesis , Sarcopenia/metabolismo , Envejecimiento/patología , Animales , Senescencia Celular , Modelos Animales de Enfermedad , Masculino , Músculo Esquelético/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Phenotypic compound screens can be used to identify novel targets in signaling pathways and disease processes, but the usefulness of these screens depends on the ability to quickly determine the target and mechanism of action of the molecules identified as hits. One fast route to discovering the mechanism of action of a compound is to profile its properties and to match this profile with those of compounds of known mechanism of action. In this work, the Novartis collection of over 12,000 pure natural products was screened for effects on early zebrafish development. The largest phenotypic class of hits, which caused developmental arrest without necrosis, contained known electron transport chain inhibitors and many compounds of unknown mechanism of action. High-throughput transcriptional profiling revealed that these compounds are mechanistically related to one another. Metabolic and biochemical assays confirmed that all of the molecules that induced developmental arrest without necrosis inhibited the electron transport chain. These experiments demonstrate that the electron transport chain is the target of the natural products manassantin, sesquicillin, and arctigenin. The overlap between the zebrafish and transcriptional profiling screens was not perfect, indicating that multiple profiling screens are necessary to fully characterize molecules of unknown function. Together, zebrafish screening and transcriptional profiling represent sensitive and scalable approaches for identifying bioactive compounds and elucidating their mechanism of action.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/efectos de los fármacos , Furanos/farmacología , Lignanos/farmacología , Membranas Mitocondriales/efectos de los fármacos , Naftalenos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Furanos/química , Perfilación de la Expresión Génica , Lignanos/química , Estructura Molecular , Naftalenos/química , Pez CebraRESUMEN
R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/ß-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.
Asunto(s)
Receptores Wnt/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Polaridad Celular/fisiología , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retroalimentación Fisiológica , Femenino , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Vía de Señalización Wnt , Xenopus , Pez Cebra , beta Catenina/metabolismoRESUMEN
The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.
Asunto(s)
Músculo Esquelético/metabolismo , Miocardio/metabolismo , Isoformas de Proteínas/metabolismo , Troponina C/metabolismo , Animales , Hibridación in Situ , Microscopía Electrónica de Transmisión , Músculo Esquelético/ultraestructura , Miocardio/ultraestructura , Isoformas de Proteínas/genética , Troponina C/genética , Pez CebraRESUMEN
The molecular pathways by which newly formed, immature endothelial cell tubes remodel to form a mature network of vessels supported by perivascular mural cells are not well understood. The zebrafish iguana (igu) genetic mutant has a mutation in the daz-interacting protein 1 (dzip1), a member of the hedgehog signaling pathway. Loss of dzip1 results in decreased size of the cranial dorsal aortae, ultrastructural defects in perivascular mural cell recruitment and subsequent hemorrhage. Although hedgehog signaling is disrupted in igu mutants, we find no defects in vessel patterning or artery-vein specification. Rather, we show that the loss of angiopoietin1 (angpt1) expression in ventral perivascular mesenchyme is responsible for vascular instability in igu mutants. Over-expression of angpt1 or partial down-regulation of the endogenous Angpt1 antagonist angpt2 rescues hemorrhage. This is the first direct in vivo link between hedgehog signaling and the induction of vascular stability by recruitment of perivascular mural cells through angiopoietin signaling.
Asunto(s)
Angiopoyetina 1/fisiología , Vasos Sanguíneos/embriología , Proteínas Hedgehog/metabolismo , Transducción de Señal , Pez Cebra/embriología , Animales , Hibridación in Situ , Microscopía Confocal , Microscopía ElectrónicaRESUMEN
Cilia defects have been implicated in a variety of human diseases and genetic disorders, but how cilia motility contributes to these phenotypes is still unknown. To further our understanding of how cilia function in development, we have cloned and characterized two alleles of seahorse, a zebrafish mutation that results in pronephric cysts. seahorse encodes Lrrc6l, a leucine-rich repeat-containing protein that is highly conserved in organisms that have motile cilia. seahorse is expressed in zebrafish tissues known to contain motile cilia. Although mutants do not affect cilia structure and retain the ability to interact with Disheveled, both alleles of seahorse strongly affect cilia motility in the zebrafish pronephros and neural tube. Intriguingly, although seahorse mutations variably affect fluid flow in Kupffer's vesicle, they can have very weak effects on left-right patterning. Combined with recently published results, our alleles suggest that the function of seahorse in cilia motility is separable from its function in other cilia-related phenotypes.
