Mechanism Of Prostaglandin E2 Signal Pathway Regulating Cilia In Zebrafish

Cilia are tubular organelles specially formed with microtubules as the basic structure.They are widely distributed on the cell surface of human body's retina,nasal cavity,renal tubules,spinal tube,fallopian tube,some other tissues and organs.They are important for embryonic development,organ formation,cell signal transduction,and Maitaing the physiological homeostasis of the human body and other aspects play a key role.Because cilia undertake a variety of physiological functions in human life activities,when cilia structure and function defects often lead to a variety of human diseases,called ciliopathy,including congenital heart disease,polycystic kidney,retinitis pigmentosa,Ciliary immobility syndrome and infertility,etc.Therefore,exploring the mechanism and signal transduction function of cilia can provide new research ideas and methods for the treatment of related diseases in the future.Our previous studies have found that prostaglandin E₂(PGE₂)is transported out ofcells through the transmembrane transporter ABC family ABCC4,and acts on the cilia-located G protein-coupled receptor EP4 to regulate cell cilia growth and asymmetric development of zebrafish internal organs.The zebrafish EP4 receptor family has three subtypes EP4a,EP4b and EP4c.Which EP4 subtype is located in embryonic cilia,what are their key positioning sequences and what are their functions,all these questions are still unclear.In this study,we hope to determine the EP4 receptor subtybes which located of cilia in different organs of embryos,and explore the key amino acid sequence of EP4c receptor to locate cilia.In this study,we used zebrafish as an animal model to prove for the first time that in the PGE₂-EP4signaling pathway,only EP4c subtype receptor is located in the,otic vesicles,spinal tube,body surface,retina,etc.EP4c receptor belongs to the family of G protein coupled receptors.The protein family generally contains 7 transmembrane and 4intracellular regions.Previous studies have found that most of the key sequences that affect the localization of G protein coupled receptors in cilia are concentrated in the third Intracellular region and fourth intracellular region,We analyzed the amino acid sequences between the three subtypes of the EP4 family of zebrafish and found that the amino acid sequences of the third and fourth intracellular regions of EP4c are the most different from the amino acid sequences of EP4a,EP4b and other EP families.We choose these two sequences as our exploration target.We use Tol2-ep4c(i3 del)-cherry(fusion protein with deletion of the third intracellular segment of EP4c)and Tol2-ep4c(i4 del)-cherry(fusion protein with deletion of the fourth intracellular segment of EP4c))Plasmid,Analyzing the location of cilia after EP4c mutation,it is preliminarily clear that EP4c will not be able to locate cilia after the third or fourth intracellular region of EP4c is missing.In order to further determine the cilia location function of the third and fourth intracellular segments of EP4c,we replaced this sequence to the same position of the non-cilia-located EP4a subtype,and constructed Tol2-ep4a(i3 del+ep4c i3)-cherry and Tol2-ep4a(i4 del+ep4c i4)-cherry plasmids,and injected into zebrafish embryos,it was found that the replaced EP4a can be located on the cilia.Therefore,we believe that the key sequences that EP4c can locate in cilia mainly exist in the third intracellular region and the fourth intracellular region.Next,we studied the function of ciliated EP4c in the development of zebrafish embryonic retina,and down-regulated the expression of EP4c through morpholine-mediated gene knockdown technology,and found that the length of the retinal connecting cilia was significantly shorter.It indicates that EP4c plays an important role in the growth and development of retinal cilia.In order to better explore the function of EP4c,we used large fragment deletion technology to construct EP4c homozygous mutants.The PGE₂-EP4 signal mainly activates adenylate cyclase(AC)to increase the concentration of the second messenger c AMP in the cell,and then transmits the signal downstream.For a long time,few scholars have deeply explored the location of adenylate cyclase downstream of the PGE₂-EP4 signal.There are 9 subtypes of AC enzymes in mammals.Zebrafish currently has 12 subtypes.Which AC enzymes are located in the cilia and whether AC enzymes could receive PGE₂-EP4 signals in the cilia are still unclear.These problems are all worthwhile to study further.The subtypes of adenylate cyclase in zebrafish are including AC1a,AC1b,AC2a,AC2b,AC3a,AC3b,AC5,AC6a,AC6b,AC7,AC8,AC9.Through subcellular localization experiments,we found that only three subtypes of adenylate cyclase are localized in cilia,namely AC3a,AC5,AC6b;In order to further explore its special role in cilia,we chose to down-regulate the expression of AC6b protein in the zebrafish homozygous mutant AC3^-/-AC5^-/-that has been constructed in the laboratory.The results showed that embryos have typical abnormal cilia developmental Phenotypes.We also found through embryo immunostaining experiments that in the absence of AC3a,AC5,and AC6b,the number of embryonic cilia reduced significantly,which also shows that the adenylate cyclase located in the cilia plays a key role in the growth and development of cilia.In order to better explore the function of PGE₂-EP4-AC signaling pathway on cilia growth and development,we used CRISPR/Cas9 technology to construct AC3^-/-AC5^-/-AC6b^-/-mutants.Through the above research,we have preliminarily determined the key amino acid sequence of the zebrafish EP4c receptor to locate the cilia,and further explored the location and preliminary function of adenylate cyclase downstream of the EP4receptor.This provides theoretical support for our subsequent exploration of how the prostaglandin signaling pathway affects the development of vertebrates by regulating cilia.