Netrin-1-Mediated Axon Guidance in Mouse Embryonic Stem Cells Overexpressing Neurogenin-1

Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98473/1/scd%2E2011%2E0437.pd

Lineage differentiation of embryonic stem cells.

Understanding events that regulate the generation of multiple cell and tissue types during mammalian development is complicated by the relative inaccessibility of the early embryo. Embryonic stem cells (ESC) are derived from and are thought to share a similar gene expression profile as the pluripotent inner cell mass (ICM) of blastocyst-staged embryos. ESC can be maintained as a self-renewing population indefinitely in culture, and yet, like the ICM, retain the potential to differentiate into all cell types of the body. These properties, along with the ability to manipulate these cells in vitro, constitute a powerful model system with which to study the roles of genes involved in maintaining pluripotency as well as those involved in lineage segregation and differentiation. Based on the premise that manipulating the expression of genes in ESC can provide insight into the regulation of cell fate choice during early development, a loss-of-function approach utilizing RNA interference (RNAi) was employed to knock down expression of the pluripotency factor, Oct4, in ESC resulting in trophectodermal differentiation even in culture conditions that inhibit differentiation. These results demonstrate the critical role of Oct4 in maintaining pluripotency and establish RNAi as a viable loss-of-function approach to study gene function in ESC. In subsequent studies, an inducible gain-of-function approach was taken to evaluate whether forced expression of a pro-neural bHLH gene, Neurogenin1, is sufficient to promote neuronal differentiation in ESC. Transient expression of Ngn1 in ESC resulted in widespread neuronal differentiation, even in conditions that inhibit differentiation. However, the induced cells were demonstrably sensitive to patterning factors including retinoic acid, BMP4, noggin, Shh, and FGFs, indicating that neurogenesis induced by Ngn1 expression likely proceeds through intermediate progenitor cell stages. Induced cells were also implanted into the neural tubes of chick host embryos, and preliminary results suggest that they integrated into domains of the peripheral and central nervous system where Ngn1 is expressed in vivo. Together, these studies show that RNAi, inducible transgene expression, and transplantation of ESC are powerful techniques to study factors regulating cell fate choice. These studies form the basis for future work to better understand lineage segregation during embryogenesis.Ph.D.Biological SciencesCellular biologyMorphologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125530/2/3192805.pd