Tadayoshi Watanabe

EphrinB2 coordinates the formation of a morphological boundary and cell epithelialization during somite segmentation

During early morphogenesis, tissue segregation is often accompanied by changes in cell shape. To understand how such coordination is regulated, somitogenesis was used as a model. When a somite forms in the anterior end of the presomitic mesoderm, an intersomitic boundary (gap) emerges, and it is rapidly followed by cell epithelialization at this border. It has been known that the gap formation is regulated by intercellular signals. We here demonstrate that cMeso-1, the chicken homolog of mouse Mesp2, up-regulates EphA4 in the cells located posteriorly to a forming boundary. This in turn activates EphrinB2-reverse signals in the anteriorly juxtaposed cells, where the EphrinB2 signal is sufficient to cause a gap formation and cell epithelialization cell-autonomously. During these processes, Cdc42 needs to be repressed via tyrosine phosphorylation of EphrinB2. This is the first demonstration that Ephrin-reverse signal acts as a platform that couples distinct morphogenetic changes in cell polarity and tissue shape.

Notch Mediates the Segmental Specification of Angioblasts in Somites and Their Directed Migration toward the Dorsal Aorta in Avian Embryos

We studied, using avian embryos, mechanisms underlying the three-dimensional assembly of the dorsal aorta, the first-forming embryonic vessel in amniotes. This vessel originates from two distinct cell populations, the splanchnic and somitic mesoderms. We have unveiled a role for Notch signaling in the somitic contribution. Upon activation of Notch signaling, a subpopulation of cells in the posterior half of individual somites migrates ventrally toward the primary dorsal aorta of splanchnic origin. After reaching the primary aorta, these somitic cells differentiate into the definitive aortic endothelial cells. This Notch-induced ventral migration is mediated by EphrinB2 and by an attractant action of the primary aorta. Furthermore, long-term chasing of cells by transposon-mediated gene transfer reveals that the segmentally provided endothelial cells of somitic origin in the dorsal aorta ultimately populate the entire region of the vessel. We demonstrate the molecular and cellular mechanisms underlying the formation of embryonic blood vessels from mesenchymal cells.

Chapter 14 Transposon-Mediated Stable Integration and Tetracycline-Inducible Expression of Electroporated Transgenes in Chicken Embryos

Publisher Summary This chapter describes the tetracycline (Tet)-dependent inducible expression system, optimized for manipulations in chickens and a novel technology that exploits transposon-mediated gene transposition to allow stable integration of transgenes into the genome. The combination of these two methods allows a stably integrated transgene to be conditionally expressed in cells in a temporally controlled manner at relatively late stages in development, including during organogenesis. The method of Tet-dependent inducible expression is used for diverse studies, ranging from in vitro cell cultures to mouse embryos. Both the reverse Tet-controlled transcriptional activator and the Tet-controlled transcriptional activator act on the cis-element promoter, tetracycline-responsive element. The Tol2-mediated transposition approaches are novel techniques for the molecular manipulation of chicken embryos by which an exogenous gene can be integrated into the host genome. The integrated gene can be stably expressed when driven by a ubiquitous promoter. This method is used for conditional expression when combined with the Tet-on system to achieve temporal control. The Tol2-transposition method allows for expression at stages even later than 3 days postelectroporation (E5), which otherwise could not be analyzed with the conventional electroporation technique. These developmental stages are critical for organogenesis, where numerous tissues interact to generate the complex structures of functional organs. Thus, the techniques presented are useful for understanding the way cells contribute to organogenesis at the molecular level.

Newly raised anti-VAChT and anti-ChAT antibodies detect cholinergic cells in chicken embryos

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O‐acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.

Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.