Rajeev Samtani

The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport.

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.

Developmental Atlas of the Early First Trimester Human Embryo

Rapid advances in medical imaging are facilitating the clinical assessment of first‐trimester human embryos at increasingly earlier stages. To obtain data on early human development, we used magnetic resonance (MR) imaging and episcopic fluorescence capture (EFIC) to acquire digital images of human embryos spanning the time of dynamic tissue remodeling and organogenesis (Carnegie stages 13 to 23). These imaging data sets are readily resectioned digitally in arbitrary planes, suitable for rapid high‐resolution three‐dimensional (3D) observation. Using these imaging datasets, a web‐accessible digital Human Embryo Atlas (http://apps.devbio.pitt.edu/humanatlas/) was created containing serial 2D images of human embryos in three standard histological planes: sagittal, frontal, and transverse. In addition, annotations and 3D reconstructions were generated for visualizing different anatomical structures. Overall, this Human Embryo Atlas is a unique resource that provides morphologic data of human developmental anatomy that can accelerate basic research investigations into developmental mechanisms that underlie human congenital anomalies. Developmental Dynamics 239:1585–1595, 2010.

Human Cardiac Development in the First Trimester A High-Resolution Magnetic Resonance Imaging and Episcopic Fluorescence Image Capture Atlas

With rapid advances in medical imaging, fetal diagnosis of human CHD is now technically feasible in the first trimester. Although the first human embryologic studies were recorded by Hippocrates in 300–400 BC, present day knowledge of normal human cardiac development in the first trimester is still limited. In 1886, two papers by Dr His described development of the heart based on dissections of young human embryos. Free hand wax models were made that illustrated the external developmental anatomy. These wax plate reconstruction methods were used by many other investigators until the early 1900s1. Subsequently serial histological sections of human embryos have been used to further investigate human cardiac development2–6. Based on analysis of histological sections and scaled reproductions of human embryos, Grant showed a large cushion in the developing heart at 6 6/7 weeks (CS 14) and separate AV valves at 9 1/7 weeks (CS 22)2. At the end of the 8th week (CS 8), separate aortic and pulmonary outflows were observed. Orts-Llorca used three dimensional reconstructions of transverse sections of human embryos to define development of the truncus arteriosus and described completion of septation of the truncus arteriosus in 14–16mm embryos, equivalent to EGA 8 weeks (CS18)5. Given the complex tissue remodeling associated with cardiac chamber formation and inflow/outflow tract and valvular morphogenesis, the plane of sectioning often limited the information that can be gathered on developing structures in the embryonic heart. These technical limitations in conjunction with limited access to human embryo specimens have meant that much of our understanding of early cardiac development in the human embryo is largely extrapolated from studies in model organisms7–10. With possible species differences in developmental timing and variation in cardiovascular anatomy, characterization of normal cardiac development in human embryos is necessary for clinical evaluation and diagnosis of CHD in the first trimester. This will be increasingly important, as improvements in medical technology allow earlier access to first trimester human fetal cardiac imaging and in utero intervention. Recent studies have shown the feasibility of using magnetic resonance imaging (MRI) to obtain information on human embryo tissue structure11, 12. MRI imaging data can be digitally resectioned for viewing of the specimen in any orientation, and three-dimensional (3D) renderings can be obtained with ease. Similarly, episcopic fluorescence image capture (EFIC), a novel histological imaging technique, provides registered two-dimensional (2D) image stacks that can be resectioned in arbitrary planes and also rapidly 3D rendered10. With EFIC imaging, tissue is embedded in paraffin and cut with a sledge microtome. Tissue autofluorescence at the block face is captured and used to generate registered serial 2D images of the specimen with image resolution better than MRI. Data obtained by MRI or EFIC imaging can be easily resectioned digitally or reconstructed in 3D to facilitate the analysis of complex morphological changes in the developing embryonic heart. In this manner, the developing heart in every embryo can be analyzed in it entirety with no loss of information due to the plane of sectioning. Using MRI and EFIC imaging, we conducted a systematic analysis of human cardiovascular development in the first trimester. 2D image stacks and 3D volumes were generated from 52 human embryos from 6 4/7 to 9 3/7 weeks estimated gestational age (EGA), equivalent to Carnegie stages (CS) 13–23. These stages encompass the developmental window during which all of the major milestones of cardiac morphogenesis can be observed. Using the MRI and EFIC imaging data, we constructed a digital atlas of human heart development. Data from our atlas were used to generate charts summarizing the major milestones of normal human heart development through the first trimester. MRI and EFIC images obtained as part of this study can be viewed as part of an online Human Embryo Atlas. To view the Human Embryo Atlas content, visit http://apps.nhlbi.nih.gov/HumanAtlas/home/login.aspx?ReturnUrl=%2fhumanatlas%2fDefault.aspx.

