On the Enigma of the Human Neurenteric Canal.

Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped "hinge of involuting notochordal cells" within Hensen's node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.

Paraxial Nodal Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species.

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel 'parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.

Modulation of cell surface architecture in gastrulating chick embryo in response to altered fibroblast growth factor (FGF) signaling.

Gastrulation is a fundamental process that results in formation of the three germ layers in an embryo. It involves highly coordinated cell migration. Cell to cell communication through cell surface and the surrounding molecular environment governs cell migration. In the present work, cell surface features, which are indicative of the migratory status of a cell, of an early gastrulating chick embryo were studied using scanning electron microscopy. The distinct ultrastructural features of cells located in the various regions of the epiblast are described. Differences in the surface features of cells from distinct embryonic regions indicate differences in their migratory capacities. Further, the dynamic nature of these cell surface features by their response to altered fibroblast growth factor (FGF) signaling, experimentally created by using either excess FGF or inhibition of FGF signaling are demonstrated.

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo.

Motile monocilia play a pivotal role in left-right axis determination in mouse and zebrafish embryos. Cilia with 9+0 axonemes localize to the distal indentation of the mouse egg cylinder ("node"), while Kupffer's vesicle cilia in zebrafish show 9+2 arrangements. Here we studied cilia in a prototype mammalian embryo, the rabbit, which develops via a flat blastodisc. Transcription of ciliary marker genes Foxj1, Rfx3, lrd, polaris, and Kif3a initiated in Hensen's node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen's node anteriorly to form the notochordal plate. Cilia lengthened to about 5 mum and polarized from an initially central position to the posterior pole of cells. Electron-microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. Our data suggest that despite a highly conserved ciliogenic program, which initiates in the organizer, axonemal structures may vary widely within the vertebrates.

Morphological left-right asymmetry of Hensen's node precedes the asymmetric expression of Shh and Fgf8 in the chick embryo.

Hensen's node and the rostral part of the primitive streak of chick embryos at HH-stage 4-7 were investigated using scanning electron microscopy, a series of semithin sections, and whole-mount in situ hybridization. An asymmetric expression of Shh and Fgf8 was first found at HH-stage 5. The asymmetric expression of both laterality genes is preceded by an asymmetric morphology of the avian organizer. The right lip of the streak and the node is much more prominent than the left one and contains a cylindrical cell condensation that is connected with the head process. Since the densely packed cells in Hensen's node and in the cranial part of the primitive streak connect the epiblast with the endoderm, a cilia-generated "nodal flow" between epiblast and endoderm in the avian embryo seems to be unlikely.

Does an equivalent of the "ventral node" exist in chick embryos? A scanning electron microscopic study.

The internal organs of vertebrates show species-specific left-right (L-R) asymmetries. Questions on the embryonic origin of these asymmetries have been fascinating embryologists since the 19th century. During the past years, remarkable progress has been made in answering these questions. Evolutionary highly conserved molecular signaling cascades have been identified that start from Hensen's node and transfer side-specific identity to the embryonic left and right halves. However, the question of what initiates these signaling cascades has remained unanswered. Studies on mouse embryos have shown that the ventral surface of Hensen's node consists of a ciliated epithelium called the ventral node. Recent findings suggest that the monocilia of ventral nodel cells generate a leftward flow of extracellular fluid possibly leading to the accumulation of an unknown morphogen at the left of the node, which then might start the signaling cascades. This hypothesis might explain the fact that gene defects causing ciliary dyskinesia are frequently associated with situs anomalies. Studies on chick embryos led to the discovery of the L-R signaling cascades. However, whether an equivalent of the ventral node exists in avian embryos remained unknown. Therefore, I examined the endoderm and epiblast of early chick embryos for the presence of monociliated cells. In the endoderm, a population of monociliated cells indeed was present. These cells, however, were neither confined to the area of Hensen's node nor did they form the predominant cell population at this location. In the epiblast, monociliated cells formed the predominant cell population at the periphery of the blastodisc but only a relatively small subpopulation of epiblast cells at Hensen's node. These findings suggest that, in the early chick embryo, an equivalent of the ventral node of mouse embryos neither exists on the ventral nor the dorsal surface of Hensen's node. It is unlikely that nodal cilia are required for initiating the L-R patterning in chick embryos.

Electrophysiological evidence for low-resistance intercellular junctions in the early chick embryo.

Electrophysiological evidence is presented for the exchange of small ions directly between cells interiors, i.e. "electrical coupling," in the early chick embryo. Experiments with intracellular marking show that coupling is widespread, occurring between cells in the same tissue, e.g. ectoderm, notochord, neural plate, mesoderm, and Hensen's node, and between cells in different tissues, e.g. notochord to neural plate, notochord to neural tube, notochord to mesoderm. The coupling demonstrates the presence of specialized low-resistance intercellular junctions as found in other embryos and numerous adult tissues. The results are discussed in relation to recent electron microscopical studies of intercellular junctions in the early chick embryo. The function of the electrical coupling in embryogenesis remains unknown, but some possibilities are considered.