J Vis Exp
April 12, 2016;
Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining.
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.
J Vis Exp
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Figure 1: Embryo arrangement and orientation in the section mould. (A) A cartoon depiction showing the mounting assembly, note the tray has been labelled with the date, stage, and the orientation of embryos. (B) An image showing the natural appearance of the mounting assembly.
Figure 2: The orientation and position of the gelatin block on the sample holding disc. (A) A cartoon depiction showing the section block assembly, note that embryos are in a "heads up" position and the gelatin block is firmly fixed onto the sample holding disc by the tissue-freezing medium at the base (see Figure 2B). (B) An image showing the natural appearance of the section block assembly, note the accumulation of the tissue-freezing medium between the gelatin block and the sample holding disc
Figure 3: Continuous sectioning. (A) Model of a section chamber. The sample holding disc should be rotated and fixed to the position that the embryo side is facing up. After a few (6-10) sections, the long strip of continuous section tiles are gently separated from the blade (blue) using a fine brush, and then turned 90° on the holding plate. Then a room-temp positively charged slide is firmly pressed onto the section strip facing down and immediately lifted up to collected finished strips. (B) Normally, each slide can accommodate 2 parallel lines of strips, as shown. Remember to make detailed record on the slide label using pencil.
Figure 4: Cross-section of stage 30 X. laevis tadpoles and staining in the neural tissue, 20X. (A) Schematics indicating the relative positions (forebrain, midbrain, hindbrain, and spinal cord) of section planes on Xenopus tadpoles. (B) Corresponding cross-sections stained with anti-Sox3 (Red), anti-acetylated-tubulin (Green), and DAPI (Blue), showing the relative locations of neural stem cell pools as well as neurofilaments within the neural tube. (C) Corresponding cross-sections stained with anti-MyT1 (Red) and DAPI (Blue), showing the relative locations of differentiated primary neurons within the neural tube. Scale bar = 20 µm.
X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation.