Investigation Of The Spatiotemporal Expression Pattern Of Slc5a8 In Mouse Cornea

Limbal stem cells(LSCs)reside in the limbus region.During ontogeny,LSCs differentiate into mature corneal epithelial cells while they migrate concentrically.The study of such process implies a theoretical basis for the localization and identification of LSCs from mature corneal epithelial cells.Mice is one of the important experimental animal models,but the studies of the differentially expressed genes in mouse limbus have not been reported yet.This project integrated molecular biology techniques and high-throughput sequencing techniques to explore the difference of different regions in mouse limbus.This study will help us understand the differentially expressed genes of mouse limbus.SLC5A8 is a sodium-coupled monocarboxylic acid transporter(also known as SMCT1)involved in the delivery of short-chain fatty acids and lactic acid.Such transporter can be expressed not only in oocytes,but also in other somatic cells.We speculate that it will also be expressed in mice cornea.In this study,we firstly used a number of experimental techniques including hematoxylin-eosin(HE)staining and immunohistochemistry staining for morphological determination of the boundary of mouse limbus.In this study,hematoxylin-eosin(HE)staining and immunohistochemistry staining were used for morphological determination of the boundary of mouse limbus.Based on the morphological results,the tissue samples from mice superior(S),Inferior(I),nasal(N),and Temporal(T)cornea containing limbus(L)were collected,and the central cornea(CC)and the conjunctiva(Cj)were also used as control groups for transcriptome sequencing.Subsequently,the differential expression,Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed.Based on differential expression analysis,the differentially expressed genes profile of S/I/N/T compared to CC and Cj was established.Moreover,we selected the key genes for further study to explore their expression pattern in mouse cornea at different level.In this study,we defined the boundary of mouse limbus.Of 18 samples,the minimum Q20 was 95.79%,the minimum Q30 was 88.71%,and the minimum clean reads ratio was 94.89%,which means that the quality of these clean data met the recognized standards,Q20?95%,Q30?85%.For differential expression analysis,the key gene,Slc5a8,is differentially expressed in 18 samples.Therefore,it was selected for further study by using qRT-PCR and Western Blotting for quantitative and semiquantitative determination.Then,in situ detection of individual RNA and immunohistochemistry staining were used for spatial distribution of Slc5a8 in mouse cornea at the transcriptome and protein levels.The results of qRT-PCR and in situ detection of individual RNA showed that Slc5a8 is highly expressed in limbus epithelium.In contrast,the results of Western Blotting and immunohistochemistry staining showed that SLC5A8 is highly expressed in cornea epithelium.It demonstrated that Slc5a8 can be used as a candidate marker for the identification of limbal epithelial cells at the transcriptome level and for the identification of corneal epithelial cells at protein level.