Assessing Primary Neurogenesis in <em>Xenopus</em> Embryos Using Immunostaining

Abstract

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

RNA helicase Mov10 is essential for gastrulation and central nervous system development

“…The ventricular zone, which, at this stage (NP36) is one to four cells thick, encloses the ventricle of the CNS, and contains neural precursor cells (NPCs) (Noctor et al, 2001; Rakic, 2009; Thuret et al, 2015), which can be identified by expression of Sox3 [SRY (sex determining region Y)-box 3] (Koyano et al, 1997; Macedoni-Luksic et al, 2009; Zhang et al, 2016) (Fig. 7).…”

“…We also examined the localization of MyT1 (Myelin Transcription factor 1), a marker for primary differentiated neurons (Bellefroid et al, 1996; Zhang et al, 2016), which are located more distally to the ventricle and are non-overlapping with the Sox3 positive NPCs (Thuret et al, 2015). In control embryos, neurons are localized outside of the ventricular zone and in particular, seem to be more lateral on the dorsal side (Fig.…”

“…For immunohistochemistry, the sections were deparaffinized using xylene and rehydrated before Heat-induced Antigen retrieval in 1X citrate buffer (pH-6.0). The slides were stained using the following primary antibodies at 0.5ug/ml- Tor-70 (notochord cells, a kind gift from Prof. Ray Keller), 12/101 (somitic mesoderm, Developmental Studies Hybridoma Bank, Iowa), at 1:500 dilution for Sox3 (neural precursor cells) and MyT1 (differentiated neurons, kind gift from Prof. Klymkowsky and Prof. Papalapoulo respectively) (Zhang et al, 2016). The secondary antibodies were Alexa Flour 488 (Jackson Immunoresearch, West Grove, PA) and RITC-conjugated IgM specific antibody (Jackson Immuoresearch) used at a concentration of 1:200.…”