Outflow tract cushions perform a critical valve-like function in the early embryonic heart requiring BMPRIA-mediated signaling in cardiac neural crest

Neural crest-specific ablation of BMP type IA receptor (BMPRIA) causes embryonic lethality by embryonic day (E) 12.5, and this was previously postulated to arise from a myocardial defect related to signaling by a small population of cardiac neural crest cells (cNCC) in the epicardium. However, as BMP signaling via cNCC is also required for proper development of the outflow tract cushions, precursors to the semilunar valves, a plausible alternate or additional hypothesis is that heart failure may result from an outflow tract cushion defect. To investigate whether the outflow tract cushions may serve as dynamic valves in regulating hemodynamic function in the early embryo, in this study we used noninvasive ultrasound biomicroscopy-Doppler imaging to quantitatively assess hemodynamic function in mouse embryos with P0-Cre transgene mediated neural crest ablation of Bmpr1a (P0 mutants). Similar to previous studies, the neural crest-deleted Bmpr1a P0 mutants died at approximately E12.5, exhibiting persistent truncus arteriosus, thinned myocardium, and congestive heart failure. Surprisingly, our ultrasound analyses showed normal contractile indices, heart rate, and atrioventricular conduction in the P0 mutants. However, reversed diastolic arterial blood flow was detected as early as E11.5, with cardiovascular insufficiency and death rapidly ensuing by E12.5. Quantitative computed tomography showed thinning of the outflow cushions, and this was associated with a marked reduction in cell proliferation. These results suggest BMP signaling to cNCC is required for growth of the outflow tract cushions. This study provides definitive evidence that the outflow cushions perform a valve-like function critical for survival of the early mouse embryo.

A Unique Set of Centrosome Proteins Requires Pericentrin for Spindle-Pole Localization and Spindle Orientation

Majewski osteodysplastic primordial dwarfism type II (MOPDII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre- and postnatal growth retardation. As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition. To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt(-/-) mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150(glued), aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

A detailed comparison of mouse and human cardiac development

Background:Mouse mutants are used to model human congenital cardiovascular disease. Few studies exist comparing normal cardiovascular development in mice vs. humans. We carried out a systematic comparative analysis of mouse and human fetal cardiovascular development.Methods:Episcopic fluorescence image capture (EFIC) was performed on 66 wild-type mouse embryos from embryonic day (E) 9.5 to birth; 2-dimensional and 3-dimensional datasets were compared with EFIC and magnetic resonance images from a study of 52 human fetuses (Carnegie stage 13–23).Results:Time course of atrial, ventricular, and outflow septation were outlined and followed a similar sequence in both species. Bilateral venae cavae and prominent atrial appendages were seen in the mouse fetus; in human fetuses, atrial appendages were small, and a single right superior vena cava was present. In contrast to humans with separate pulmonary vein orifices, a pulmonary venous confluence with one orifice enters the left atrium in mice.Conclusion:The cardiac developmental sequences observed in mouse and human fetuses are comparable, with minor differences in atrial and venous morphology. These comparisons of mouse and human cardiac development strongly support that mouse morphogenesis is a good model for human development.

Ventricular Rotation Is Independent of Cardiac Looping: A Study in Mice With Situs Inversus Totalis Using Speckle-Tracking Echocardiography

