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Family 26 with persistent truncus arteriosus and craniofacial anomalies. (A) A pup that died at birth had a short snout, low set ears, rounded head, and short neck. (B) A normal neonatal C57BL6/J pup. Alcian blue staining (E,F) showed the abnormal pup (E) had a shortened premaxilla (PM), maxilla (M) and nasal bone (N), while its frontal bone (F) was expanded. The shape of the mandible (MN) was also altered. The heart exhibited persistent truncus arteriosus (PTA in D), when compared with normal septated outflows (C), consisting of an aortic (Ao) and pulmonary (P) trunk. Histological sections of the abnormal heart (G,H) revealed a single outflow positioned over the RV and VSD. This vessel gave rise to the ascending aorta (AAo) with its brachiocephalic artery (BCA), the right and left pulmonary arteries (RPA, LPA), and the coronary arteries (LCA, RCA). A VSD with inlet extension can be seen in (H). Asterisk in H denotes abnormal AV valve. Panels A and B, E and F, and G and H are shown at the same magnification.
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We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen...
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Context 1
... septation defects in a number of ENU families. In family 26, the original ultrasound scan had indicated one fetus in a litter of six with outflow regurgitation. At birth, six pups were delivered, with one born dead. The dead pup exhibited craniofacial dysmorphism that included a pug nose, dome- shaped head, low-set ears, and short neck and limbs (Fig. 3A). Necropsy revealed that the fetus had PTA (Fig. 3D, compare with 3C) and also hypoplastic thymus. Histological analysis revealed a single great artery that gave rise to the coronary arteries, aorta and pulmonary arteries, with a short main pulmonary artery segment ('type 1 1/2' persistent truncus arteriosus). This heart also had an AVC ...
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... 26, the original ultrasound scan had indicated one fetus in a litter of six with outflow regurgitation. At birth, six pups were delivered, with one born dead. The dead pup exhibited craniofacial dysmorphism that included a pug nose, dome- shaped head, low-set ears, and short neck and limbs (Fig. 3A). Necropsy revealed that the fetus had PTA (Fig. 3D, compare with 3C) and also hypoplastic thymus. Histological analysis revealed a single great artery that gave rise to the coronary arteries, aorta and pulmonary arteries, with a short main pulmonary artery segment ('type 1 1/2' persistent truncus arteriosus). This heart also had an AVC with a primum ASD, VSD and atrioventricular valve ...
Context 3
... with 3C) and also hypoplastic thymus. Histological analysis revealed a single great artery that gave rise to the coronary arteries, aorta and pulmonary arteries, with a short main pulmonary artery segment ('type 1 1/2' persistent truncus arteriosus). This heart also had an AVC with a primum ASD, VSD and atrioventricular valve abnormalities (Fig. 3G,H). The skeletal preparation showed micrognathia due to reduction in the premaxilla, maxilla, mandible and nasal bones. By contrast, the frontal bone was enlarged, accounting for the dome-shaped appearance of the head (Fig. 3E, compare with control in ...
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... truncus arteriosus). This heart also had an AVC with a primum ASD, VSD and atrioventricular valve abnormalities (Fig. 3G,H). The skeletal preparation showed micrognathia due to reduction in the premaxilla, maxilla, mandible and nasal bones. By contrast, the frontal bone was enlarged, accounting for the dome-shaped appearance of the head (Fig. 3E, compare with control in ...
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During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a
strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow
(BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This str...
Citations
... A mutant mouse line, 220, harboring an Ift140 splicing defect mutation was previously recovered from an ethylnitrosourea (ENU) mutagenesis screen and found to be homozygous neonatal lethal with a wide spectrum of SBDs (Table 1 and Fig 1) [25,26]. The neonatal lethality is in sharp contrast to the prenatal lethality seen in most other IFT mutant mice [2,27], including the Cauli Ift140 mutant [22]. ...
... The Ift140 220 mutant line was recovered from a large-scale mouse ENU mouse mutagenesis screen as previously described [26]. The Ift140 null1 and Ift140 flox alleles were generated from ES cells with an Ift140 targeted allele generated by KOMP [60] as described [23] Cre lines used include CAGGCre-ER [61], Wnt1-Cre [41,42], Tbx18-Cre [43], Foxa2Cre-ER [37], Mef2c-Cre [39,62], and Tie2-Cre [40] (S2 Table). ...
Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140 -deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.
