FKH-6 is a forkhead transcriptional regulator that is necessary for proper gonadal development in C. elegans. A previous expression study using a transcriptional reporter showed that the fkh-6 transcript was initially expressed in the gonad from mid-L1 larval stage until around the L1/L2 molt in both sexes (Chang et al. 2004). Transcriptional reporters and partial gene fusions indicated that FKH-6 was also expressed from the L3 larval stage through adult in the hermaphrodite gonad in the spermatheca and sheath cells (Hope et al.2003, Chang et al. 2004). In this current study, the endogenous locus for fkh-6 was C-terminally fused with GFP using CRISPR-Cas. In addition to recapitulating previously reported expression patterns, this new construct revealed expression of FKH-6::GFP in 2-4 male gonadal cells in the L3 larval stage. This is the first report of FKH-6 expression in males after the L1/L2 molt. The expression of FKH-6::GFP in the gonad of the L3 male is reproducible but short-lived, and coincides with the period when the proximal region of the migrating male gonad is extending past the distal tip of the gonad. This work shows that FKH-6 is expressed in both sexes during the early mitotic period of the gonad in L1 as well as the late mitotic period of the gonad in L3.
The fkh-6::gfp fusion was generated using CRISPR-Cas (Dickinson et al.2015). Sequencing confirmed the integration of GFP at the 3’ end of the fkh-6 locus prior to the stop codon. The MBK9 strain (fkh-6(ndz1[fkh-6::gfp::3xflag]) II; him-8(e1489) IV was used for imaging. Differential interference contrast (DIC) and fluorescent images were acquired on a Zeiss Axiovert 135 microscope using a Lumenera Infinity 3S-1UR monochrome camera and Infinity Analyze and Capture software.
University of South Alabama faculty start-up funds
Reviewed ByJennifer Wolff
HistoryReceived: November 29, 2018
Accepted: December 31, 2018
Published: December 31, 2018