DOI: 10.5061/dryad.xgxd254j4
title: Serial thin section movie of every third section from the DTC to the distal extensions of Sh1 in a young adult hermaphrodite posterior gonad arm

description: 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.
description: 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.