EXPLORING THE ROLE OF FGFS ON RADIAL PATTERNING OF THE EMBRYONIC CHICKEN COCHLEA
Proper development of the inner ear, including the cochlea, is necessary for normal hearing. Development of the inner ear requires many signaling molecules under both spatial and temporal control. These signaling molecules include the wingless-related integration site (Wnts) and the fibroblast growth factors (Fgfs) gene families. The embryonic chick inner ear was chosen as the model to study cochlear development due to its homology with the mammalian cochlea and the ease of access to the inner ear in ovo. Both the mammalian cochlea and the homologous chick basilar papilla contain two domains with their own type of hair cells and innervation. The neural side of the basilar papilla contains the tall hair cells innervated by the afferent axons which takes the noise signal to the brain. The abneural side of the basilar papilla contains the short hair cells innervated by the efferent axons which receive signals from the brain to turn down added gain.
Previous research showed that virally induced cWnt9a overexpression within the basilar papilla generated a neural side phenotype across the basilar papilla (Munnamalai et al., 2017). These basilar papillas contained more tall hair cells and increased innervation at embryonic day 18 (E18) than their wild-type counterparts. Additionally, there were many differentially expressed genes found to be downstream of cWnt9a including cFgf3 and cFgf19. This project focused on determining the role of cFgf19 in inducing a neural side phenotype in the basilar papilla. First, in situ hybridization was used to determine the cFgf3 and cFgf19 mRNA transcript location with cWnt9a overexpression. Both Fgfs were found across the basilar papilla. Next, a possible cWnt9a receptor, cFzd4, which was upregulated with cWnt9a overexpression, was found in the neural side of the basilar papilla. cFgf19 was then overexpressed using one of two different vectors: RCAS(A)/EGFP-P2A-Fgf19 or RCAS(B)/Fgf19-P2A-EGFP in which the order of cFgf19 transcription was altered. RCAS(B)/Fgf19-P2A-EGFP was found to produce less GFP when transfected into DF-1 cells than RCAS(A)/EGFP-P2A-Fgf19. Additionally, RCAS(B)/Fgf19-P2A-EGFP transfected cells produced secreted fusion proteins of GFP and Fgf19, compared to RCAS(A)/EGFP-P2A-Fgf19 transfected cells which produced secreted individual proteins. The viruses were injected into the otocyst at E3 and the embryos harvested several days later including at E6, E10, and E14. Inner ears injected with either virus showed no changes in innervation, hair cells, proliferation, cartilage formation around the cochlear duct, cFgf3 expression, or phosphorylation of ERK. To determine understand where Fgf19 could be producing an effect, the location of a possible receptor, Fgfr4, was determined in wild-type embryos. At E6 and E8, cFgfr4 was found within the basilar papilla, but many more transcripts were found surrounding the cochlear duct. Overall, the role of Fgf19 in neural side fate of the basilar papilla was not determined. Possible reasons for the lack of phenotypic changes include nonfunctional Fgf19 being secreted which could not bind and induce downstream signaling, Fgf19 being responsible for an untested aspect of the cWnt9a overexpression model, or other misregulated genes would be needed for the phenotypic change to occur.