Shigehito Yamada

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.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.

Assisted reproductive technologies and birth defects.

ABSTRACT  In vitro fertilization (IVF) and other assisted reproductive technologies (ART) are effective treatments for infertility and are widely provided at infertility clinics. Although IVF and related ART procedures are generally considered safe, some studies have suggested an excess occurrence of major malformations, low birth‐weight and other perinatal complications in babies conceived by ART. Further, it was recently reported that IVF and intracytoplasmic sperm injection (ICSI) are associated with imprinting disorders in the offspring such as Beckwith‐Wiedemann syndrome and Angelman syndrome. Here we review the human and animal studies investigating the potential risks of ART, and discuss the need for further investigation.

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.

Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time‐consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer‐based self‐learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos. Developmental Dynamics 235:468–477, 2006.

Fetal brain development in chimpanzees versus humans

Summary It is argued that the extraordinary brain enlargement observed in humans is due to not only the human-specific pattern of postnatal brain development, but also to that of prenatal brain development [1,2]. However, the prenatal trajectory of brain development has not been explored in chimpanzees ( Pan troglodytes ), even though they are our closest living relatives. To address this lack of information, we tracked fetal development of the chimpanzee brain from approximately 14 to 34 weeks of gestation (just before birth) in utero using three-dimensional ultrasound imaging. The results were compared with those obtained for the human brain during approximately the same period. We found that the brain volume of chimpanzee fetuses was only half that of human fetuses at 16 weeks of gestation. Moreover, although the growth velocity of brain volume increased until approximately 22 weeks of gestation in both chimpanzees and humans, chimpanzee fetuses did not show the same accelerated increase in brain volume as human fetuses after that time. This suggests that maintenance of fast development of the human brain during intrauterine life has contributed to the remarkable brain enlargement observed in humans.

Visualization of human prenatal development by magnetic resonance imaging (MRI).

It is essential to visualize the structures of embryos and their internal organs three‐dimensionally to analyze morphogenesis; this used to rely solely on serial histological sectioning and solid reconstruction, which were tedious and time‐consuming. We have applied imaging with a magnetic resonance (MR) microscope equipped with a 2.35 T superconducting magnet to visualize human embryos; we were successful in acquiring high‐resolution sectional images and in identifying the detailed structures of major organs. The imaging process was facilitated by using a super‐parallel MR microscope. A dataset of MR images of more than 1,000 human embryos, now collected, will be important for future biomedical research and for education.

Early pathogenesis of holoprosencephaly

Holoprosencephaly (HPE) is one of the most common malformations encountered in early human embryos. It is assumed that more than 90% of HPE embryos die in utero and are eliminated by spontaneous abortion. Embryonic HPE displays some characteristic craniofacial phenotypes, which are not necessarily comparable to those in postnatal HPE cases. In this article, we summarize our studies on HPE in human embryos and discuss the pathogenesis of HPE malformations.

Embryogenesis of holoprosencephaly.

Holoprosencephaly (HPE) is a malformation of the human brain caused primarily by incomplete division of the prosencephalon into two halves and is often associated with various facial anomalies. Although HPE is rather rare in newborns (1/10,000–15,000 births), it is frequently encountered in therapeutic abortuses (>1/250). To date, nine gene mutations responsible for human HPE have been identified, but the pathogenetic mechanisms of the craniofacial anomalies in HPE have just begun to be understood. Here, we summarize our studies on human embryos with HPE and discuss the embryogenesis and the underlying molecular mechanisms of HPE malformations under the following headings: pathology, pathogenesis, and critical period of development.

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.