MRNA-Seq Analysis Of Developing Bat Wings

Bats(order Chiroptera)occupy more than 20%of extant mammalian di-versity,which are the only mammals capable of self-powered flight.A series of morphological and structural changes are associated with bat flight.Critical adaptations for flight include a pair of dramatically elongated hands with broad wing membranes and the foot of the bat has evolved a long calcar that spread the interfemoral membrane.Unlike the avian win gs with feathers,the bat wings are membranous,elastic and thin and supported by skeletal elements in the form of elongated forearm and digit bones(digits II-V).While the four posterior fingers of bats are dramatically elongated,the thumb(forelimb digit I)and hindlimb digits remain short and are similar in length and width to each other.The dramatic morphological change in bat forelimb required a combination of changes in the expression of many genes.However,so far,targeted candidate gene approaches have identified only several genes with differences in expres-sions in bat forelimb compared with hindlimb and mouse limbs during devel-opment.We perform genomewide mRNA sequencing to study the molecular mechanisms of bat wing evolution and investigate how the transcription profiles were altered during bat wing evolution.We performed a transcriptome sequenc-ing approach(mRNA-Seq)for bat's fore-and hindlimbs at a series of develop-mental stages.We validated these results in comparison with other mammals by in situ hybridization(ISH)for embryonic bats and mice.The mRNA-seq data were normalized by the trimmed mean of M values(TMM)method using the edgeR package,and the DEGseq package was used to identify differentially expressed genes between samples.The cut-off of signifi-cantly differential expression is a q-value less than 0.0001.Bat forelimb digits condense at approximately stage 15 and elongate during subsequent stages.On one hand,to find genes likely to contribute to digit elongation,we applied mRNA-Seq to bat fore-and hindlimb digits at stages 15-17.We compared the mRNA-Seq data of the elongating forelimb digits with that of digits that remain short(thumb and hindlimb digits).To limit subsequent analysis to those key genes that may play a prominent role in evolution of morphology,we sorted the genes by expression fold change and looked for genes with high expression in the elongating or short digits.We chose a set of transcription factors important for limb development.Hoxd9-12 and Tbx3 are in the top eight genes that are expressed at a significantly higher level in the elongating forelimb digits than in the short digits.Fam5c is the top gene that is expressed at a significantly higher level in the short digits than in the elongating forelimb digits.Another 5'HoxD gene Hoxdl3 that is expressed at a significantly higher level in the elongating forelimb digits than in the short digits.One the other hand,to find genes likely to contribute to the interdigital membrane of elongating forelimb digits re-mained.We compared the mRNA-Seq data of interdigital tissues between elongating forelimb digits at stages 15-17,which are retented during.embryonic development with that of interdigital tissue between forelimb digit I and digit II that are not completed disappeared and that of interdigital tissue between hindlimb digits which are disappeared during embryonic development.We find transcription factors related to limb development;Meis2 is most highly expressed in the interdigital tissues of the elongating forelimb digits(digits II-V),where tissues are retained and form the wing membrane in the adult bat.Through these analyses,we have identified a subset of highly differentially expressed genes included all 5'HoxD genes(Hoxd9-13),Tbx3,Fam5c and Meis2.We identify these eight key genes that display unique expression patterns in embryonic bat wings and feet,compared with mouse fore-and hindlimbs.The expression of all 5'HoxD genes(Hoxd9-13)and Tbx3,six known crucial tran-scription factors for limb and digit development,is extremely high and pro-longed in the elongating wing area.Unique expression patterns of 5'HoxD genes(Hoxd9-13)and Tbx3 suggest an integral role of these genes for bat digit elon-gation.Meanwhile,Tbx3 may also play an important role in inhibiting the re-gression of forelimb interdigital tissues and promoting the growth of wing membranes in bats.The expression of Fam5c,a tumour suppressor,in bat limbs is bat-specific and significantly high in all short digit regions(the thumb and foot digits).Potentially restricts endochondral growth and promotes osteoblast differentiation.Meis2 is known to play diverse roles in morphogenesis and promote cellular proliferation and differentiation.The prolonged and high ex-pression of Meis2 in the interdigital tissues between the elongated bat digits suggests that it participates in the sculpting of the bat wings.These results suggest multiple genetic changes occurred independently during the evolution of bat wings to elongate the hand digits,promote mem-brane growth and keep other digits short.Our findings also indicate that the evolution of limb morphology depends on the complex integration of multiple gene regulatory networks and biological processes that control digit formation and identity,chondrogenesis,and interdigital regression or retention.