A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

Diverse contribution of amniogenic somatopleural cells to cardiovascular development: With special reference to thyroid vasculature.

BACKGROUND: The somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra-embryonic ASCs (hereafter referred to as iASCs) remain largely unknown. RESULTS: By quail-chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra-thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single-cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast-like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression. CONCLUSION: The present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives.

Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development.

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1+ lineage-specific deletion of Prox1 resulted in disrupted LYVE1+ vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3+ and/or Prox1+ cells that overlapped with the Isl1+ pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis.

Postotic and preotic cranial neural crest cells differently contribute to thyroid development.

Thyroid development and formation vary among species, but in most species the thyroid morphogenesis consists of five stages: specification, budding, descent, bilobation and folliculogenesis. The detailed mechanisms of these stages have not been fully clarified. During early development, the cranial neural crest (CNC) contributes to the thyroid gland. The removal of the postotic CNC (corresponding to rhombomeres 6, 7 and 8, also known as the cardiac neural crest) results in abnormalities of the cardiovascular system, thymus, parathyroid glands, and thyroid gland. To investigate the influence of the CNC on thyroid bilobation process, we divided the CNC into two regions, the postotic CNC and the preotic CNC (from the mesencephalon to rhombomere 5) regions and examined. We found that preotic CNC-ablated embryos had a unilateral thyroid lobe, and confirmed the presence of a single lobe or the absence of lobes in postotic CNC-ablated chick embryos. The thyroid anlage in each region-ablated embryos was of a normal size at the descent stage, but at a later stage, the thyroid in preotic CNC-ablated embryos was of a normal size, conflicting with a previous report in which the thyroid was reduced in size in the postotic CNC-ablated embryos. The postotic CNC cells differentiated into connective tissues of the thyroid in quail-to-chick chimeras. In contrast, the preotic CNC cells did not differentiate into connective tissues of the thyroid. We found that preotic CNC cells encompassed the thyroid anlage from the specification stage to the descent stage. Finally, we found that endothelin-1 and endothelin type A receptor-knockout mice and bosentan (endothelin receptor antagonist)-treated chick embryos showed bilobation anomalies that included single-lobe formation. Therefore, not only the postotic CNC, but also the preotic CNC plays an important role in thyroid morphogenesis.

Adult mice expressing a Braf Q241R mutation on an ICR/CD-1 background exhibit a cardio-facio-cutaneous syndrome phenotype.

Activation of the RAS pathway has been implicated in oncogenesis and developmental disorders called RASopathies. Germline mutations in BRAF have been identified in 50-75% of patients with cardio-facio-cutaneous (CFC) syndrome, which is characterized by congenital heart defects, distinctive facial features, short stature and ectodermal abnormalities. We recently demonstrated that mice expressing a Braf Q241R mutation, which corresponds to the most frequent BRAF mutation (Q257R) in CFC syndrome, on a C57BL/6J background are embryonic/neonatal lethal, with multiple congenital defects, preventing us from analyzing the phenotypic consequences after birth. Here, to further explore the pathogenesis of CFC syndrome, we backcrossed these mice onto a BALB/c or ICR/CD-1 genetic background. On a mixed (BALB/c and C57BL/6J) background, all heterozygous Braf(Q241R/+) mice died between birth and 24 weeks and exhibited growth retardation, sparse and ruffled fur, liver necrosis and atrial septal defects (ASDs). In contrast, 31% of the heterozygous Braf(Q241R/+) ICR mice survived over 74 weeks. The surviving Braf(Q241R/+) ICR mice exhibited growth retardation, sparse and ruffled fur, a hunched appearance, craniofacial dysmorphism, long and/or dystrophic nails, extra digits and ovarian cysts. The Braf(Q241R/+) ICR mice also showed learning deficits in the contextual fear-conditioning test. Echocardiography indicated the presence of pulmonary stenosis and ASDs in the Braf(Q241R/+) ICR mice, which were confirmed by histological analysis. These data suggest that the heterozygous Braf(Q241R/+) ICR mice show similar phenotypes as CFC syndrome after birth and will be useful for elucidating the pathogenesis and potential therapeutic strategies for RASopathies.

Distinct effects of Hoxa2 overexpression in cranial neural crest populations reveal that the mammalian hyomandibular-ceratohyal boundary maps within the styloid process.

Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures.

