FGF and Notch signaling hierarchy in sensory neurogenesis

FGF and Notch signaling hierarchy in sensory neurogenesis Jacob Voelkel 1, Agostino Molteni 1 Jamison Harvey 2, Jason Adams 2, Rhonda Lassiter 2, Michael Stark 2 1 UMKC School of Medicine, 2 Brigham Young University Introduction - Canonical Wnt, Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), and Notch pathways are involved in ophthalmic trigeminal placode (op. V) sensory neurogenesis - FGF is necessary for placode cell delamination - Notch inhibition leads to premature neuronal differentiation Purpose - To investigate regulatory relationship between FGF and Notch in sensory neurogenesis Methods - FGF signaling was blocked globally with the FGF receptor antagonist SU 5402 in 12 -14 ss embryos from fertilized chicken eggs (Gallus gallus) - Notch signaling was simultaneously inhibited with the Notch chemical inhibitor DAPT - Embryos were incubated for 24 hr to the 24 -28 ss then prepared, cryosectioned, and stained with the op. V marker Pax-3 and the proneural marker Islet-1. - Notch signally was blocked with DAPT in 6 -9 ss embryos and FGFR 4 m. RNA expression was evaluated after 4 or 12 hr by in situ hybridization Conclusion Results Figure 1. Global suppression of FGF and Notch inhibits delamination and differentiation. Transverse sections through the op. V placode region of 24 -28 ss embryos Coexpressing Pax 3/Islet 1 cells are shown in yellow. Figure 2. FGFR 4 m. RNA is not upregulated by Notch inhibtion after 4 and 12 hr. Assay of FGFR 4 m. RNA expression in brightfield microscopy. Figure 3. FGF activation coupled with Notch inhibition disrupts basement membrane integrity. Transverse sections through the op. V placode region of 24 -28 ss. Arrows indicate areas of disruption in basement membrane integrity. Figure 4. Proposed interactions of FGF and Notch signaling leading to neurogenesis - FGF activation does not downregulate Notch signaling leading to neurogenesis (A) - Notch downregulation does not lead to upregulation of FGFR 4 expressiong and increased FGF signaling leading to neurogenesis (B) - FGF activation and Notch downregulation act in parallel to promote neurogenesis (C) - Notch downregulation disrupts basement membrane integrity Future Direction Next step - Determine the role of Notch signaling in disruption of cell-cell and/or cell-extracellular matrix protein interactions Clinical Significance - Notch downregulation leading to basement membrane fragmentation may be a mechanism involved in malignant transformation References 1. 2. 3. 4. 5. Begbie J, et al. Early steps in the production of sensory neurons by the neurogenic placodes. Mol Cell Neurosci- 2002; vol 21: 502 -11. Lassiter RN, et al. Sensory neuron differentiation is regulated by notch signaling in the trigeminal placode. Dev Biol- 2010; vol 344: 836 -48. Lassiter RN, et al. FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation. Dev Dyn- 2009; vol 238: 107382. Scaal M, et al. In ovo electroporation of avian somites. Dev Dyn- 2004; vol 229: 643 -50. Schimmang T. Expression and functions of FGF ligands during early otic development. Int J Dev Biol- 2007; vol 51: 473 -81.