Germline sex determination is a complex process that is necessary for the formation of sexually dimorphic gametes. C. elegans hermaphrodites switch from generating sperm to oocytes in the fourth larval stage. Mutations in genes in this pathway results in an increase or decrease the number of sperm that are generated. We have identified a family of microRNAs that regulate the switch from producing sperm to producing oocytes in C. elegans. The miR-44 family comprises four miRNAs: miR-44, miR-45, miR-61, and miR-247 that share a common seed sequence and thus are predicted to regulate shared target mRNAs. Interestingly, miR-44 and miR-45 share an identical mature sequence and are located only ~9kb apart on chromosome II. This close proximity precluded the generation of double mutants through conventional genetic crosses. Using CRISPR/Cas9, we generated worms that lacked both
mir-44 and
mir-45:
mir-45(
xw11)
mir-42/43/44(nDf49), which are referred to as "
mir-44/45" mutants.
mir-44/45 mutants have a significantly decreased brood size and generate an increased number of unfertilized oocytes. The low number of progeny and high number of unfertilized oocytes can be accounted for by a decreased number of sperm. To determine if the decreased number of sperm was due to a premature switch from producing sperm to producing oocytes, we analyzed worms at L4 molt+5 hours and saw that few wild-type worms had any embryos compared to 66% of
mir-44/45 mutants at the same time point that have at least one embryo, indicating that
mir-44/45 mutants switch earlier. To understand the genetic relationship between
mir-44/45 and the genes that regulate the switch from producing sperm to oocytes, we examined a component of the pathway which modulates sperm number, FBF-1.
fbf-1(
ok91) mutants generate an increased number of sperm and show a delayed switch to producing oocytes (Crittenden et al. 2002). In a
mir-44/45;
fbf-1 multiply mutant strain, we saw that loss of
fbf-1 activity suppressed the decreased sperm and early embryo phenotypes observed in
mir-44/45 mutants, indicating that
mir-44/45 likely functions upstream of
fbf-1 to regulate the germline sex determination pathway.