Asunto(s)
Tubo Neural/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/fisiología , Cilios/fisiología , Datos de Secuencia Molecular , Mutación , Tubo Neural/fisiología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Myelin, the isolating sheath around large diameter axons, is formed in the central nervous system (CNS) by oligodendrocytes. We isolated the zebrafish ortholog of olig1, a bHLH transcription factor, and describe the origin and development of oligodendrocytes in the zebrafish brain. Olig1:mem-eGFP transgenic animals demonstrate the highly dynamic nature of oligodendrocyte membrane processes, providing a tool for studying in vivo oligodendrocyte development. Formation of oligodendrocytes and initiation of olig1 expression are under the control of long-range hedgehog and notch signaling while maintenance of olig1 expression only depends on hedgehog. Over-expression of olig1 did not affect myelin formation in the brain and combined over-expression of olig1 and olig2 could not rescue loss of hedgehog signaling, indicating that critical factors other than olig1 and olig2 are necessary. Lastly, knockdown of Olig1 in an Olig2-sensitized background did result in defects in CNS myelination, indicating a functional overlap between Olig1 and Olig2 proteins.
Asunto(s)
Proteínas Hedgehog/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Receptores Notch/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular , Secuencia Conservada , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas/genética , Alineación de Secuencia , Transducción de Señal , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
The Bone morphogenetic proteins (BMPs) act in many key regulatory processes during development, including dorsoventral axis specification and organ development and are part of a conserved signal pathway. Specifically, BMP7 is a vital signaling molecule for normal development in the mammalian system. The zebrafish mutant snailhouse (snh) was originally isolated as being strongly dorsalized and the mutation was determined to lie within the bmp7 gene. We report here the cloning and expression of a second bmp7 homolog, which we term bmp7b. Sequence alignments show that bmp7b is more closely related to human, mouse and non-mammalian BMP7 than is snh. We further show that bmp7b is strongly expressed in developing organ systems such as the eyes, the ears, the pronephric kidney and the gastrointestinal system.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Clonación Molecular , Sistema Digestivo/embriología , Sistema Digestivo/metabolismo , Oído/embriología , Ojo/embriología , Ojo/metabolismo , Riñón/embriología , Riñón/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
The epicardium is the last layer of the vertebrate heart to form, surrounding the heart muscle during embryogenesis and providing signaling cues essential to the continued growth and differentiation of the heart. This outer layer of the heart develops from a transient structure, the proepicardial organ (PEO). Despite its essential roles, the early signals required for the formation of the PEO and the epicardium remain poorly understood. The molecular markers wt1 and tcf21 are used to identify the epicardial layer in the zebrafish heart, to trace its development and to determine genes required for its normal development. Disruption of lateral plate mesoderm (LPM) migration through knockdown of miles apart or casanova leads to cardia bifida with each bilateral heart associated with its own PEO, suggesting that the earliest progenitors of the epicardium lie in the LPM. Using a gene knockdown approach, a genetic framework for PEO development is outlined. The pandora/spt6 gene is required for multiple cardiac lineages, the zinc-finger transcription factor wt1 is required for the epicardial lineage only and finally, the cell polarity genes heart and soul and nagie oko are required for proper PEO morphogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Organogénesis , Pericardio/embriología , Proteínas de Pez Cebra/metabolismo , Animales , Biomarcadores , Linaje de la Célula , Polaridad Celular/genética , ADN Complementario , Embrión no Mamífero , Femenino , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microinyecciones , Modelos Biológicos , Mutación , Miocardio/metabolismo , Oligonucleótidos Antisentido/farmacología , Pericardio/citología , Pericardio/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Factores de Transcripción SOX , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knock-down of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation.
Asunto(s)
Cilios/fisiología , Mutación , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Animales , Clonación Molecular , Embrión no Mamífero/fisiología , Glomérulos Renales/fisiología , Túbulos Renales/fisiología , Microscopía por Video , Mutagénesis , Nefronas/embriología , Nefronas/fisiología , Nefronas/fisiopatología , Fenotipo , Pez Cebra/genéticaRESUMEN
The vasculature tailors to the needs of different tissues and organs. Molecular, structural, and functional specializations are observed in different vascular beds, but few genetic models give insight into how these differences arise. We identify a unique cerebrovascular mutation in the zebrafish affecting the integrity of blood vessels supplying the brain. The zebrafish bubblehead (bbh) mutant exhibits hydrocephalus and severe cranial hemorrhage during early embryogenesis, whereas blood vessels in other regions of the embryo appear intact. Here we show that hemorrhages are associated with poor cerebral endothelial-mesenchymal contacts and an immature vascular pattern in the head. Positional cloning of bbh reveals a hypomorphic mutation in betaPix, a binding partner for the p21-activated kinase (Pak) and a guanine nucleotide exchange factor for Rac and Cdc42. betaPix is broadly expressed during embryonic development and is enriched in the brain and in large blood vessels. By knockdown of specific betaPix splice variants, we show that they play unique roles in embryonic vascular stabilization or hydrocephalus. Finally, we show that Pak2a signaling is downstream of betaPix. These data identify an essential in vivo role for betaPix and Pak2a during embryonic development and illuminate a previously unrecognized pathway specifically involved in cerebrovascular stabilization.
Asunto(s)
Proteínas de Ciclo Celular/genética , Circulación Cerebrovascular/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Pez Cebra/fisiología , Empalme Alternativo , Animales , Vasos Sanguíneos/fisiología , Encéfalo/patología , Hemorragia Cerebral/genética , Mapeo Cromosómico , Clonación Molecular , Endotelio Vascular/fisiología , Etilnitrosourea , Exones , Variación Genética , Factores de Intercambio de Guanina Nucleótido/deficiencia , Datos de Secuencia Molecular , Factores de Intercambio de Guanina Nucleótido Rho , Pez Cebra , Proteínas de Pez Cebra/genética , Quinasas p21 ActivadasRESUMEN
During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step, demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of morpholino pairs indicate that san, vtn and heg are in a genetic pathway, and san and vtn contain protein motifs, NPxY and PTB domain, respectively, known to interact. This suggests that concentric growth of the myocardium, crucial for blood pressure generation, is dictated by a heg-san-vtn signaling pathway.
Asunto(s)
Cardiopatías Congénitas/embriología , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Recuento de Células , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Endocardio/citología , Endocardio/embriología , Regulación de la Expresión Génica , Genes Letales , Corazón/embriología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/genética , Mutación , Miocardio/citología , Miocardio/metabolismo , ARN Mensajero/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central ;slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.
Asunto(s)
Corazón/embriología , Miocardio/metabolismo , Neurregulinas/metabolismo , Receptor Notch1/metabolismo , Animales , Endotelina-1/genética , Endotelina-1/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neurregulinas/genética , Especificidad de Órganos , Receptor Notch1/genética , Circulación Renal , Transducción de Señal , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoAsunto(s)
Neovascularización Fisiológica/fisiología , Pez Cebra/embriología , Fosfatasa Alcalina/análisis , Inhibidores de la Angiogénesis/farmacología , Animales , Animales Modificados Genéticamente , Evaluación Preclínica de Medicamentos/métodos , Endotelio Vascular/química , Endotelio Vascular/embriología , Endotelio Vascular/crecimiento & desarrollo , Angiografía con Fluoresceína/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Coloración y Etiquetado/métodos , Factor A de Crecimiento Endotelial Vascular/fisiología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
The local presence of endothelial cells seems necessary for proper embryonic development of several organs. However, the signals involved are unknown. The glomerulus is generated by the coalescence of podocytes around an ingrowing capillary and is the site of blood ultrafiltration. In the absence of vessels, glomerular assembly does not occur. We describe mutations in the zebrafish that prevent glomerulogenesis. All mutants display cardiac dysfunction. Pharmacological interference with cardiac output and focal laser occlusion of the vessel similarly prevent glomerular formation. The unifying feature of all these perturbations is absence of blood flow. We find that expression of matrix metalloproteinase-2 (MMP-2), known in other systems to be regulated in a stretch-responsive manner, is in renal endothelial cells and is regulated by flow, suggesting that an MMP-2-sensitive event may be downstream of the flow-related signal. In support of this, blockade of MMP-2 activity by injection of TIMP-2 does not perturb circulation but does prevent glomerular assembly. Thus, vascular flow is required for glomerular assembly, most probably acting via a stretch-responsive signaling system in the vessel wall.