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Draft #4, week 15, figure/methods analysis for sensory neurobiology

Submitted by vvikhrev on Tue, 05/01/2018 - 12:20

FIG 1 - before the actual experiment, they needed to make sure that the tau_lacZ would be transported down the OSN axons and be visualized
- replace the exon OMP (unknown function) with tau_lacZ during homologous recombination
- use cre Recombinase to cut at the loxP sites to removes tkneo
- before they began this experiment, what was known? what kind of topographic mapping? the unknown?
- receptor activation in the bulb evidence derives from ISH experiments that detect the presence of specific receptor mRNAs at
convergent loci in the OB, but this doesn’t permit the visualization of the projection pathways but only identifies sites of axonal
convergence, also w/ this technique, can’t see single axons
FIG 2 – since OMP is expressed in all OSNs, you see intense X-gal staining in OSN axons and in OB glomeruli
- conclusion = tau_lacZ fusion protein is efficiently transported down axons
FIG3 – generate P2 tau_lacZ mice, restrict tau_lacZ to subset of OSNs and use those that express the P2 receptor
- if you just replaced P2 coding sequence w/ tau_lacZ then P2 expression would be eliminated from the modified allele but OSNs express a receptor from only one of the 2 alleles therefore cells expressing the modified P2 allele would express tau_lacZ but not the receptor and cells expressing the WT P2 allele would express the receptor but not tau_lacZ
- to assure that cells expressing tau_lacZ also express a fnuctional P2 receptor, they designed a targeting vector that would result in a
bicistronic mRNA
FIG 4 - see expression of lacZ in P2-IRES-tau_lacZ mice as early as E12.5, also no wandering axons (they saw that these results matched w/ the ISH experiment mentioned above), only a subset of OSNs express tau_lacZ = two distinct glomeruli receive projections from P2-tau_lacZ OSNs
FIG 5 – do P2-tau_lacZ OSNs project to the same glomeruli as WT P2 neurons?
- b/c in the P2-tau_lacZ mouse the other allele is WT
- 50% of P2 OSNs express the WT and the P2-tau_lacZ = monoallelic
FIG 7 – Receptor Swap replace P2 w/ M12 receptor (into P2 locus)
- model 1: OR is sole determinant of axon targeting therefore replacing P2 w/ M12 should target these OSN axons to the “M12 glomeruli”
- model 2: OR plays no role in targeting therefore replacing P2 w/ M12 won’t effect OSN targeting and the axons will go to “P2 glomeruli”
Results: project v. close to P2 (not M12) glomeruli (true in heter- and homo-)
- M12 to P2 projections about 200 micrometers posterior to WT P2 projections
- this suggest that OR is not the sole determinant of axonal targeting but OR is an important factor
So what is the role of OR in axon targeting? - data suggest that OR plays an instructive role in guidance process
- OR expressed in noth axons and OR may recognize guidances cues presented by OB cells independently of cilia OR activation
- activity is important – synchronous firing of subset of OSN expressing a specific OR could result in the segregation of M12P2
OSNs at sites close to but distant from P2 OSNs

source: (Mombaerts, Peter et al. "Visualizing an Olfactory Sensory Map." Cell, Vol. 87, 675–686, 15 Nov. 1996.)