Vertebrate central nervous system develops from a neural tube derived from the embryonic ectoderm. In mouse, the neural tube around embryonic day 10 primarily consists of neural precursor cells (NPCs). During the development of embryonic central nervous system, NPCs proliferate and migrate outward; thus later stages show NPCs toward the lumen of the neural tube and neurofilament-positive differentiated cells toward the periphery. In conventional liquid culture, NPCs isolated from mouse on embryonic day 10 proliferate and differentiate into neurofilament-positive neurons. In the present communication, we show that fragments of neural tubes and aggregates of NPCs, when placed into collagen gel matrix, form three-dimensional structures which resemble the neural tube formed in vivo in the developing embryos. Even dissociated NPCs form the three-dimensional structures in the collagen gel matrix. Our results indicate that individual NPCs or fragments of neural tubes carry morphogenetic information which allows them to reconstruct neural tube-like structures in vitro.Vertebrate central nervous system (CNS) derives from a neural tube, in which pluripotent neural precursor cells (NPCs) proliferate and differentiate into various types of neuronal and glial cells (1-4). The neural tube is initially consisted of pseudostratified NPCs, then increases in thickness as NPCs proliferate, and finally becomes multilayered. In mouse, neurons become detectable in the hindbrain region around embryonic day 10 (E10) and are localized only in the marginal zone of the tube (5). The spatial as well as temporal control of CNS development requires the precise proliferation, migration, and differentiation of NPCs. Although the dynamic morphogenesis in the early CNS development has attracted investigators for near a century (6-9), the mechanisms controlling the programmed development of NPCs remain poorly understood, primarily due to technical difficulties associated with the study of mammalian CNS development.As the first step to circumvent these difficulties we have established a simple method to isolate NPCs from embryonic mouse head at E10 (10). In primary cultures, NPCs differentiate into neurons and glia by following a time schedule similar to that observed in embryos. These findings prompted us to develop models of the CNS development in vitro. Collagen gel has been used as a matrix for studies of threedimensional growth, differentiation, and morphogenesis of epithelial cells (11-16). In this study, we have utilized collagen gel culture of NPCs to observe their morphogenetic behavior three-dimensionally. NPCs were found to proliferate, extend neurites, and reconstruct neural tube-like structures in collagen gel matrix.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
MATERIALS AND METHODSNPC Preparation and Culture. An enzymatic method for preparation of NPCs from E10 em...