BACKGROUND The authors conducted an ultrasound interrogation of a mutant mouse model with a Dnah5 mutation to determine whether cardiac mechanics may be affected by reversal of cardiac situs. This mutant is a bona fide model of primary ciliary dyskinesia, with surviving homozygous mice showing either situs solitus (SS) or situs inversus totalis (SI). METHODS High-frequency ultrasound interrogations of 27 neonatal and infant Dnah5 mutant mice, 16 with SS and 11 with SI, were conducted using an ultra-high-frequency biomicroscope. Electrocardiographic and respiratory gating were used to reconstruct high-resolution two-dimensional cines at 1,000 Hz, with speckle-tracking echocardiography used to further analyze midchamber and apical rotation. RESULTS All SS mice exhibited the expected counterclockwise apical rotation as viewed caudocranially, and surprisingly, the same counterclockwise motion was also observed in SI mice. Speckle-tracking analysis confirmed counterclockwise systolic rotation in both SS and SI mice, and this increased in magnitude from the subepicardium to the endocardium and from the papillary muscles to the apex. The magnitude of apical endocardial rotation was not different for SS and SI mice (5.64+/-0.75 degrees and 5.76+/-1.90 degrees, respectively, P=.93). The anatomic segments responsible for the largest components of apical endocardial systolic rotation differed between the SS and SI hearts (P=.004). In both, the two largest contributors to rotation were offset 180 degrees from each other, but the anatomic regions differed between them. In SS hearts, maximal regional rotation occurred at the anterior mid-septum and posterolateral free wall, while in SI hearts, it was derived from the posterior septum and the anterolateral free wall. Analysis by episcopic fluorescence image capture histology of representative SI and SS mice showed normal intracardiac and segmental anatomy ({S,D,S} or {I,L,I}) without intracardiac defects. CONCLUSIONS These results show that mirror-image cardiac looping did not result in mirror-image rotation of the morphologic left ventricle. These findings suggest that further studies are warranted to evaluate whether fiber orientation and cardiac mechanics may be abnormal in individuals with reversal of cardiac situs. The results of this study indicate that cardiac looping and myofiber orientation may be independently regulated.

Human Cardiac Development in the First Trimester

With rapid advances in medical imaging, fetal diagnosis of human CHD is now technically feasible in the first trimester. Although the first human embryologic studies were recorded by Hippocrates in 300–400 BC, present day knowledge of normal human cardiac development in the first trimester is still limited. In 1886, two papers by Dr His described development of the heart based on dissections of young human embryos. Free hand wax models were made that illustrated the external developmental anatomy. These wax plate reconstruction methods were used by many other investigators until the early 1900s1. Subsequently serial histological sections of human embryos have been used to further investigate human cardiac development2–6. Based on analysis of histological sections and scaled reproductions of human embryos, Grant showed a large cushion in the developing heart at 6 6/7 weeks (CS 14) and separate AV valves at 9 1/7 weeks (CS 22)2. At the end of the 8th week (CS 8), separate aortic and pulmonary outflows were observed. Orts-Llorca used three dimensional reconstructions of transverse sections of human embryos to define development of the truncus arteriosus and described completion of septation of the truncus arteriosus in 14–16mm embryos, equivalent to EGA 8 weeks (CS18)5. Given the complex tissue remodeling associated with cardiac chamber formation and inflow/outflow tract and valvular morphogenesis, the plane of sectioning often limited the information that can be gathered on developing structures in the embryonic heart. These technical limitations in conjunction with limited access to human embryo specimens have meant that much of our understanding of early cardiac development in the human embryo is largely extrapolated from studies in model organisms7–10. With possible species differences in developmental timing and variation in cardiovascular anatomy, characterization of normal cardiac development in human embryos is necessary for clinical evaluation and diagnosis of CHD in the first trimester. This will be increasingly important, as improvements in medical technology allow earlier access to first trimester human fetal cardiac imaging and in utero intervention. Recent studies have shown the feasibility of using magnetic resonance imaging (MRI) to obtain information on human embryo tissue structure11, 12. MRI imaging data can be digitally resectioned for viewing of the specimen in any orientation, and three-dimensional (3D) renderings can be obtained with ease. Similarly, episcopic fluorescence image capture (EFIC), a novel histological imaging technique, provides registered two-dimensional (2D) image stacks that can be resectioned in arbitrary planes and also rapidly 3D rendered10. With EFIC imaging, tissue is embedded in paraffin and cut with a sledge microtome. Tissue autofluorescence at the block face is captured and used to generate registered serial 2D images of the specimen with image resolution better than MRI. Data obtained by MRI or EFIC imaging can be easily resectioned digitally or reconstructed in 3D to facilitate the analysis of complex morphological changes in the developing embryonic heart. In this manner, the developing heart in every embryo can be analyzed in it entirety with no loss of information due to the plane of sectioning. Using MRI and EFIC imaging, we conducted a systematic analysis of human cardiovascular development in the first trimester. 2D image stacks and 3D volumes were generated from 52 human embryos from 6 4/7 to 9 3/7 weeks estimated gestational age (EGA), equivalent to Carnegie stages (CS) 13–23. These stages encompass the developmental window during which all of the major milestones of cardiac morphogenesis can be observed. Using the MRI and EFIC imaging data, we constructed a digital atlas of human heart development. Data from our atlas were used to generate charts summarizing the major milestones of normal human heart development through the first trimester. MRI and EFIC images obtained as part of this study can be viewed as part of an online Human Embryo Atlas. To view the Human Embryo Atlas content, visit http://apps.nhlbi.nih.gov/HumanAtlas/home/login.aspx?ReturnUrl=%2fhumanatlas%2fDefault.aspx.