Prdm1 functions in the mesoderm of the second heart field, where it interacts genetically with Tbx1, during outflow tract morphogenesis in the mouse embryo.

Congenital heart defects affect at least 0.8% of newborn children and are a major cause of lethality prior to birth. Malformations of the arterial pole are particularly frequent. The myocardium at the base of the pulmonary trunk and aorta and the arterial tree associated with these great arteries are derived from splanchnic mesoderm of the second heart field (SHF), an important source of cardiac progenitor cells. These cells are controlled by a gene regulatory network that includes Fgf8, Fgf10 and Tbx1. Prdm1 encodes a transcriptional repressor that we show is also expressed in the SHF. In mouse embryos, mutation of Prdm1 affects branchial arch development and leads to persistent truncus arteriosus (PTA), indicative of neural crest dysfunction. Using conditional mutants, we show that this is not due to a direct function of Prdm1 in neural crest cells. Mutation of Prdm1 in the SHF does not result in PTA, but leads to arterial pole defects, characterized by mis-alignment or reduction of the aorta and pulmonary trunk, and abnormalities in the arterial tree, defects that are preceded by a reduction in outflow tract size and loss of caudal pharyngeal arch arteries. These defects are associated with a reduction in proliferation of progenitor cells in the SHF. We have investigated genetic interactions with Fgf8 and Tbx1, and show that on a Tbx1 heterozygote background, conditional Prdm1 mutants have more pronounced arterial pole defects, now including PTA. Our results identify PRDM1 as a potential modifier of phenotypic severity in TBX1 haploinsufficient DiGeorge syndrome patients.

New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

Hesr2 knockout mice develop aortic valve disease with advancing age.

OBJECTIVE: Acquired heart diseases, such as valve disease, are major causes of human morbidity and mortality. However, the pathological mechanisms underlying these diseases are largely unknown. Our aim is to identify the role of the hairy and enhancer of split-related (Hesr)-2 gene in the adult heart. METHODS AND RESULTS: Echocardiography detected heart dysfunctions indicative of aortic valve anomalies, stenosis, and regurgitation, in ≈59% of >12-month-old Hesr2 knockout survivor mice. Morphological and histological analyses revealed thickened semilunar valves with increased fibrotic areas, indicating that sclerotic degeneration of valves is the main cause of aortic valve disease. The expression of osteogenic genes, such as osteopontin and sclerostin, were upregulated in the mutants, and the overexpression of sclerostin in endothelial cells resulted in thickened semilunar valves with increased fibrotic areas, similar to that seen in the Hesr2 knockout mice, suggesting that Hesr2 can regulate osteogenic gene expression in valves. Reduced left ventricular function, which may be caused by increased ventricular interstitial fibrosis, and enlarged myocardial cell size without ventricular wall thickening were found in both aortic valve stenosis/regurgitation-positive (33%) and aortic valve stenosis/regurgitation-negative (38%) subpopulations in 12-month-old survivor mice. Dilated left ventricular internal dimensions were specifically detected in the aortic valve stenosis/regurgitation-positive subpopulation, thus suggesting that the degeneration of cardiomyocytes is influenced by irregular hemodynamics. CONCLUSIONS: These data revealed that survivor mice lacking the Hesr2 gene exhibit fibrosis in the aortic valve and ventricle in adulthood, thus suggesting that Hesr2 plays an important role in maintaining the homeostasis of the aortic valve and ventricle.

Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation.

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.

Coronary artery established through amniote evolution.

Coronary arteries are a critical part of the vascular system and provide nourishment to the heart. In humans, even minor defects in coronary arteries can be lethal, emphasizing their importance for survival. However, some teleosts survive without coronary arteries, suggesting that there may have been some evolutionary changes in the morphology and function of coronary arteries in the tetrapod lineage. Here, we propose that the true ventricular coronary arteries were newly established during amniote evolution through remodeling of the ancestral coronary vasculature. In mouse (Mus musculus) and Japanese quail (Coturnix japonica) embryos, the coronary arteries unique to amniotes are established by the reconstitution of transient vascular plexuses: aortic subepicardial vessels (ASVs) in the outflow tract and the primitive coronary plexus on the ventricle. In contrast, amphibians (Hyla japonica, Lithobates catesbeianus, Xenopus laevis, and Cynops pyrrhogaster) retain the ASV-like vasculature as truncal coronary arteries throughout their lives and have no primitive coronary plexus. The anatomy and development of zebrafish (Danio rerio) and chondrichthyans suggest that their hypobranchial arteries are ASV-like structures serving as the root of the coronary vasculature throughout their lives. Thus, the ventricular coronary artery of adult amniotes is a novel structure that has acquired a new remodeling process, while the ASVs, which occur transiently during embryonic development, are remnants of the ancestral coronary vessels. This evolutionary change may be related to the modification of branchial arteries, indicating considerable morphological changes underlying the physiological transition during amniote evolution.

