A Study Of Filopodia In The Feather Branching System

Feather is an excellent model to research of branch,with precise configuration distinct bifurcate structure.In the process of branch,the flat epithelial layer is transformed into periodic arrangement of barbs simultaneously.Several signaling molecules such as FGF,Notch,BMP,Wnt,etc,can regulate the feather branch.However,during the process,the accurate molecular mechanisms and cellular behavior remain unclear.In Drosophila tracheal system or in the vertebrate vascular system,branch epithelial cells extended filopodia under mesenchymal factors.Through filopodia,activated signaling pathway regulate the branch formation.We also found the filopodia in feather branch system.The filopodia in feather branch system is composed by F-actin,with the expression of VASP and Fscnl.Immunofluorescence and TEM revealed that,from proximal to distal of rachis,there are filopodia on basal epithelial cells.Filopodia were also found in the proximal of barbs,disappeared after branch forming.So,there are certain relations between filopodia and the formation of branch.Filopodia can promote cell adhesion with the rich location of cellular adhesive molecular NCAM,LCAM and ?-cat.When double stained with a mesenchymal marker Tenascin C(Tn),inter-digitation of ?-catenin and Tn can be seen,suggesting the filopoida extends into the mesenchyme.Rho family can regulate the cytoskeletonand filopodia.In Hela,Rho A and Cdc 42 regulate filopodia.By means of lentivirals infection,the overexpression of DN Rho A and Cdc 42 in the feather follicles,produced the tips of feathers with supernumerary branches in the rachis region.Local overexpression of DN Rho A and Cdc 42 resulted in ectopic branching in the rachis.Therefore,under the regulation of Rho,filopodia can affect the formation of branch.Studies show that from DP to pulp,there are FGF gradient,and the overexpression of FgflO can inhibit the formation of the feather branch.By RT-PCR,we found that from DP to pulp the expression of Fgf2 and Fgf 10 gradually diminishing.The FGF gradient were confirmed by in situ.Local overexpression of Fgf 10 blocks epithelial branching,In this ectopic rachis,filopodia is also induced.In contrast,inhibition of FGF signaling with SU5402 resulted in ectopic branching formation in the rachis,and filopodia were disappeared.Therefore,FGF can regulate the formation of filopodia and branch.pERK1/2 activated in the proximal areas and rachis,but not on barbs.Inhibition of pERK with PD98059 resulted in similar phenotype to SU5402.It shows that pERK participates in the regulation of filopodia and branch.In situ hybridization showed that Notchl expressed in barbule plate,The expression of Ser1 similar to Notchl,Ser2 epressed in Marginal plate.Overexpression of Notchl led to ectopic rachis,without filopodia appear.RNAi knockdown of Notchl resulted in ectopic epithelial branching in the rachis,meanwhile filopodia disappear.Notch signal regulate the branching formation,but it can't directly induce filopodia.There are higher exprssion levels of E-cad and P-cat in a barbules plate,mediating cell aggregation to form the core of branch.Notchl directly bind to ?-cat,participates in the regulation of the core formation.RNAi-Ecad resulted in ectopic epithelial branching in the rachis,phenotype similar to RNAi-Notchl.RNAi-?-cat resulted in side branch missing.Together,in the proximal feather follicles,basal epithelial cells extended filopodia,to probe the mesenchymal FGF signal,with suppression to branch.With growth upward,FGF signaling diminishing in mesenchymal,and basal filopodia reduced or disappear,meanwhile branch form Notch signal also participates in the regulation of branching.We preliminary explored the filopodia structure,regulation and its role in the feather branch,provides a new hypothesis for feathers branch mechanism.