Dedicated macrophages organize and maintain the enteric nervous system.

Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMϕ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMϕ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMϕ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-ß produced by the ENS; depletion of the ENS and disruption of transforming growth factor-ß signalling result in a decrease in neuron-associated MMϕ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell-cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.

Mechanics of ascending aortas from TGFß-1, -2, -3 haploinsufficient mice and elastase-induced aortopathy.

Transforming growth factor-beta (TGFß-1, -2, -3) ligands act through a common receptor complex yet each is expressed in a unique and overlapping fashion throughout development. TGFß plays a role in extra-cellular matrix composition with mutations to genes encoding TGFß and TGFß signaling molecules contributing to diverse and deadly thoracic aortopathies common in Loeys-Dietz syndrome (LDS). In this investigation, we studied the TGFß ligand-specific mechanical phenotype of ascending thoracic aortas (ATA) taken from 4-to-6 months-old Tgfb1+/-, Tgfb2+/-, and Tgfb3+/- mice, their wild-type (WT) controls, and an elastase infusion model representative of severe elastolysis. Heterozygous mice were studied at an age without dilation to elucidate potential pre-aortopathic mechanical cues. Our findings indicate that ATAs from Tgfb2+/- mice demonstrated significant wall thickening, a corresponding decrease in biaxial stress, decreased biaxial stiffness, and a decrease in stored energy. These results were unlike the pathological elastase model where decreases in biaxial stretch were found along with increases in diameter, biaxial stress, and biaxial stiffness. ATAs from Tgfb1+/- and Tgfb3+/-, on the other hand, had few mechanical differences when compared to wild-type controls. Although aortopathy generally occurs later in development, our findings reveal that in 4-to-6 month-old animals, only Tgfb2+/- mice demonstrate a significant phenotype that fails to model ubiquitous elastolysis.

Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGFß2.

In the bone marrow (BM) microenvironment, where breast cancer (BC) disseminated tumour cells (DTCs) can remain dormant for decades, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here, we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can also instruct BC DTCs to enter dormancy. NG2+/Nestin+ MSCs produce TGFß2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27 induction. Genetic depletion of MSCs or conditional knock-out of TGFß2 in MSCs using an NG2-CreER driver led to bone metastatic outgrowth of otherwise dormant p27+/Ki67- DTCs. Also ER+ BC patients without systemic recurrence displayed higher frequency of TGFß2 and BMP7 detection in the BM. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche homeostasis may result in a break from dormancy and BC bone relapse.

Transforming Growth Factor Beta3 is Required for Cardiovascular Development.

Transforming growth factor beta3 (TGFB3) gene mutations in patients of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD1) and Loeys-Dietz syndrome-5 (LDS5)/Rienhoff syndrome are associated with cardiomyopathy, cardiac arrhythmia, cardiac fibrosis, cleft palate, aortic aneurysms, and valvular heart disease. Although the developing heart of embryos express Tgfb3, its overarching role remains unclear in cardiovascular development and disease. We used histological, immunohistochemical, and molecular analyses of Tgfb3-/- fetuses and compared them to wildtype littermate controls. The cardiovascular phenotypes were diverse with approximately two thirds of the Tgfb3-/- fetuses having one or more cardiovascular malformations, including abnormal ventricular myocardium (particularly of the right ventricle), outflow tract septal and alignment defects, abnormal aortic and pulmonary trunk walls, and thickening of semilunar and/or atrioventricular valves. Ventricular septal defects (VSD) including the perimembranous VSDs were observed in Tgfb3-/- fetuses with myocardial defects often accompanied by the muscular type VSD. In vitro studies using TGFß3-deficient fibroblasts in 3-D collagen lattice formation assays indicated that TGFß3 was required for collagen matrix reorganization. Biochemical studies indicated the 'paradoxically' increased activation of canonical (SMAD-dependent) and noncanonical (MAP kinase-dependent) pathways. TGFß3 is required for cardiovascular development to maintain a balance of canonical and noncanonical TGFß signaling pathways.

TGF-ß2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism.