Coronary arteries are tasked with supplying the heart with oxygenated blood and nutrients. Any blockage or developmental problem in these blood vessels can have severe and sometimes lethal consequences. Due to their importance for health, researchers have extensively studied how coronary arteries form in humans and mice; a more limited range of studies have also looked at their equivalent in zebrafish. However, little is known about these structures develop in animals such as birds, amphibians, or other groups of fish. This makes it difficult to retrace the evolutionary processes that have given rise to the coronary arteries we are familiar with in mammals. To address this knowledge gap, Mizukami et al. set out to compare blood vessel development around the heart of mammals, birds, amphibians, and fish. To do this, they performed detailed anatomical studies of blood vessel structure at different stages of development in mice as well as quail, frogs and newts, zebrafish and sharks. In both mice and quail, small arterial subepicardial vessels (or ASVs) emerged early in development around the heart; these subsequently reorganised and remodelled themselves to give rise to the 'true' coronary arteries characteristic of the mature heart. Frogs and newts also developed similar ASV-like structures; however, unlike their mammalian and bird equivalents, these vessels did not reorganise, instead being retained into adulthood. In fish, blood vessel development resembled that of amphibians, suggesting that the coronary artery-like structures seen in some fish are an 'ancestral' form of ASVs, rather than the equivalent of the mature coronary arteries in mammals and birds. This work sheds light on the evolutionary processes shaping essential structures in the heart. In the future, Mizukami et al. hope that this knowledge will help develop a greater range of experimental animal models for studying heart disease and potential treatments.

Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries.

RATIONALE: The genes encoding fibroblast growth factor (FGF) 8 and 10 are expressed in the anterior part of the second heart field that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects. OBJECTIVES: Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole. METHODS AND RESULTS: We generated mesodermal Fgf8; Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance; however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to pharyngeal arch artery phenotypes and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches. CONCLUSIONS: These results show functional overlap of FGF8 and FGF10 signaling from second heart field mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during pharyngeal arch artery formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development.

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.

The cardiopharyngeal mesoderm contributes to lymphatic vessel development in mouse.

Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1Cre/+ and Isl1CreERT2/+ mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

Atresia of the right coronary arterial ostium with a left ventricular fistula: A case report.

We describe the case of a young patient with atresia of the right coronary arterial ostium with left ventricular fistula. This was suspected from the abnormality detected on a 12­lead electrocardiogram (ECG) during a school examination at the time of admission to junior high school and echocardiography findings. This disease may occur due to abnormalities in several molecules that are essential for coronary artery development. In cases of ECG abnormality, and unexplained aortic valve regurgitation and coronary artery abnormalities on echocardiography are detected, this disease should be suspected. Learning objective: Coronary artery anomalies have been identified in coronary angiograms. Herein, we describe the case of a young patient with atresia of the right coronary arterial ostium with left ventricular fistula. This disease may be caused by abnormalities in several molecules that are essential molecule for coronary artery development.

Effects of transforming growth factor-ß3 and matrix metalloproteinase-3 on the pathogenesis of chronic mitral valvular disease in dogs.

OBJECTIVE: To investigate the roles of transforming growth factor-ß (TGF-ß) isoforms and matrix metalloproteinases (MMPs) in development of chronic mitral valvular disease (CMVD) in dogs. SAMPLE POPULATION: 12 mitral valve leaflets collected from cadavers of 5 clinically normal dogs and from 7 dogs with CMVD. PROCEDURES: Expression of TGF-ß isoforms 1, 2, and 3; MMPs 1, 2, 3, and 9; TGF-ß receptor II (TßR-II); and α smooth muscle actin (αSMA) in mitral valves of dogs with CMVD was compared with that in mitral valves from clinically normal dogs. Additionally, responses of valvular interstitial cells (VICs) to TGF-ß3, MMP-3, and angiotensin-converting enzyme inhibitor (ACEI) as a suppressor of TGF-ß3 were examined in vitro. RESULTS: Expression of TGF-ß3, TßR-II, αSMA, and MMP-3 was only detected in mitral valves of dogs with CMVD. Concentrations of αSMA and proteoglycans in cultured VICs were significantly increased following incubation with TGF-ß3; treatment with MMP-3 resulted in increased amounts of active and total TGF-ß3, and total TGF-ß3 in VICs was significantly decreased by incubation with ACEI. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that increased TGF-ß3 and MMP-3 contribute to the pathogenesis of valvular degeneration associated with CMVD. In addition, it is possible that the use of ACEI could effectively block pathological alterations in VICs associated with CMVD in vitro. Impact on Human Medicine-CMVD is associated with primary mitral valve prolapse and Marfan syndrome in humans. Results of the study reported here will help to elucidate the molecular mechanisms of CMVD in dogs and humans.

