standard Cas9 Mutagenesis in Houseflies Confirms Male Determining Factor Identification

File:Musca domestica mating.jpg

Musca domestica. Image Credit: Muhammad Mahdi Karim

Sharma et al. (2107) report in a recent issue of Science the identification of a male determining factor in the housefly Musca domestica and provide convincing reverse genetic confirmatory evidence using dsRNA-mediated gene silencing and Cas9-mediated mutagenesis.

Alternative splicing of the tra transcript to produce male- and female-specific gene products is a common element in insect sex determination. What is not common among insects are the genetic signals upstream of tra that regulate this alternative splicing.

Conserved terminal steps in insect sex determination. Signals upstream of tra vary from insect to insect.

In houseflies M-factors are a source of upstream signals that leads to male development.

M-factors in males can be located on any of the autosomes, X or Y chromosomes.

Senior investigator, Dr. Daniel Bopp (an IGTRCN Participant), has invested heavily over many years in studying the sex determination system of Musca and those efforts have resulted in a solution to a significant portion of the Musca M-factor puzzle.  RNAseq on male embryos resulted in the identification an excellent candidate transcript/gene that they call Musca domestica male determinerMdmd.  The timing of zygotic expression among other things were consistent with Mdmd being a bona fide male determining factor.  Mdmd is a paralog of Mdncm (Musca domestica nucompholin), a protein associated with the spliceosome and required for the formation of the exon junction complex – an interesting connection to a process of major importance in insect sex determination.

Genetic Technology Delivery by Embryo Microinjection

Sharma et al. used two reverse genetic technologies to provide compelling evidence that Mdmd is a male determining factor.  Double-stranded RNA-based gene silencing was use to silence Mdmd early in development by injecting Mdmd dsRNA into preblastoderm embryos.  The results were flies with male and female characteristics; such as a fly with male genitalia and well developed ovaries.  Fully penetrant male to female conversions were unlikely given the transient nature of the dsRNA silencing signal and the results reported supported the hypothesis that Mdmd was an M-factor.

More compelling were data from a Cas9 mutagenesis effort.  Cas9 protein complexed with a guide RNA was injected into preblastoderm embryos and Sharma et al. were able to recover almost 60 fertile males.  At least 10 produced females with loss-of-function mutations in the Mdmd locus.

Cas9-mediated double strand DNA breaks leading to inaccurate repair resulting in indels.

While not the first insect male determining factor to be identified – two have been identified in mosquitoes – Mdmd bares no resemblance to either.  Interestingly, Sharma et al report data that this is only one of at least two M-factors in M. domesticaMdmd could not be detected in lines of M. domestica with an M-factor on chromosome 1 suggesting that there is yet another way that M. domestica sex differentiation is initiated.

This is a wonderful example of the use of reverse genetic technologies in a genetically tractable insect system to reveal some very interesting biology.

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