Evolution In Regulating Cardiac Function Of Members Of POPDC Family

Studies on the POPDC family have been more than 10 years and during this period,genetically modified models have been used to explore the functions of the POPDC family,which shows that knockout of Drosophila POPDC 1(Bves)led to abnormal extension of the germ band,and zebrafish POPDC2 knockdown led to muscle morphological abnormalities,craniofacial muscle defect,pericardial cavity enlargement,and arrhythmia.However,all of their homozygous embryos were not lethal.In normal cases,mouse POPDC1(Bves)and POPDC2 were not shown with mutant phenotypes and the phenotypes of both mouse genes with bradycardia under pressure seemed to be perfectly duplicated in the zebrafish,suggesting that there is a functional redundancy between POPDC genes.However,the research on the function of POPDC3 has been carried out very late,and so far,it still stays on the disease correlation.We used different animal models to attempt systematically to understand the evolution of POPDC family in the heart function at different levels of evolution by knockout strategy for Drosophila POPDC1(Bves)and vertebrates and mammals POPDC3 genes.It is well known that knockout of a gene causes the death of the homozygous embryos indicates that the gene has important biological functions.Drosophila POPDC1(Bves)is the initial gene in evolution and the only gene in Drosophila.Therefore,we questioned the conclusions made by others for knockout of fly POPDC1 gene without death,and without role in cardiac development.To this,we have established Drosophila Bves gene knockout lines using CRISPR/cas9 targeting technology.The results showed that knockout of Bves expression in flies resulted in the death of homozygous embryos.Also this gene plays a role in regulating the development of the heart.Our results negate the conclusions of the international counterparts.In the same period in our lab,we found that knockout of the zebrafish POPDC3 gene expression resulted in a homozygous lethal(death rate of about 80%).We also established a mouse POPDC3 knockout strain,and the preliminary result showed that no homozygous embryos was found but it needs further study.Thus,POPDC3 seems to be different from other members of the POPDC family and it is of important biological functions which is irreplaceable.So we suggest that POPDC3 is a key gene in the POPDC family.We also need to find out their origin at an evolutionary point of view.The three members,POPDC1,POPDC2 and POPDC3,encode a protein size of 300-360 amino acids,with a conserved transmembrane domain in the C-terminal,a conservative Popeye domain,and 1-2 glycosylation sites in N-terminal transmembrane domain.The similarity of its structure suggests that there exists a similar function among them.POPDC3 and POPDC1 seems to appear at the same time of evolution,and both are closely located,only 2KB and 4KB,on chromosome 6 in invertebrates and vertebrates.POPDC1 is currently been considered a single gene in vertebrates and invertebrates.Our results showed that in Drosophila POPDC1 knockout caused embryonic lethality,which is actually the functional representative of the entire POPDC family.POPDC2 has been considered to exist only in vertebrates,due to its protein homology of 50%when compared with POPDC3,but POPDC1 is only 25%homology to POPDC2 and POPDC3.POPDC2 has been considered an ancestry replica from vertebrate POPDC3.From the evolution of fish,POPDC2 could be detected in Xenopus,chicken,mice and humans,which reflects the demand for higher-order structure of biological complexity at the molecular regulation.The essence of life lies in the steady state,mutual compensation among their members and the function of the links,so one will not be part of the problem that lead to the collapse of the entire system.Indeed,severe phenotypes were observed in Drosophila POPDC1 knockout flies,which could be observed when simultaneously knockout POPDC1 and POPDC2.This is why knockout POPDC1 or POPDC2 in zebrafish and mice showed no malformations,separately.This mechanism will show greater tolerance and adaptability to environmental changes.However,how to achieve this compensation between POPDC1 and POPDC2 remains to be explored.To understand why the POPDC3 show different role in heart development to other POPDC family members,and that the function redundancy for compensatory between members of POPDC family,we analyzed the functions in heart development of POPDC genes.Our studies show that the Drosophila bves knockout led to embryonic heart deformity.In vertebrate zebrafish model,the low concentration of MO for knockdown of POPDC1 and 2 expression had no significant effect on heart development,but low concentration of MO for knockdown of POPDC3 expression caused significant cardiac linear phenotype.International counterparts seem to be more concerned on the studies of POPDC2,which is actually impossible for understanding the function of the POPDC protein family.It has been shown that Zebrafish POPDC1 knockdown did not affect heart development,and POPDC2 seems to play an important role in regulating heart rhythm,but gave not much contribution to the heart shape,which show a differentiation begins from the original and important functional role of Drosophila POPDC.The original role of Drosophila POPDC1 seems to be passed to zebrafish POPDC3.Zebrafish POPDC2 knockdown did not affect the expression of POPDC1 and POPDC3.The POPDC3 knockout caused cardiac malformations,suggesting that POPDC1 and POPDC2 could not compensate POPDC3.It is possible that POPDC1 and POPDC2 is functionality involved in specific regulation of cardiac rhythm.Whether the other species of POPDC1 and POPDC3 has a similar function remains unknown.To sum up,international counterparts and our results show that Drosophila POPDC1 plays an important role,and with evolution,a number of independent features appear on different POPDC genes.POPDC3 has obtained important functions from the Drosophila POPDC genes,and become a key gene in regulation of cardiac function in the POPDC family.