Roles of Wnt signalling during neural differentiation of embryonic stem (ES) cells

• Main Author: Nordin, Norshariza
• Published: University of Edinburgh 2006
• Subjects: 611
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660044

In order to investigate how Wnt signalling affects the differentiation of ES cells into neurons <i>in vitro. </i>RNA expression of all 19 Wnt genes together with two Wnt antagonists, <i>Dkk1 </i>and <i>sFRP2, </i>during the process was first determined by RT-PCR. Of 19 Wnt genes, the expression of 12 with particularly interesting patterns was subsequently analysed by quantitative RT-PCR (qRT-PCR). Neural differentiation of ES cells was induced through the formation of embryoid bodies (EBs) and the addition of retinoic acid (RA). The gene expression was determined in five different stages of the process including undifferentiated ES cells, early EBs prior to the addition of RA, EBs in the presence of RA, attached EBs after withdrawal of RA and finally neuron-like cells grown on an adhesive substratum. Many <i>Wnt </i>genes showed dynamic alterations in expression levels during neural differentiation. In order to test the effect of expressing Wnts at different stages, we developed an inducible expression system. The system is based on cre-<i>loxP</i> strategy and allows for stimulation or inhibition of Wnt signalling at specific steps of the differentiation process. Overexpression of <i>Wnt1-</i>HA and <i>Wnt3a </i>at early and late stages of the process in addition to constitutive expression of these genes was carried out. Additionally, inhibition of Wnt signalling by overexpressing <i>Dkk1 </i>at early and late stages as well as its constitutive expression during the differentiation process was also conducted. The effects of altered Wnt signalling on the formation of neural precursor cells (npc) and neuronal cells was analysed using specific protein markers by Immunocytochemistry and fluorescent-activated cell sorting (FACS) analysis. It was found that overexpression of <i>Wnt1 </i>and <i>Wnt3a </i>at the early stages of the differentiation process significantly reduced the formation of npc while inhibition of Wnt signalling at this stage by induction of <i>Dkk1 </i>significantly increased the number of npc. Interestingly, constitutive expression of each of these genes reduced the number of npc. Formation of neurons was stimulated by overexpression of <i>Wnt3a </i>at late stages of neural differentiation process, whereas induction of <i>Wnt1 </i>at this stage enhanced the percentage of neurons in cultures. Overexpression of <i>Dkk1 </i>at this stage however significantly decreased the formation of npc. A significant increase in the percentage of neuron formation was observed when <i>Dkk1 </i>and <i>Wnt1</i> were overexpressed at early stages of the process. In contrast, overexpression of <i>Wnt3a </i>at this stage significantly reduced the number of neurons. Additionally, constitutive stimulation or inhibition of Wnt signalling during the process was observed to reduce the ability of ES to differentiate into neurons. These observations therefore highlight the complexity of probable function of stage-dependent response to Wnt signalling during neural differentiation.