| The correct asymmetrical arrangement of visceral organs is essential to the normal driving function of each organ in vertebrates.Studies in vertebrates have confirmed that Pitx2 is the key point involved in the asymmetric development of visceral organs.Because the asymmetric organ morphogenesis is delicate and complex,and there are differences in the morphogenesis of different organs,as a result,how Pitx2 regulates asymmetrical organogenesis remains unclear.However,to date,the study of Pitx gene in invertebrates has been limited and the evolutionary origin of Pitx function is still unknown.Amphioxus is a transitional group between invertebrates and vertebrates,and has clearly discernible left-right asymmetrical tissues and organs,therefore,amphioxus is a valuable material to study the mechanism of asymmetrical organogenesis and the evolutionary origin of gene function.Similar to vertebrates,amphioxus Pitx gene is also regulated by asymmetrical cascade Dand5-Nodal-Lefty,but it is unknown whether Pitx is involved in asymmetric organ morphogenesis and the mechanism by which it regulates remains unclear in amphioxus.In this study,we generated amphioxus Pitx mutants using TALEN method.Through phenotype observation and detection of asymmetric organ related genes by in situ hybridization,we found that in Pitx-/-,left-side organs such as mouth,preoral pit and Hatschek’s nephridium were lost,and right-side organs such as endostyle,club-shaped gland,hematopoietic domain and Hatschek’s right diverticulum were ectopic formed on the left side and tended to be symmetrical on left and right sides.In addition,asymmetric arrangement of somites and peripheral nerves were weakened but not abolished in Pitx-/-.Further research showed that larvae with partial loss of Pitx function exhibited milder phenotype compared with complete loss of Pitx,These results suggested that Pitx gene was essential to asymmetrical organ morphogenesis and asymmetrical organs have different sensitivities to Pitx activity in amphioxus.To investigate the mechanism of Pitx regulating asymmetrical organ morphogenesis,we first analyzed the expressional position relationship between Pitx gene and asymmetric organ-specific genes.During the development of 3-somite and 6-somite neurula,Pitx expression on the left side was gradually expanding from anterior to posterior and also from dorsal to ventral pharyngeal endoderm,meanwhile,the expression signal of left organ-specific gene Lhx3 and Prop1 overlapped with Pitx expression in the preoral pit region.The expression signal of right organ-specific gene FoxE4,Nkx2.1 and Hex on the left side was gradually reduced from anterior to posterior and from dorsal to ventral,however,the expression pattern was opposite to Pitx.In Pitx-/-,Lhx3 and Prop1 expression on the preoral pit was lost,however,the right-side genes FoxE4,Nkx2.1 and Hex retained high expression levels on the left side.In embryos overexpressing Pitx,Lhx3 expression on the preoral pit were activated ectopically on the right side the while the expression of the right-side genes were inhibited.These results indicated that Pitx induced left organ morphogenesis through activating the expression of left organ-specific genes and regulated right organ asymmetrical morphogenesis by inhibiting the expression of right organ-specific gene in the left pharyngeal endoderm.Luciferase assay revealed that Pitx probably functions through inducing the development of left-side organs and inhibiting that of right-side organs directly.Further studies revealed that the regulation of Pitx on the right organ morphogenesis needed to work together with another unknown factor X,while Pitx was a dominant determinant in this process.In a word,this study not only uncovered the important role of amphioxus Pitx gene in the development of asymmetric organs and proposed a regulatory model of Pitx involved in asymmetric morphogenesis,this study also provided new ideas for vertebrate research on asymmetric organ mechanism.Pharyngeal endoderm plays an important role in pharyngeal gill slit morphogenesis in chordates.Multiple genes Pax1,Pax9,Tbx1,Eya1 and Six1 have been found to be involved in the early development of pharyngeal pouches and their derivative organs.The functions of these genes in pharyngeal gill slit morphogenesis are different among species and many questions remain about the regulatory relationship between these genes.Amphioxus is the most primitive chordate,having orthologs of above genes.Amphioxus is the optimal model for studying the functional origin of Pax1,Pax9,Tbx1,Eya1 and Six1 genes and the mechanism of pharyngeal organogenesis.Our lab has obtained Pax1/9 homozygous mutant.In this study,we generated amphioxus Tbx1/10,Eya and Six1/2 homozygous mutants using TALEN method,respectively.In Tbx/1/10-/-,the first gill slit developed normally,while the second gill slit was smaller,even only the primordium existed and the third gill slit was completely lost.These results revealed that Tbx1/10 was necessary for the development of the second gill slit and formation of third gill slit.By scanning electron microscopy,semi-thin section,electron microscopy observation and cell proliferation detection,we found that in Eya-/-,the first two gill slits were outward,the number of gill lip cells in the first gill slit was greatly reduced,the cell types were changed and the cell proliferation ability was significantly reduced.Meanwhile,the number of gill lip cells in the second gill slit was unchanged,the cells in undifferentiated state and the cell proliferation ability was not affected.In Six1/2-/-,the number of first gill lip cells was reduced,the cell type did not change,and the cell proliferation ability was decreased,meanwhile,the number of second gill lip cells was also unchanged,the cells were in undifferentiated state and the cell proliferation ability was not affected.These results indicated that Eya was involved in proliferation-differentiation process of first gill lip cells,while Six1/2 was responsible for the proliferation process.In addition,Both Eya and Six1/2 did not affect the proliferation but differentiation process of the second gill lip cells.To further investigate the regulatory relation between Pax1/9,Tbx1/10,Eya and Six1/2,we analyzed the expression pattern of the above genes in pharyngeal endoderm and also detected other gene expression in their mutants.Pax1/9 was initially expanded from the ventral pharyngeal endoderm to dorsal region and subsequently expressed throughout the pharyngeal endoderm.Together with our lab previous studies,above results further confirmed that Pax1/9 was located upstream of the other three genes and played a leading role in the development of gill slits.Tbx1/10 expression was the last initiated in pharyngeal endoderm and Tbx1/10 knockout resulted in the delayed and reduced expression of Eya and Six1/2 in the second pharyngeal gill slit primordium.It was indicated that Tbx1/10 partially regulated Eya and Six1/2 gene.The expressional pattern of Eya and Six1/2 was similar,moreover,in Eya-/-,Tbx1/10 expression was unchanged while Six1/2 expression was almost eliminated.In addition,Tbx1/10 and Eya expression did not changed in Six1/2-/-.All these results showed that Pax1/9 was upstream of the regulatory network,Tbx1/10 partially regulated Eya and Six1/2 gene,Eya regulated the expression of Six1/2,which was downstream of Pax1/9-Tbx1/10-Eya gene regulation network.In this study,we identified the function of Tbx1/10,Eya and Six1/2 gene in gill slit formation and preliminarily illuminated the mechanism of pharyngeal gill slit development.It is valuable to understand the functional origin of pharyngeal gill slits related genes in chordates and the mechanism of pharyngeal pouches development in vertebrate. |
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