| Abstract |
Embryonic olfactory neuroepithelium provides a useful experimental system
for the study of olfactory neurogenesis. As a substrate for experimental
neural cell biology, olfactory neuroepithelium is of unique interest
since, unlike other neural cells, olfactory neurons are continually
replaced -a feature that is dictated by their direct exposure to the
damaging external environment. Basal cells in the olfactory placode are
the source of this replacement. Each olfactory neuron expresses only one
or a few of the many olfactory receptors that are encoded by the large
array of olfactory genes. Despite this limited cellular display of
receptors, vertebrates are able to distinguish many thousands of different
odorants, implying a complicated need for perceptive neurological
processing of signals coming from individual olfactory neurons. To study
the events that take place during the differentiation of neuronal
precursors - a process that sustains a diverse receptor repertoire - I felt
that lines of conditionally immortalised cells that could be induced to
differentiate would provide useful reagents. In this thesis I describe my
successful attempts to immortalise olfactory cell lines from the
neuroepithelium of E 10.5 mouse embryos. I used a conditionally
immortalising retrovirus that included the coding sequence for the
temperature-sensitive SY 40 large T antigen. Integration of this
retrovirus into the genome of cells allowed continuous proliferation at
the permissive temperature of 33 Celsius. A shift to the nonpermissive
temperature of 39 Celsius inactivated the SV40 large T antigen, the cells ceased
proliferation and differentiation commenced. Sixty cell lines were derived
of which four were chosen for further characterisation. These four cell
lines (OP6, OP27, OP47 and OP55) were clonally derived and were
immortalised rather than transformed. They continued to express the SV40
large T antigen at 33 Celsius but lost expression at 39 Celsius concomitant with
cessation of proliferation. When the OP cells were shifted to 39 Celsius in the
absence or presence of the morphogen, retinoic acid, morphological changes
ensued that were consistent with the development of neuronal
characteristics. The OP6, OP27 and the OP47 cells became phase-bright with
neuritic extensions. The OP55 cells were the exception in that they did
not develop extensions but instead differentiated to form compact
epithelial islands when grown in DM-10 medium but not in RA medium.
Differentiation of the OP cells at 39 Celsius was further documented by the
induced expression of a number of markers demonstrated by RT-PCR and/or
immunocytochemistry. The OP cells differentiated at 39 Celsius in DM-10 and
in retinoic acid-containing medium to express olfactory receptor transcripts.
Cloning and sequencing showed that each cell line expressed a single
receptor type but that different receptors were expressed by different
cell lines. Sequencing revealed that the receptors cloned from the OP27
cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55
had greatest homology to rat-Olf3 olfactory receptor. The transcripts
induced in OP6 and the OP47 cells showed greatest homology with Gus58 -a
taste receptor homologous to olfactory receptors. Sequences obtained from
OP6, OP47 and OP55 cells were not 100% identical to published receptors
and could thus represent members of different subfamilies. Interestingly,
induced OP55 cells also expressed mRNA for clusterin - a molecule that has
no homology with olfactory receptor transcripts but is involved in
differentiation during embryogenesis.
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