ETD

Digital archive of theses discussed at the University of Pisa

 

Thesis etd-06162014-153316


Thesis type
Tesi di laurea magistrale
Author
KOCIAJ, ANITA
URN
etd-06162014-153316
Thesis title
Studing the role of Lmx1a during midbrain dopamine neuron specification using NesE-Lmx1a gain of function cell line
Department
BIOLOGIA
Course of study
BIOLOGIA MOLECOLARE E CELLULARE
Supervisors
relatore Prof. Pasqualetti, Massimo
correlatore Ori, Michela
correlatore Dott.ssa Garcia Gil, Maria De Las Mercedes
Keywords
  • RT-qPCR
  • dopamine
  • cell differentiation
  • EpiSC
  • ESC
  • mDA neuron
  • Transcriptome profiles
  • RNAseq
Graduation session start date
21/07/2014
Availability
Full
Summary

The midbrain dopaminergic (mDA) neurons constitute about 75% of dopaminergic neurons in the adult brain. These neurons are involved in the control of voluntary movements and in the regulation of emotion - related behaviour. Loss of mDA neurons can cause Parkinson’s disease and other neurological disorders as well. In order to understand the selective degeneration of these neurons, insight in the pathways and factors involved in the development and maintenance of this subset of dopaminergic neurons is needed. On the other hand, the prospect of using stem–cell derived DA neurons has emphasized the requirement of understanding the normal pathway of DA neuron development. Among the factors implicated in mDA neuron development are Wnt1, Wnt5a, En1/2, Otx2, Foxa1, Foxa2, Ngn2, Nurr1, Pitx3, Msx1 and LIM homeodomain transcription factors Lmx1a and Lmx1b.
Several studies suggested a role for Lmx1a in establishing a mDA neuronal phenotype (Andersson et al., 2006; Chung et al.,2009). Gain and loss of function studies in chick revealed that Lmx1a is required for the specification of mDA neurons. Furthermore Lmx1a can induce mouse embryonic stem (mES) cells into DA neurons. Together these experiments in chick and in mES cells suggest an essential role for Lmx1a in determination of mDA neurons.
Beside several studies suggesting a role for Lmx1a in proliferation and neurogenesis, the precise role of Lmx1a in the mouse mDA is still not fully understood.
Therefore, to understand the Lmx1a phenotype in depth we studied the gain of function of Lmx1a in NesE-Lmx1a gain of function cell line.
In this cell line Lmx1a is under the control of the enhancer of Nestin. Using NesE-Lmx1a cell line Andersson and colleagues (Andersson et al., 2006) showed that Lmx1a functions as DA neuron determinant for midbrain dopamine neurons in mES cells.
We were interested to know the molecular network that Lmx1a activates to do that. To elucidate the molecular programming we performed qPCR analysis on d5 and d9 NesE-Lmx1a cells.
RT-qPCR analysis at d5 showed that several genes involed in midbrain dopamine neuron specification, differentiation and migration such as Ngn2, Foxa2, Nurr1, Rspo2, Slit2, Aldh1a1 were upregulated.
At d9 qPCR results showed that Tuj1, which is used as marker for postmitotic neuronal cells was upregulated in NesE-Lmx1a compared with control cells and so Nurr1.
Altogether these data indicate that Lmx1a is essential for the correct development of mDA neurons and it does that by regulating the expression of of several genes involved in dopamine neuron differentiation and migration.
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