Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions – lacking somatic gonadal cell coverage of germ cells – are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic poisonous gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

Wild-type N2 young adults were analyzed using high-pressure freezing/freeze-substitution freezing (HPF/FS; Hall, 2012). Four animals from chemical immersion and four animals from HPF/FS were collected in serial sections on slot grids, ranging from 80 nm–100 nm thickness on an RMC PowerTome XL ultramicrotome (Eden Instruments, Valence, France). Sections were post-stained in 2% uranyl acetate in H2O for 20 min and in Reynold’s lead for 3 min. Sections from each animal were viewed in the gonad region using Digital Micrograph software (Gatan, Pleasanton, CA) for the JEOL JEM-1400Plus M (Jeol USA, Peabody, MA), using a Gatan Orius SC1000B digital camera.

We selected the most optimally positioned and resolved HPF/FS-fixed animal (2% osmium tetroxide, 0.1% uranyl acetate, and 2% H2O in acetone) to collect high-resolution montages of the gonad arm from every third section covering 80 µm, including the DTC and sheath filopodia of a posterior distal gonad arm. This series was collected from the tail to the vulva region to encompass the whole posterior gonad arm, but we did not collect full images closer to the gonad reflection in order to save effort and expense. The pixel size for the images was 3.23 nm. The images were aligned with TrakEM2 software (Cardona et al., 2012) and traced on a Wacom DTZ 2100D tablet (Wacom, Portland, OR) to build the 3D model.

We followed the DTC processes and their fragments and the filopodial extensions of Sh1 due to their increased translucency compared with the germ cells and by the fact that these somatic cellular extensions contained many small mitochondria. We observed what we infer to be shed pieces of the DTC beginning at approximately 30 µm from the distal end, as has been observed using light microscopic methods (Byrd et al., 2014). However, some DTC and Sh1 processes may appear discontinuous due to the fact that micrographs of every third section were used to generate the reconstructed model (Video 5). Regardless of the effect that this sampling technique has on our ability to resolve contiguous versus shed bits of somatic gonadal cells, this TEM analysis is in agreement with our light microscopic observations that a large subset of distal germ cell progenitors are not in contact either with the DTC or Sh1.