... Cx43 −/− heart disease obstruction of right ventricular outflow tract and abnormal coronary deployment [15] CMV43 heart disease malformation of the conotruncus [16] α-MHC-Cre;Cx43 flox/flox sudden arrhythmic death spontaneous ventricular arrhythmias, reduced ventricular conduction velocity [17] MLC2v-Cre;Cx43 flox/flox sudden arrhythmic death spontaneous ventricular arrhythmias [17] Wnt1-Cre;Cx43 flox/flox heart disease abnormal development of coronary, normal formation of OFT [18] P3pro-Cre;Cx43 flox/flox heart disease infundibular bulging and coronary anomalies [18] Cx43KI32 spontaneous ventricular arrhythmias morphological defects, Spontaneous ventricular arrhythmias [19] Cx43KI40 spontaneous ventricular arrhythmias mild hypertrophy of heart [19] Cx43KI31 heart disease Malformation in the subpulmonary outlet of the right ventricle, low voltage of the QRS complex [20] Cx43KI26 heart disease slowed ventricular conduction [21] Cx43 +/I130T heart disease Reduced Cx43 protein level, conduction velocity, and junctional conductance [22] Gja1 W45X heart disease Conotruncal malformation, coronary aneurysms. [23] Cx43 K258stop defect of the heart and the epidermal barrier Impaired differentiation of keratinocytes, dilatation of the right ventricular outflow tract [24] α-MHC-Cre;Cx43 floxD378stop/floxD378stop severe ventricular arrhythmias impaired cardiac sodium and potassium currents [25] α-MHC-Cre;Cx43 +/floxG138R Spontaneous arrhythmias loss of the phosphorylated forms of Cx43 [26] Cx43 +/jrt ODDD dominant-negative effect, syndactyly, enamel hypoplasia, craniofacial anomalies, cardiac dysfunction. ...
Inherited diseases caused by connexin mutations are found in multiple organs and include hereditary deafness, congenital cataract, congenital heart diseases, hereditary skin diseases, and X-linked Charcot–Marie–Tooth disease (CMT1X). A large number of knockout and knock-in animal models have been used to study the pathology and pathogenesis of diseases of different organs. Because the structures of different connexins are highly homologous and the functions of gap junctions formed by these connexins are similar, connexin-related hereditary diseases may share the same pathogenic mechanism. Here, we analyze the similarities and differences of the pathology and pathogenesis in animal models and find that connexin mutations in gap junction genes expressed in the ear, eye, heart, skin, and peripheral nerves can affect cellular proliferation and differentiation of corresponding organs. Additionally, some dominant mutations (e.g., Cx43 p.Gly60Ser, Cx32 p.Arg75Trp, Cx32 p.Asn175Asp, and Cx32 p.Arg142Trp) are identified as gain-of-function variants in vivo, which may play a vital role in the onset of dominant inherited diseases. Specifically, patients with these dominant mutations receive no benefits from gene therapy. Finally, the complete loss of gap junctional function or altered channel function including permeability (ions, adenosine triphosphate (ATP), Inositol 1,4,5-trisphosphate (IP3), Ca2+, glucose, miRNA) and electric activity are also identified in vivo or in vitro.
... Therefore, to prevent any mutations of interest from being lost, all mutants would need to be bred prior to screening, which would simply require the use of too many fish. To overcome this major hurdle in regenerative screens, it would be interesting to determine whether genes critical to cardiac regeneration could be learnt from mutagenesis of other zebrafish regenerative organs or from screens of impaired cardiac development [76,77]. Alternatively, the larval zebrafish heart can be injured using laser/needle-stick injuries [78]. ...
The adult human heart cannot repair itself after injury and, instead, forms a permanent fibrotic scar that impairs cardiac function and can lead to incurable heart failure. The zebrafish, amongst other organisms, has been extensively studied for its innate capacity to repair its heart after injury. Understanding the signals that govern successful regeneration in models such as the zebrafish will lead to the development of effective therapies that can stimulate endogenous repair in humans. To date, many studies have investigated cardiac regeneration using a reverse genetics candidate gene approach. However, this approach is limited in its ability to unbiasedly identify novel genes and signalling pathways that are essential to successful regeneration. In contrast, drawing comparisons between different models of regeneration enables unbiased screens to be performed, identifying signals that have not previously been linked to regeneration. Here, we will review in detail what has been learnt from the comparative approach, highlighting the techniques used and how these studies have influenced the field. We will also discuss what further comparisons would enhance our knowledge of successful regeneration and scarring. Finally, we focus on the Astyanax mexicanus, an intraspecies comparative fish model that holds great promise for revealing the secrets of the regenerating heart.
... In normal development, SEMA3C is thought to play a role in development and migration of cardiac neural crest cells. Mice lacking Sema3c exhibited interruption of the aortic arch and persistent truncus arteriosis as well as defects in migration of cardiac neural crest cells towards the outflow tract [90]. Interestingly, in some animals, heart defects were accompanied by ectopic pigmentation in the heart, lung and other tissues, and hypopigmentation of the skin suggesting that SEMA3C also plays a role in differentiation and migration of neural crest-derived melanocytes [90]. ...