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-ß2 (TGF-ß2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-ß2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-ß2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2-/- mice, into sedentary mice improves glucose tolerance. TGF-ß2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-ß2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-ß2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-ß2-signaling cycle.

Effects of the isothiocyanate sulforaphane on TGF-ß1-induced rat cardiac fibroblast activation and extracellular matrix interactions.

An important step in many pathological conditions, particularly tissue and organ fibrosis, is the conversion of relatively quiescent cells into active myofibroblasts. These are highly specialized cells that participate in normal wound healing but also contribute to pathogenesis. These cells possess characteristics of smooth muscle cells and fibroblasts, have enhanced synthetic activity secreting abundant extracellular matrix components, cytokines, and growth factors, and are capable of generating contractile force. As such, these cells have become potential therapeutic targets in a number of disease settings. Transforming growth factor ß (TGF-ß) is a potent stimulus of fibrosis and myofibroblast formation and likewise is an important therapeutic target in several disease conditions. The plant-derived isothiocyanate sulforaphane has been shown to have protective effects in several pathological models including diabetic cardiomyopathy, carcinogenesis, and fibrosis. These studies suggest that sulforaphane may be an attractive preventive agent against disease progression, particularly in conditions involving alterations of the extracellular matrix and activation of myofibroblasts. However, few studies have evaluated the effects of sulforaphane on cardiac fibroblast activation and their interactions with the extracellular matrix. The present studies were carried out to determine the potential effects of sulforaphane on the conversion of quiescent cardiac fibroblasts to an activated myofibroblast phenotype and associated alterations in signaling, expression of extracellular matrix receptors, and cellular physiology following stimulation with TGF-ß1. These studies demonstrate that sulforaphane attenuates TGF-ß1-induced myofibroblast formation and contractile activity. Sulforaphane also reduces expression of collagen-binding integrins and inhibits canonical and noncanonical TGF-ß signaling pathways.

Tak1, Smad4 and Trim33 redundantly mediate TGF-ß3 signaling during palate development.

Transforming growth factor-beta3 (TGF-ß3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-ß3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-ß activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-ß signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-ß signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-ß signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.

Generation of mice carrying a knockout-first and conditional-ready allele of transforming growth factor beta2 gene.

Transforming growth factor beta2 (TGFß2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFß2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell-matrix remodeling, epithelial-mesenchymal transition, and wound healing in a highly context-dependent and tissue-specific manner. Tgfb2(-/-) mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2(ßgeo) (knockout-first lacZ-tagged insertion) gene-trap allele and Tgfb2(flox) conditional allele. Tgfb2(ßgeo/ßgeo) or Tgfb2(ßgeo/-) mice died at perinatal stage from the same congenital heart defects as Tgfb2(-/-) mice. ß-galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2(ßgeo) fetal tissue sections. Tgfb2(flox) mice were produced by crossing the Tgfb2(+/ßgeo) mice with the FLPeR mice. Tgfb2(flox/-) mice were viable. Tgfb2 conditional knockout (Tgfb2(cko/-) ) fetuses were generated by crossing of Tgfb2(flox/-) mice with Tgfb2(+/-) ; EIIaCre mice. Systemic Tgfb2(cko/-) embryos developed cardiac defects which resembled the Tgfb2(ßgeo/ßgeo) , Tgfb2(ßgeo/-) , and Tgfb2(-/-) fetuses. In conclusion, Tgfb2(ßgeo) and Tgfb2(flox) mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFß2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis

Tgfß-Smad and MAPK signaling mediate scleraxis and proteoglycan expression in heart valves.

