Geminin Coordinates The Molecular Mechanism Of Somite Formation By Regulating Fgf8 And Notch Signalings

Somitogenesis is a critical developmental event whereby pairs of epithelial spheres,named somites,form periodically from the mesenchymal presomitic mesoderm(PSM)in vertebrates(e.g.zebrafish).The “clock and wavefront” model was proposed to explain the mechanism of somite formation.This model postulates interactions between the wavefront of gradients(e.g.,those of Fgf8)and the segmentation clock(cyclic genes)in the PSM that gate cells into potential somites in vertebrates.During somitogenesis,Fgf8 maintains the pre-differentiation stage of presomitic mesoderm(PSM)cells and its retraction gives a cue for somite formation.Delta/Notch initiates the expression of oscillation genes in the tail bud and subsequently contributes to somite formation in a periodic way.Whether there exists a critical factor coordinating Fgf8 and Notch signaling pathways is largely unknown.Geminin is a crucial cell cycle regulator and it expressed specifically in the tail bud,PSM,and newly formed somite during somitogenesis in zebrafish.The expression pattern of the geminin is in the tail suggests a possible role of it in somitogenesis.To confirm this,we have synthesized gemininMO to block the translation of geminin in zebrafish.When geminin is knocked down,the embryos did not show any obvious defect except for the shortened AP axis during gastrulation.At the early somite stage,the embryos have been displayed developmental delay,cell apoptosis,deformed somite shape,and blurry somitic boundary.Fgf8 plays an important role in maintaining PSM cell fate in previous papers.Transplantation of Fgf8 beads into PSM regions maintains their posterior axis and gives rise to shorter somites.RA and Fgf8 have been reported to serve as antagonistic gradients that control somite symmetric patterning and segmentation boundary formation in mouse.Here,in zebrafish,we demonstrate that the loss of function of Geminin have gave rise to narrower somites because of de-repressed Fgf8 gradient in the PSM and tail bud.The disturbance of somitogenesis is caused by Notch signaling deficiency has been demonstrated in many species.In geminin morphants,the somite boundary could not form properly but the oscillation of cyclic genes was normal,displaying the blurry somitic boundary and disturbed somite polarity along the AP axis.In zebrafish,by the result of ChIP ?luciferase report and qPCR experiment,these manifestations were mediated by the disrupted association of the Geminin/Brg1 complex with intron 3 of mib1.The latter interaction was found to positively regulate mib1 transcription,Notch activity,and sequential somite segmentation during somitogenesis.In addition,Geminin was also shown to regulate the expression of deltaD in a mib1-independent way in zebrafish.In summary,our data for the first time showed a critical role of geminin in regulating Fgf8 and Notch signaling during somitogenesis in zebrafish.On the one hand,in geminin morphants,the expression levels of deltaD and mib1 were simultaneously downregulated,concomitantly with the downregulation of Notch activity and consequential somite boundary defect.This effect was likely mediated by the interaction of geminin,together with Brg1,with intron 3 of mib1 and subsequent positive regulation of mib1 transcription.On the other hand,geminin loss of function also resulted in the upregulation of fgf8 and downregulation of raldh2 via a direct or indirect way,which led to delayed retraction of Fgf8 activity along the PSM to tail bud and resulted in the formation of narrower somites compared to those in control morphants.We conclude that geminin regulates Fgf8 and Notch signaling and thereby coordinates somite segmentation during somitogenesis.