Investigating the role of Neuronatin in neural development

Neural induction is the process that occurs during embryogenesis whereby ectoderm is converted into neural tissue. However, the cellular and molecular mechanisms that govern this process are still not completely understood. For instance, in Xenopus, calcium signalling is known to play a pivotal role in neural induction but whether a similar mechanism occurs in mammals is unknown. As part of a wider study on genes regulating neural stem cell (NSC) development, we have discovered a novel role for Neuronatin (Nnaf), a regulator of intracellular Ca2+ concentration. In my thesis, I describe the use of a mouse embryonic stem cell (ESC)-derived neural differentiation model to dissect the function of Nnat in neural development. Using gain- and loss-of function experiments I show that ESCs in which Nnat expression had been knocked-down have a dramatically decreased ability to generate NSCs, and subsequently neurons, while ESCs that over-expressed Nnat generate an excess of NSCs and neurons. I reveal that Nnat interacts directly with the sarcoendoplasmic reticulum Ca2+-ATPase 2 (Apt2a2/SERCA2), and that Apt2a/SERCA inhibitors cause an increase in cytosolic calcium levels rescuing the ability of Nnat knockdown cells to generate neural cell-types. From these experiments I conclude that Apt2a/SERCA inhibition mimics the function of Nnat and, via the increase of cytosolic calcium, causes neural induction in Nnat knocked-down ESCs. I go on to show that this increase in cytosolic Ca2+, caused by inhibiting Apt2a/SERCA, activates Erk signalling and that inhibition of Erk signalling dramatically reduces the ability of ESCs to generate NSC. Together these results suggest that Nnat functions by increasing cytosolic Ca2+ levels, which in turn causes activation of Erk signalling leading to neural induction. In line with this, the expression of BMP4, which is an inhibitor of neural induction, was found to be up-regulated by Nnat knocked-down.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Lin, Hsuan-Hwai

Advisor dc:contributor.advisor

• Williams, Brenda Patricia

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/c7fad85a-bdaf-4d98-bc6b-8b465c8675bb

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/c7fad85a-bdaf-4d98-bc6b-8b465c8675bb