Abstract-Descriptive Analysis of Cells in Xenopus laevis Embryonic Central Nervous System during Secondary Neurogenesis

This project will be an analysis of the distribution and lineage of proliferative embryonic cells during secondary neurogenesis in the Central Nervous System (CNS) of the African Clawed Frog, Xenopus laevis. The mechanism of embryonic neurogenesis in Xenopus laevis is a major focus in developmental neurobiology. While the first wave of neurogenesis, known as primary neurogenesis, occurs between gastrulation and stage 22 and is widely studied, much less is known about the later waves of neurogenesis, commonly referred to as secondary neurogenesis.

The project investigates the distribution of different cells in CNS by looking at the co-localization of different types of cell markers in double in situ hybridization (ISH). NBT, the neuronal differentiation marker, Sox2, the neural progenitor cell marker and PCNA, the proliferating cell marker are the three types of mRNA probes that will be used in this aspect of the project. The double ISHs will be performed using the probe combination of NBT/Sox2 and PCNA/Sox2, each on stage 20, 25 and 30 embryos. After the double ISH, embryos will be sectioned and imaged. For NBT/Sox2 embryos, it provides information on the distribution of neural progenitors and differentiated neural cells in the CNS; for PCNA/Sox2 embryos, the region of co-localization indicates the proliferating neural progenitor cell population. The number of cells expressing each/both markers will also be counted to obtain a quantitative analysis. If time permits, a lineage analysis using a plasmid construct will also be performed. The plasmid construct is a fluorescent protein reporter driven by repeats of Sox2 binding domain (Bestman et al., 2012). The construct will be expressed in the cells using electroporation during earlier stages of development. As the embryo grows, only the cells that express Sox2 will be fluorescently labeled. By doing in vivo time lapse imaging of the embryo, it not only reveals how neural progenitor cell population in one single embryo changes over time (which can’t be done using ISH), but also allows us to trace individual cells and document their developmental pathways.

Although secondary neurogenesis is really common in “lower level” vertebrates, it doesn’t exist in humans. By discovering the mechanisms of secondary neurogenesis in Xenopus laevis as well as other model organisms, we can compare that to the cells in human CNS and figure out why humans don’t have secondary neurogenesis. The process of secondary neurogenesis is likely related to the ability of amphibians to regenerate neural tissue, a process that does not readily occur in humans.  On top of that, we might even be able to manually induce secondary neurogenesis using the neural progenitor cells in human brain. If the measure is successful, it would have huge applications in neurological and regenerative medicine.