Mature heart valves are complex structures consisting of three highly organized extracellular matrix layers primarily composed of collagens, proteoglycans and elastin. Collectively, these diverse matrix components provide all the necessary biomechanical properties for valve function throughout life. In contrast to healthy valves, myxomatous valve disease is the most common cause of mitral valve prolapse in the human population and is characterized by an abnormal abundance of proteoglycans within the valve tri-laminar structure. Despite the clinical significance, the etiology of this phenotype is not known. Scleraxis (Scx) is a basic-helix-loop-helix transcription factor that we previously showed to be required for establishing heart valve structure during remodeling stages of valvulogenesis. In this study, we report that remodeling heart valves from Scx null mice express decreased levels of proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), while overexpression in embryonic avian valve precursor cells and adult porcine valve interstitial cells increases CSPGs. Using these systems we further identify that Scx is positively regulated by canonical Tgfß2 signaling during this process and this is attenuated by MAPK activity. Finally, we show that Scx is increased in myxomatous valves from human patients and mouse models, and overexpression in human mitral valve interstitial cells modestly increases proteoglycan expression consistent with myxomatous mitral valve phenotypes. Together, these studies identify an important role for Scx in regulating proteoglycans in embryonic and mature valve cells and suggest that imbalanced regulation could influence myxomatous pathogenesis.

Hyperactive transforming growth factor-ß1 signaling potentiates skeletal defects in a neurofibromatosis type 1 mouse model.

Dysregulated transforming growth factor beta (TGF-ß) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-ß1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-ß1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-ß1 in bone, overexpress TGF-ß1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-ß1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-ß1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-ß1 expression levels and reduced Smad phosphorylation in response to TGF-ß1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-ß receptor 1 (TßRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-ß1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-ß signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.

Transforming growth factor Beta2 is required for valve remodeling during heart development.

Although the function of transforming growth factor beta2 (TGFß2) in epithelial mesenchymal transition (EMT) is well studied, its role in valve remodeling remains to be fully explored. Here, we used histological, morphometric, immunohistochemical and molecular approaches and showed that significant dysregulation of major extracellular matrix (ECM) components contributed to valve remodeling defects in Tgfb2(-/-) embryos. The data indicated that cushion mesenchymal cell differentiation was impaired in Tgfb2(-/-) embryos. Hyaluronan and cartilage link protein-1 (CRTL1) were increased in hyperplastic valves of Tgfb2(-/-) embryos, indicating increased expansion and diversification of cushion mesenchyme into the cartilage cell lineage during heart development. Finally, Western blot and immunohistochemistry analyses indicate that the activation of SMAD2/3 was decreased in Tgfb2(-/-) embryos during valve remodeling. Collectively, the data indicate that TGFß2 promotes valve remodeling and differentiation by inducing matrix organization and suppressing cushion mesenchyme differentiation into cartilage cell lineage during heart development.

Origin of cardiac fibroblasts and the role of periostin.

Cardiac fibroblasts are the most populous nonmyocyte cell type within the mature heart and are required for extracellular matrix synthesis and deposition, generation of the cardiac skeleton, and to electrically insulate the atria from the ventricles. Significantly, cardiac fibroblasts have also been shown to play an important role in cardiomyocyte growth and expansion of the ventricular chambers during heart development. Although there are currently no cardiac fibroblast-restricted molecular markers, it is generally envisaged that the majority of the cardiac fibroblasts are derived from the proepicardium via epithelial-to-mesenchymal transformation. However, still relatively little is known about when and where the cardiac fibroblasts cells are generated, the lineage of each cell, and how cardiac fibroblasts move to reside in their final position throughout all four cardiac chambers. In this review, we summarize the present understanding regarding the function of Periostin, a useful marker of the noncardiomyocyte lineages, and its role during cardiac morphogenesis. Characterization of the cardiac fibroblast lineage and identification of the signals that maintain, expand and regulate their differentiation will be required to improve our understanding of cardiac function in both normal and pathophysiological states.

Generation of mice with a conditional allele for transforming growth factor beta 1 gene.

Transforming growth factor beta1 (TGFbeta1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFbeta1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity.

Ligand-specific function of transforming growth factor beta in epithelial-mesenchymal transition in heart development.

The ligand specificity of transforming growth factor beta (TGFbeta) in vivo in mouse cardiac cushion epithelial-to-mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFbeta in cushion EMT, we analyzed Tgfb1(-/-), Tgfb2(-/-), and Tgfb3(-/-) mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1(-/-) and Tgfb3(-/-) mice. However, analysis of Tgfb2(-/-) AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2(-/-) embryos, and not Tgfb1(-/-) or Tgfb3(-/-) embryos, develop enlarged cushions at E14.5 with elevated levels of well-validated indicators of EMT. Collectively, these data indicate that TGFbeta2, and not TGFbeta1 or TGFbeta3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT.

Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor-2 in cardiovascular development and disease.

Fibroblast growth factor 2 (FGF2) consists of multiple protein isoforms (low molecular weight, LMW, and high molecular weight, HMW) produced by alternative translation from the Fgf2 gene. These protein isoforms are localized to different cellular compartments, indicating unique biological activity. FGF2 isoforms in the heart have distinct roles in many pathological circumstances in the heart including cardiac hypertrophy, ischemia-reperfusion injury, and atherosclerosis. These studies suggest distinct biological activities of FGF2 LMW and HMW isoforms both in vitro and in vivo. Yet, due to the limitations that only the recombinant FGF2 LMW isoform is readily available and that the FGF2 antibody is nonspecific with regards to its isoforms, much remains to be determined regarding the role(s) of the FGF2 LMW and HMW isoforms in cellular behavior and in cardiovascular development and pathophysiology. This review summarizes the activities of LMW and HMW isoforms of FGF2 in cardiovascular development and disease.

Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes.

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

Transforming growth factor beta-SMAD2 signaling and aortic arch development.

Molecular processes underlying the remodeling of the symmetric system and the already muscularized pharyngeal arch arteries into an asymmetric aortic arch are slowly becoming unraveled. In normal arch remodeling, selective apoptosis of part of the right dorsal aorta and sixth pharyngeal arch is seen, whereas in the common arch malformations in Tgfbeta2 knockout mice, comprising type B interruption and an aberrant right subclavian artery, selective upregulation of apoptosis is additionally found in the left and/or right fourth artery segment. All pharyngeal arch arteries derive transforming growth factor beta2-expressing smooth muscle cells from the neural crest. The marked high vulnerability of specifically the fourth arch arteries can be linked to a localized reduced vascular SMAD2 signaling and related expression of fibronectin and neural cell adhesion molecule, providing a link between disturbed arteriogenesis and innervation. The marked correlation between intracardiac and aortic arch malformations in Tgfbeta2 mutants can also be understood as a combination of neural crest and flow-related mechanisms. A relation between apoptosis and flow is postulated, in which a role for genes with a shear stress-responsive element, including the endothelially expressed transforming growth factor beta1, is implied.

A signaling pathway involving TGF-beta2 and snail in hair follicle morphogenesis.

In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor beta2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis, Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.

Transforming growth factor beta-SMAD2 signaling regulates aortic arch innervation and development.

Aortic arch interruptions in humans and animal models are mainly caused by aberrant development of the fourth pharyngeal arch artery. Little is known about the maturation of this vessel during normal and abnormal development, which is the subject of this study. Tgfbeta2 knockout mice that present with fourth artery defects have been associated with defective neural crest cell migration. In this study, we concentrated on pharyngeal arch artery development during developmental days 12.5 to 18.5, focusing on neural crest cell migration using a Wnt1-Cre by R26R neural crest cell reporter mouse. Fourth arch artery maturation was studied with antibodies directed against smooth muscle alpha-actin and neural NCAM-1 and RMO-270. For diminished transforming growth factor beta (TGF-beta) signaling, SMAD2 and fibronectin have been analyzed. Neural crest migration and differentiation into smooth muscle cells is unaltered in mutants, regardless of the cardiovascular defect found; however, innervation of the fourth arch artery is affected. Absent staining for nuclear SMAD2, NCAM-1, and RMO-270 in the fourth artery in mutant coincides with severe defects of this segment. Likewise, fibronectin expression is diminished in these cases. From these data we conclude the following: (1) neural crest cell migration is not a common denominator in cardiovascular defects of Tgfbeta2-/- mice; (2) fourth arch artery maturation is a complex process involving innervation; and (3) TGF-beta2 depletion diminishes SMAD2-signaling in the fourth arch artery and coincides with reduced vascular NCAM-1 expression and neural innervation of this artery. We hypothesize that disturbed maturation of the fourth pharyngeal arch artery, and especially abrogated vascular innervation, will result in fourth arch interruptions.