Slit/Robo signaling underlies the spatial patterning of spiral ganglion neurons to shape the peripheral auditory circuitry assembly

During development, proliferating neuroblasts delaminate from the otocyst to generate spiral ganglion neurons which settled down in Rosenthal’s canal medial to the cochlea sensory epithelium. Subsequently spiral ganglion neurons extend their peripheral axons back to the cochlea by penetrating through the spiral limbus and Kolliker’s organ and ultimately form the tonotopically organized innervations with cochlea hair cells. How spiral ganglion neurons territory is defined along the medial-lateral axis is largely unknown. In this study, we performed microarray analysis and identified Slit molecules as potential candidates involved in this process. Analysis of Slit2 mutant mouse embryos showed that a significant number of spiral ganglion neuron soma are not restrained in Rosenthal’s canal but rather spread randomly over the cochlea tissue, some even go beyond the hair cells. These mis-positioned spiral ganglion neurons extend their merits largely along the longitudinal axis and randomly travel within the cochlea tissue without innervating hair cells. Similar phenotypes are also observed in embryos with mutations for Robo, the receptors for Slit molecules. Furthermore, the spiral ganglion territory is dramatically expanded towards the sensory epithelium in Robo mutant in addition to those individually displaced cells. In situ hybridization showed Robo1 and Robo2 are expressed in spiral ganglion neurons and Slit 2 and Slit3 are expressed in the greater epithelium ridge region (Kolliker’s organ) as well as spiral limbus region during the time spiral ganglion neurons innervate cochlea hair cells. Developmental studies revealed that these SGNs progressively moved to ectopic positions from their normal locations in the Rosenthal’s canal during the time SGNs innervate HCs. We propose that this disruption of spatial patterning of SGNs is attributed to the loss of the restriction force imposed by Slit/Robo signaling, which serves to refrain SGNs from invading the cochlear epithelium and to ensure the formation of precise innervation patterns in the peripheral auditory circuitry. ❧ In the second part of the thesis, we want to dissect the cell lineage relationship between cochlear hair cells and their neighboring supporting cells, which is largely elusive primarily due to the lack of a straightforward genetic method to perform the lineage tracing in the inner ear. To address this need, we developed a method termed STARS: stochastic gene activation with genetically regulated sparseness. The stochastic expression was achieved by two cross-linked, mutually-exclusive Cre-mediated recombinations. The stochastic level was further controlled by regulating Cre/lox reaction kinetics through varying the intrachromosomal distance between the lox sites mediating one of the recombinations. We further explore the possibility to extend the sparseness level by mutagenizing lox sites employed in the STARS transgene. In mammalian cell lines stably transfected with a single copy of different STARS transgenes, the activation/knockout of reporter genes was specifically controlled to occur in from 5% to 50% of the cell population and further down to 1% when combined with lox variants with lower recombination efficiency. STARS can potentially provide a convenient way for genetic labeling as well as gene expression/knockout in a population of cells with a desired sparseness level and hence will provide a useful tool to the dissection of the cell lineage in the inner ear.