Use Of DC Macrochaetes As A Model To Study The Mechanism Of Morphological Difference Between Dipteran Species During Evolutionary Development

Evolutionary developmental biology(evo-devo)is the combination of three disciplines which are evolutionary biology,developmental biology and genetics that explore the origin and evolution of embryonic development and morphological change in organisms over time.It also studies the developmental plasticity during evolution and how the environment influences the evolution,homoplasy and homology.D.melanogaster has 2 pairs of Dorsocentral(DC)bristles,while M.domestica has 6(DC)bristles.This large difference between them can be used as an excellent model for evo-devo study.Here,we take DC bristle as a model system to study how evo-devo affects two dipteran species,M.domestica and D.melanogaster.The main results are as follows.(1)The protein sequences of SCUTE,PNRp,EMC,HAIRY AND USH(USH-D)between M.domestica and D.melanogaster has high conservation,up to 42.7%,51.0%,53.1%,69.2%and 42.9%respectively.The M.domestica and D.melanogaster SCUTE and HAIRY both have bHLH domain,the similarities are 94.9%and 100%respectively.EMC encodes HLH domain,the similarity is 95.5%,PNR? and USH both have two zinc finger DNA binding domains,and the similarities are 97.9%and 71.4%respectively.All these results indicated that the proteins regulating bristles from M.domestica and D.melanogaster have conserved functional domains,and probably conserved function as well.(2)By overexpressing M.domestica scute,pnr?,emc and hairy in D.melanogaster.We found that M.domestica scute could induce a large number of macrochaetes in D.melanogaster notum.Overexpression of pnr? exhibited various of phenotypes in DC bristles,among them,51%had two or three DC bristles,17%had five bristles,and the rest of 32%had no change in bristle number.Upon the M.domestica emc expression in D.melanogaster,the microchaetes in AC and DC were absent entirely,and macrochaete in DC domain had five phenotypes:0,1,2,3 and 4.This result showed that the function of M.domestica emc is highly conserved with D.melanogaster,which can negatively regulate the development of bristles.Similarly,the expression of M.domestica hairy with MD237-Gal4,like the emc,resulted in inhibition of all the AC and DC microchaetes,and the DC macrochaete number exhibited a dramatic decrease,where over 70%of them lost all of four DC bristles.Together,these results demonstrated gene scute,emc and hairy in M.domestica have the conserved function in D.melanogaster.However,pnr? displayed different function in regulating bristle development,thus,it requires a further study.(3)The similarities of H.sapiens ASCL1 protein with M.domestica and D.melanogaster SCUTE is 32.7%,and all has the bHLH function domain.The functional domain of H.sapiens ID2 is HLH,which is conserved with D.melanogaster and M.domestica,up to 59.5%and 61.9%receptively.EMC and ID2 form non-functional hetero-oligomers with bHLH and prevent transcription.EMC and ID2 have 9 dimerization interfaces,but only 4 are conserved.These data incidated that the genetic information of H.sapiens ASCL1 and ID2 changed greatly compared to D.melanogaster and M.domestica,but they also encoded conserved functional domain.Upon expressing H.sapiens ascl1,we found a large number of extra bristles in D.melanogaster AC and DC area,which indicated that ascl1 can promote the development of bristles.Overexpressing human Id2,the DC macrochaete number exhibited a dramatic decrease:about 55%of them lost two of four DC bristles,and 45%lost one bristle.The effect of Id2 to bristles development is weeker but also conserved with emc in D.melanogaster and M.domestica.(4)The expressions of scute,pnr,emc,hairy and ush changed with time.The expression of all the five genes had low levels at 1st and 2nd,and up to their highest level at 3rd(3rd2 and 3rd3)stage.At 3rd stage,there were some difference between M.domestica and D.melanogaster.The period 3rd3 stage had highest expression level in D.melanogaster of all the five genes,but it is different in M.domestica.Only scute,pnr and emc expressed highest at the period 3rd3,while hairy and ush expressed highest at 3rd2.In this study,we found that at the 3rd larval stage,the expression of D.melanogaster scute was raised gradually,and up to its highest at the 3rd3 larval stage,but the expression of M.domestica scute kept at a high level throughout 3rd stage.The different expression patterns between D.melanogaster and M.domestica may explain why they have different bristle pattern.(5)By in situ examination of the expression patterns of scute,pnr,emc,hairy and ush,we found that they had species specificity.The D.melanogaster and M.domestica scute expressed at the presumptive notum of DC,SC,SA,NP and PS.pnr and ush are expressed strongly in a conserved pattern of notum domains.Over the dorsal notum,the expression of emc differs significantly between the two species.emc is expressed in three transverse bands over the dorsal notum of M.domestica.The first is at the anterior area of the prescutum,second band is midway down the scutum,and band three is at the posterior edge of the future scutellum where the scutellar lever is joined to the postnotum.In D.melanogaster,emc is expressed ubiquitously but at low levels throughout the disc.The expression pattern of hairy is conserved in both M.domestica and D.melanogaster,and both strongly expressed over the scutellum and posterior edge of the future supra and postalar,and at last extended to the presumptive wing area.In conclusion,these findings reveal that the function of scute,emc and hairy in M.domestica is conserved at regulating the development of macrochaete,but the spatial expression led to species-specificity.This demonstrated that the evolution of gene spatial expression during development is very important in morphologic evolution.Our results have provided a greater understanding of an experimental paradigm for evolutionary developmental biology.