Human Cardiac Development in the First Trimester: A High Resolution MRI and Episcopic Fluorescence Image Capture Atlas

With rapid advances in medical imaging, fetal diagnosis of human CHD is now technically feasible in the first trimester. Although the first human embryologic studies were recorded by Hippocrates in 300–400 BC, present day knowledge of normal human cardiac development in the first trimester is still limited. In 1886, two papers by Dr His described development of the heart based on dissections of young human embryos. Free hand wax models were made that illustrated the external developmental anatomy. These wax plate reconstruction methods were used by many other investigators until the early 1900s1. Subsequently serial histological sections of human embryos have been used to further investigate human cardiac development2–6. Based on analysis of histological sections and scaled reproductions of human embryos, Grant showed a large cushion in the developing heart at 6 6/7 weeks (CS 14) and separate AV valves at 9 1/7 weeks (CS 22)2. At the end of the 8th week (CS 8), separate aortic and pulmonary outflows were observed. Orts-Llorca used three dimensional reconstructions of transverse sections of human embryos to define development of the truncus arteriosus and described completion of septation of the truncus arteriosus in 14–16mm embryos, equivalent to EGA 8 weeks (CS18)5. Given the complex tissue remodeling associated with cardiac chamber formation and inflow/outflow tract and valvular morphogenesis, the plane of sectioning often limited the information that can be gathered on developing structures in the embryonic heart. These technical limitations in conjunction with limited access to human embryo specimens have meant that much of our understanding of early cardiac development in the human embryo is largely extrapolated from studies in model organisms7–10. With possible species differences in developmental timing and variation in cardiovascular anatomy, characterization of normal cardiac development in human embryos is necessary for clinical evaluation and diagnosis of CHD in the first trimester. This will be increasingly important, as improvements in medical technology allow earlier access to first trimester human fetal cardiac imaging and in utero intervention. Recent studies have shown the feasibility of using magnetic resonance imaging (MRI) to obtain information on human embryo tissue structure11, 12. MRI imaging data can be digitally resectioned for viewing of the specimen in any orientation, and three-dimensional (3D) renderings can be obtained with ease. Similarly, episcopic fluorescence image capture (EFIC), a novel histological imaging technique, provides registered two-dimensional (2D) image stacks that can be resectioned in arbitrary planes and also rapidly 3D rendered10. With EFIC imaging, tissue is embedded in paraffin and cut with a sledge microtome. Tissue autofluorescence at the block face is captured and used to generate registered serial 2D images of the specimen with image resolution better than MRI. Data obtained by MRI or EFIC imaging can be easily resectioned digitally or reconstructed in 3D to facilitate the analysis of complex morphological changes in the developing embryonic heart. In this manner, the developing heart in every embryo can be analyzed in it entirety with no loss of information due to the plane of sectioning. Using MRI and EFIC imaging, we conducted a systematic analysis of human cardiovascular development in the first trimester. 2D image stacks and 3D volumes were generated from 52 human embryos from 6 4/7 to 9 3/7 weeks estimated gestational age (EGA), equivalent to Carnegie stages (CS) 13–23. These stages encompass the developmental window during which all of the major milestones of cardiac morphogenesis can be observed. Using the MRI and EFIC imaging data, we constructed a digital atlas of human heart development. Data from our atlas were used to generate charts summarizing the major milestones of normal human heart development through the first trimester. MRI and EFIC images obtained as part of this study can be viewed as part of an online Human Embryo Atlas. To view the Human Embryo Atlas content, visit http://apps.nhlbi.nih.gov/HumanAtlas/home/login.aspx?ReturnUrl=%2fhumanatlas%2fDefault.aspx.