Surgical protocol for permanent ligation of the left anterior descending coronary artery in mice to generate a model of myocardial infarction.

Myocardial infarction (MI) is one of the most common causes of death worldwide. Animal models for MI are useful for studying the pathophysiology and developing therapies. Here, we describe a surgical protocol for permanent ligation of the left anterior descending coronary artery in mice, which mimics human acute coronary syndrome. This protocol includes descriptive step-by-step surgical procedures and high-quality surgical videos, which are useful for performing stable and highly reproducible operations. For complete details on the use and execution of this protocol, please refer to Maruyama et al. (2021).

Semaphorin3E-PlexinD1 signaling in coronary artery and lymphatic vessel development with clinical implications in myocardial recovery.

Blood and lymphatic vessels surrounding the heart develop through orchestrated processes from cells of different origins. In particular, cells around the outflow tract which constitute a primordial transient vasculature, referred to as aortic subepicardial vessels, are crucial for the establishment of coronary artery stems and cardiac lymphatic vessels. Here, we revealed that the epicardium and pericardium-derived Semaphorin 3E (Sema3E) and its receptor, PlexinD1, play a role in the development of the coronary stem, as well as cardiac lymphatic vessels. In vitro analyses demonstrated that Sema3E may demarcate areas to repel PlexinD1-expressing lymphatic endothelial cells, resulting in proper coronary and lymphatic vessel formation. Furthermore, inactivation of Sema3E-PlexinD1 signaling improved the recovery of cardiac function by increasing reactive lymphangiogenesis in an adult mouse model of myocardial infarction. These findings may lead to therapeutic strategies that target Sema3E-PlexinD1 signaling in coronary artery diseases.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

New Noonan syndrome model mice with RIT1 mutation exhibit cardiac hypertrophy and susceptibility to ß-adrenergic stimulation-induced cardiac fibrosis.

BACKGROUND: Noonan syndrome (NS) is a genetic disorder characterized by short stature, a distinctive facial appearance, and heart defects. We recently discovered a novel NS gene, RIT1, which is a member of the RAS subfamily of small GTPases. NS patients with RIT1 mutations have a high incidence of hypertrophic cardiomyopathy and edematous phenotype, but the specific role of RIT1 remains unclear. METHODS: To investigate how germline RIT1 mutations cause NS, we generated knock-in mice that carried a NS-associated Rit1 A57G mutation (Rit1A57G/+). We investigated the phenotypes of Rit1A57G/+ mice in fetal and adult stages as well as the effects of isoproterenol on cardiac function in Rit1A57G/+ mice. FINDINGS: Rit1A57G/+ embryos exhibited decreased viability, edema, subcutaneous hemorrhage and AKT activation. Surviving Rit1A57G/+ mice had a short stature, craniofacial abnormalities and splenomegaly. Cardiac hypertrophy and cardiac fibrosis with increased expression of S100A4, vimentin and periostin were observed in Rit1A57G/+ mice compared to Rit1+/+ mice. Upon isoproterenol stimulation, cardiac fibrosis was drastically increased in Rit1A57G/+ mice. Phosphorylated (at Thr308) AKT levels were also elevated in isoproterenol-treated Rit1A57G/+ hearts. INTERPRETATION: The A57G mutation in Rit1 causes cardiac hypertrophy, fibrosis and other NS-associated features. Biochemical analysis indicates that the AKT signaling pathway might be related to downstream signaling in the RIT1 A57G mutant at a developmental stage and under ß-adrenergic stimulation in the heart. FUND: The Grants-in-Aid were provided by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science KAKENHI Grant.