... Mice lacking Sema3c exhibited interruption of the aortic arch and persistent truncus arteriosis as well as defects in migration of cardiac neural crest cells towards the outflow tract [90]. Interestingly, in some animals, heart defects were accompanied by ectopic pigmentation in the heart, lung and other tissues, and hypopigmentation of the skin suggesting that SEMA3C also plays a role in differentiation and migration of neural crest-derived melanocytes [90]. Epithelial prostate cells overexpressing SEMA3C lose their cobblestone architecture and exhibit a spindle-like appearance. ...
The semaphorins represent a large family of signaling molecules with crucial roles in neuronal and cardiac development. While normal semaphorin function pertains largely to development, their involvement in malignancy is becoming increasingly evident. One member, Semaphorin 3C (SEMA3C), has been shown to drive a number of oncogenic programs, correlate inversely with cancer prognosis, and promote the progression of multiple different cancer types. This report surveys the body of knowledge surrounding SEMA3C as a therapeutic target in cancer. In particular, we summarize SEMA3C’s role as an autocrine andromedin in prostate cancer growth and survival and provide an overview of other cancer types that SEMA3C has been implicated in including pancreas, brain, breast, and stomach. We also propose molecular strategies that could potentially be deployed against SEMA3C as anticancer agents such as biologics, small molecules, monoclonal antibodies and antisense oligonucleotides. Finally, we discuss important considerations for the inhibition of SEMA3C as a cancer therapeutic agent.
... Detailed phenotypic characterization by imaging [11][12][13][14] is crucial in the development of animal models, mechanistic studies into disease pathogenesis and testing the efficacy of experimental therapeutics. With the advancement of modern transgenic technology, including CRISPR-Cas9 gene targeting, as well as several national and international programs, such as IMPC, KOMP, IPAD-MD, MGD, and EMMA, systematically knocking out every single gene in the mouse genome, generating unprecedented numbers of mutant mice, it is now possible to pursue detail mapping of genetic functions and pathways in CHD. ...
... While the detection of CHD is possible with the Acuson, its detection sensitivity is limited as is the specificity, which largely precludes the diagnosis of specific structural heart defects with 2D imaging on clinical ultrasound systems (Table 1). For example, the detection of shunts may indicate valvular abnormalities, but it is not possible to diagnose the specific CHD (Fig. 1A,I,N,S) (Yu et al., 2004;Shen et al., 2005). Rather, the assessment of cardiovascular anatomy is largely indirect, inferred from the color flow imaging and spectral Doppler assessments. ...
Mouse models are invaluable for investigating the developmental etiology and molecular pathogenesis of structural birth defects. While this has been deployed for studying a wide spectrum of birth defects, mice are particularly valuable for modeling congenital heart disease, given they have the same four-chamber cardiac anatomy as in humans. We have developed the use of noninvasive fetal ultrasound together with micro-computed tomography (micro-CT) imaging for high throughput phenotyping of mice for congenital heart defects (CHD) and other developmental anomalies. We showed the efficacy of fetal ultrasound and micro-CT imaging for diagnosis of a wide spectrum of CHD. With fetal ultrasound, longitudinal scans can be conducted to track the developmental profile of disease pathogenesis, providing both structural information with two-dimensional (2D) imaging, as well as functional data from hemodynamic assessments with color flow and spectral Doppler imaging. In contrast, with micro-CT imaging, virtual necropsies can be conducted rapidly postmortem for diagnosis of not only CHD, but also other structural birth defects. To validate the CHD diagnosis, we further showed the use of a novel histological technique with episcopic confocal microscopy to obtain rapid 3D reconstructions for accurate diagnosis of even the most complex anatomical defect. The latter histological imaging technique when combined with the use of ultrasound and micro-CT imaging provides a powerful combination of imaging modalities that will be invaluable in meeting the accelerating demands for high throughput mouse phenotyping of genetically engineered mice in the coming age of functional genomics. Birth Defects Research, 2017.© 2017 Wiley Periodicals, Inc.
... The method uses a combination of two-dimensional imaging of cardiac structures and Doppler imaging of blood flow. Fetal echocardiography is well-suited for the detection of large defects that cause abnormal patterns of blood flow and hence for screening severe mutations (Li et al., 2015;Yu et al., 2004). Common, septal defects are more difficult to detect because of the limited spatial resolution of imaging and the nature of fetal circulatory physiology. ...
