Currently, within the United States, 42,600 adults and 28,400 children have cochlear implants, which depend on the spiral ganglion neurons (SGNs) for conducting auditory information including human speech. However, the SGNs can be damaged or degenerate in the absence of stimulation from the hair cells, rendering cochlear implants less effective. Understanding how SGNs are generated and develop can inform methods for how to maintain the auditory nerve for optimal cochlear implant function. Insm1 is a zinc finger transcription factor expressed throughout the developing nervous system in neuronal progenitors and nascent, but not mature neurons. It is expressed during neurogenesis and promotes uncommitted, apically-dividing progenitors to migrate basally and commit to neuronal cell fate in both the developing cortex and olfactory epithelium. Insm1 is also expressed in nascent neurons and necessary for differentiation of certain neuron types. In the developing otocyst, some apically-dividing progenitors move basally, delaminate and become neuronally-committed progenitors of the statoacoustic ganglion (SAG). We hypothesize that (1) Insm1 in the otocyst promotes the transition from apically uncommitted progenitors to delaminated neuronal progenitors, and (2) that continued expression of Insm1 in nascent SAG neurons promotes their differentiation. We will test this hypothesis by using three specific aims.
Aim 1 : To determine the spatiotemporal expression pattern of Insm1 in the developing mouse ear. We will use in situ hybridization (ISH) to detect the expression of Insm1 in the developing ear at different time points during embryonic development. We will also use ISH followed by immunohistochemistry (IHC) for proliferation markers (Ph3, Ki67, and BrdU) to determine if Insm1 is expressed by apically dividing progenitors, delaminated progenitors, or both.
Aim 2 : To determine if Insm1 promotes a transition of apically-dividing progenitors to delaminated neuronal progenitors. We will determine the effects of Insm1 deletion on proliferation and apoptosis in the otocyst. We will compare the number of proliferating and apoptotic (ACC3 positive) cells in the ears of Insm1 knockout (KO) and wildtype (WT) littermate embryos using IHC. We will also determine the effects of Insm1 ectopic expression on delamination, proliferation, and neuron production by driving over expression using in utero electroporation.
Aim 3 : To establish if Insm1 is necessary for differentiation of SAG neurons. In the Insm1 KO we will look for changes in expression of the few factors that are either known to promote differentiation of SAG neurons or be expressed by nascent SAG neurons by IHC and ISH. We will also look at neurite outgrowth from the SAG as an indicator of differentiation, using IHC for Tuj or neurofilaments, which clearly label SAG processes. We will perform RNA Seq to compare expression patterns of the KO and WT post-proliferative SAG. This will afford a thorough and unbiased search for genes regulated by Insm1.
Currently, within the United States, 42,600 adults and 28,400 children have cochlear implants, which depend on the spiral ganglion neurons (SGNs) for conducting auditory information including human speech. Understanding how SGNs are generated and develop can inform methods for how to maintain the auditory nerve for optimal cochlear implant function. We believe that the transcription factor Insm1 promotes the production and differentiation of SGNs.
|Wiwatpanit, Teerawat; Lorenzen, Sarah M; Cantú, Jorge A et al. (2018) Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1. Nature 563:691-695|
|Lorenzen, Sarah M; Duggan, Anne; Osipovich, Anna B et al. (2015) Insm1 promotes neurogenic proliferation in delaminated otic progenitors. Mech Dev 138 Pt 3:233-45|