Generation of mouse embryonic stem cell reporter lines to identify developmental stages during induction of dopaminergic neurons
Creators: name: Bellinge, Rhys Henry Stone
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: The most fundamental questions on how neurons arise from specific developmental programs focus on how they acquire their correct synaptic connections, and how they mature and develop their distinct phenotypes. It is known in a broad sense that each group of neurons in the functional circuits of the adult brain develops according to both intrinsic programming of molecular cascades, and extrinsic influences from their environments. Here I set out to investigate the cells of the rostral region of the central nervous system, those of the mesencephalon, or midbrain, by the use of murine embryonic stem (ES) cells as an in vitro model of midbrain dopaminergic (mDA) progenitor cells. The focus of the work was to introduce genetic reporters into ES cells to allow identification and potentially isolation of neuronal progenitors during in vitro differentiation of ES cells. mDA neurons arise in the ventral midline of the midbrain, and are intrinsic in such functions as fine motor control, emotion, perception and higher cognition, as well as learning and reward. Loss of these neurons in the substantia nigra leads to the symptoms of Parkinson's disease (PD). Before ES cell therapies can attempt to treat PD, the signalling factors involved in the early specification of mDA neurons need to be thoroughly characterised. Differentiation of ES cells using existing protocols does not result in homogeneous populations of neural progenitors. Typically, mouse ES cells differentiate into neural progenitors by default once ES cell growth supplements are removed, but rarely are these mDA progenitor neurons. In this thesis, the neural induction protocol was first investigated to examine whether it was generating neural progenitors of a midbrain phenotype using an Lmx1a-eGFP reporter cell line, as a study control. The induction protocol was found to generate, at its peak, 8.1% of the total cell population expressing the fluorescent reporter. This indicated that the induction protocol was not yielding enriched populations of Lmx1a+ neural progenitors. Additionally it was considered that it may be that not all of the Lmx1a-eGFP+ cells were destined to become dopaminergic neurons, and that perhaps more reporters were needed to identify midbrain dopaminergic neurons. Following this, a series of homologous recombination experiments were performed, initially successfully producing an Lmx1b-CBR-IRES-dsRed reporter in a murine ES (mES) cell line, with 17.9% targeting efficiency. Following this a series of targeting experiments were carried out in an attempt to target a Pitx3-mCitrine reporter to an existing Lmx1a-AMP mES reporter cell line. Unfortunately, despite the expansion and screening of over 1000 antibiotic resistant colonies over a 15 month period, this targeting campaign was not successful. In an effort to assess the cause for this lack of success, an additional targeting experiment was successfully performed, using an Lmx1a-mCFP targeting vector to recombine with a wild-type mES cell line, with a 3.45% targeting efficiency. In all likelihood the failure of the Pitx3-mCitrine to target adequately lies with the design of the targeting vector and primers used; the homology of the vector to host genome was 7kb in total, which may in fact have been in excess when considering that the target, Pitx3, is expressed late in the differentiation of DA neurons, giving rise to the possibility that the DNA is tightly bound and comparatively inaccessible at this undifferentiated state. The intention for the generation of dual Lmx1a-postive/Pitx3-positive fluorescent reporter cell lines was to enable the identification of DA neuronal progenitors, with the goal of ultimately using these cells to isolate DA cells at definite specific stages of development prior to transplantation into a mouse model of PD. Although the Pitx3-mCitrine targeting did not succeed, the neural induction protocol was successfully assayed using an Lmx1a-eGFP reporter cell line, a novel Lmx1a-mCFP reporter cell line was created, as was a novel Lmx1b-CBR-dsRed cell line.