The closure of the neural tube (NT) in the human embryo has generally been described as a continuous process that begins at the level of the future cervical region and proceeds both rostrally and caudally. On the other hand, multiple initiation sites of NT closure have been demonstrated in mice and other animals. In humans, based on the study of neural tube defects (NTD) in clinical cases, van Allen et al. (1993) proposed a multisite NT closure model in which five closure sites exist in the NT of human embryos. In the present study, we examined human embryos in which the NT was closing (Congenital Anomaly Research Center, Kyoto University) grossly and histologically, and found that NT closure in human embryos initiates at multiple sites but that the mode of NT closure in humans is different from that in many other animal species. In addition to the future cervical region that is widely accepted as an initiation site of NT closure (Site A), the mesencephalic-rhombencephalic boundary was found to be another initiation site (Site B). The second closure initiating at Site B proceeds bidirectionally and its caudal extension meets the first closure from Site A over the rhombencephalon, and the rostral extension of the second closure meets another closure extending from the rostral end of the neural groove (Site C) over the prosencephalon, where the anterior neuropore closes. The caudal extension of the first closure initiating at Site A was found to proceed all the way down to the caudal end of the neural groove where the posterior neuropore is formed, indicating that in humans, NT closure does not initiate at the caudal end of the neural groove to proceed rostrally. Since there is a considerable species difference in the mode of NT closure, we should be careful when extrapolating the data from other animals to the human. It seems that the type of NTD affects the intrauterine survival of abnormal embryos. Almost all the embryos with total dysraphism appear to die by 5 weeks of gestation, those with an opening over the rhombencephalon by 6.5 weeks, and those with a defect at the frontal and parietal regions survive beyond 7 weeks.

The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.

Development of the posterior neural tube (PNT) in human embryos is a complicated process that involves both primary and secondary neurulation. Because normal development of the PNT is not fully understood, pathogenesis of spinal neural tube defects remains elusive. To clarify the mechanism of PNT development, we histologically examined 20 human embryos around the stage of posterior neuropore closure and found that the developing PNT can be divided into three parts: 1) the most rostral region, which corresponds to the posterior part of the primary neural tube, 2) the junctional region of the primary and secondary neural tubes, and 3) the caudal region, which emerges from the neural cord. In the junctional region, the axially-condensed mesenchyme (AM) intervened between the neural plate/tube and the notochord at the stage of posterior neuropore closure, while the notochord was directly attached to the neural plate/tube in the most rostral region. A single cavity was found to be formed in the AM as the presumptive luminal surface cells were radially aligned in the junctional region prior to the formation of the neural cord. The single cavity was continuous with the central cavity of the primary neural tube. In contrast, multiple or isolated cavities were frequently observed in the caudal region of the PNT. Our observation suggests that the junctional region of the PNT is distinct from other regions in terms of the relationship with the notochord and the mode of cavitation during secondary neurulation.

*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,

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.