Despite decades of progress, congenital heart disease remains a major cause of mortality and suffering in children and young adults. Prevention would be ideal, but formidable biological and technical hurdles face any intervention that seeks to target the main causes, genetic mutations in the embryo. Other factors, however, significantly modify the total risk in individuals who carry mutations. Investigation of these factors could lead to an alternative approach to prevention. To define the risk modifiers, our group has taken an "experimental epidemiologic" approach via inbred mouse strain crosses. The original intent was to map genes that modify an individual's risk of heart defects caused by an Nkx2-5 mutation. During the analysis of >2000 Nkx2-5(+/-) offspring from one cross we serendipitously discovered a maternal-age associated risk, which also exists in humans. Reciprocal ovarian transplants between young and old mothers indicate that the incidence of heart defects correlates with the age of the mother and not the oocyte, which implicates a maternal pathway as the basis of the risk. The quantitative risk varies between strain backgrounds, so maternal genetic polymorphisms determine the activity of a factor or factors in the pathway. Most strikingly, voluntary exercise by the mother mitigates the risk. Therefore, congenital heart disease can in principle be prevented by targeting a maternal pathway even if the embryo carries a causative mutation. Further mechanistic insight is necessary to develop an intervention that could be implemented on a broad scale, but the physiology of maternal-fetal interactions, aging, and exercise are notoriously complex and undefined. This suggests that an unbiased genetic approach would most efficiently lead to the relevant pathway. A genetic foundation would lay the groundwork for human studies and clinical trials.
... Live in vivo imaging with ultrasound and MRI often suffer from restrictions in field of view, signal-to-noise ratio, and differential tissue contrast ( Metscher, 2009). Ultrasound is widely used for screening hemodynamic profiles in developing embryos and couples nicely with 3D reconstructions for further computation analysis of fluid-solid tissue interactions ( Wessels and Sedmera, 2003;Yu et al., 2004;Shen et al., 2005;Yu et al., 2015). Whole specimen MRI studies are emerging as radio frequency coil design and postprocessing techniques improve, but MRI remains an expensive and a lengthy scanning process (especially for very high resolutions) requiring additional considerations for environmental control to maintain healthy, viable samples ( Gregg and Butcher, 2012). ...
Background:
Gestationally survivable congenital malformations arise during mid-late stages of development that are inaccessible in vivo with traditional optical imaging for assessing long-term abnormal patterning. MicroCT is an attractive technology to rapidly and inexpensively generate quantitative three-dimensional (3D) datasets but requires exogenous contrast media. Here we establish dose-dependent toxicity, persistence, and biodistribution of three different metallic nanoparticles in day 4 chick embryos.
Results:
We determined that 110-nm alkaline earth metal particles were nontoxic and persisted in the chick embryo for up to 24 hr postinjection with contrast enhancement levels at high as 1,600 Hounsfield units (HU). The 15-nm gold nanoparticles persisted with x-ray attenuation higher than that of the surrounding yolk and albumen for up to 8 hr postinjection, while 1.9-nm particles resulted in lethality by 8 hr. We identified spatial and temporally heterogeneous contrast enhancement ranging from 250 to 1,600 HU. With the most optimal 110-nm alkaline earth metal particles, we quantified an exponential increase in the tissue perfusion vs. distance from the dorsal aorta into the flank over 8 hr with a peak perfusion rate of 0.7 μm(2) /s measured at a distance of 0.3 mm.
Conclusions:
These results demonstrate the safety, efficacy, and opportunity of nanoparticle based contrast media in live embryos for quantitative analysis of embryogenesis. Developmental Dynamics 245:1001-1010, 2016. © 2016 Wiley Periodicals, Inc.
... CHDs affect nearly 1 in 100 births every year in the United States and are the most common type of birth defect(Hoffman & Kaplan, 2002;Reller, Strickland et al., 2008). It is a major cause of morbidity and mortality in infants and children(Van Der Bom et al., 2012;Yu et al., 2004). It occurs in approximately 0.5 to 5% of live births(Van Der Bom et al., 2012). ...
... Such loss-of-function screens are especially challenging in mice because heart dysfunction can cause early embryonic lethality. To overcome these hurdles, several groups used in utero ultrasound-based phenotyping to uncover mutations with early cardiovascular developmental defects in mice 27 . Yet, phenotype-based screens may not uncover factors that are also essential before heart formation. ...
Transcriptional regulation of thousands of genes instructs complex morphogenetic and molecular events for heart development. Cardiac transcription factors choreograph gene expression at each stage of differentiation by interacting with cofactors, including chromatin-modifying enzymes, and by binding to a constellation of regulatory DNA elements. Here, we present salient examples relevant to cardiovascular development and heart disease, and review techniques that can sharpen our understanding of cardiovascular biology. We discuss the interplay between cardiac transcription factors, cis-regulatory elements, and chromatin as dynamic regulatory networks, to orchestrate sequential deployment of the cardiac gene expression program.
© 2015 American Heart Association, Inc.
