Abstract

Background

   The two Caenorhabditis elegans somatic gonadal precursors (SGPs) are
   multipotent progenitors that generate all somatic tissues of the adult
   reproductive system. The sister cells of the SGPs are two head
   mesodermal cells (hmcs); one hmc dies by programmed cell death and the
   other terminally differentiates. Thus, a single cell division gives
   rise to one multipotent progenitor and one differentiated cell with
   identical lineage histories. We compared the transcriptomes of SGPs and
   hmcs in order to learn the determinants of multipotency and
   differentiation in this lineage.

Results

   We generated a strain that expressed fluorescent markers specifically
   in SGPs (ehn-3A::tdTomato) and hmcs (bgal-1::GFP). We dissociated cells
   from animals after the SGP/hmc cell division, but before the SGPs had
   further divided, and subjected the dissociated cells to
   fluorescence-activated cell sorting to collect isolated SGPs and hmcs.
   We analyzed the transcriptomes of these cells and found that 5912
   transcripts were significantly differentially expressed, with at least
   two-fold change in expression, between the two cell types. The
   hmc-biased genes were enriched with those that are characteristic of
   neurons. The SGP-biased genes were enriched with those indicative of
   cell proliferation and development. We assessed the validity of our
   differentially expressed genes by examining existing reporters for five
   of the 10 genes with the most significantly biased expression in SGPs
   and found that two showed expression in SGPs. For one reporter that did
   not show expression in SGPs, we generated a GFP knock-in using
   CRISPR/Cas9. This reporter, in the native genomic context, was
   expressed in SGPs.

Conclusions

   We found that the transcriptional profiles of SGPs and hmcs are
   strikingly different. The hmc-biased genes are enriched with those that
   encode synaptic transmission machinery, which strongly suggests that it
   has neuron-like signaling properties. In contrast, the SGP-biased genes
   are enriched with genes that encode factors involved in transcription
   and translation, as would be expected from a cell preparing to undergo
   proliferative divisions. Mediators of multipotency are likely to be
   among the genes differentially expressed in SGPs.

Electronic supplementary material

   The online version of this article (10.1186/s12864-019-5821-z) contains
   supplementary material, which is available to authorized users.

   Keywords: SGP, hmc, Multipotent progenitor, C. elegans, Transcriptome,
   Differential gene expression

Background

   Embryonic stem cells are pluripotent; they can generate all cell types
   of the body, including cells from all three germ layers. Adult stem and
   progenitor cells can give rise to a more limited array of cell types
   and are therefore classified as multipotent. Although progress has been
   made in understanding the determinants of pluripotency [[35]1], much
   less is known about the determinants of multipotency.

   The C. elegans somatic gonadal precursors (SGPs) are multipotent
   progenitors that generate all somatic cells of the adult reproductive
   system. The two SGPs, Z1 and Z4, are born during embryogenesis and they
   migrate to join the primordial germ cells (PGCs) to form the
   four-celled gonadal primordium [[36]2]. SGPs remain quiescent until the
   first larval stage, when they go through two periods of cell division
   to produce all 143 cells of the mature hermaphrodite somatic gonad
   (Fig. [37]1a) [[38]3]. The SGPs give rise to important regulatory
   cells, the distal tip cells (DTCs) and the anchor cell (AC), as well as
   complex multicellular tissues, including the sheath, spermatheca, and
   uterus (reviewed in [[39]4]). The sisters of the SGPs are the two head
   mesodermal cells, hmcR and hmcL. hmcR undergoes programmed cell death
   late in embryogenesis and hmcL differentiates without further division
   as the single head mesodermal cell (Fig. [40]1b) [[41]2]. The hmc cell
   extends cellular processes along the anterior-posterior and
   dorsal-ventral body axes to generate its distinctive H-shaped
   morphology [[42]5]. The function of hmc remains unknown.

Fig. 1.

   Fig. 1
   [43]Open in a new tab

   FACS sorting SGPs and hmcs from L1 larvae. (a) The SGPs (Z1 and Z4;
   red), and one hmc (green) are present in the first larval (L1) stage.
   The SGPs divide to produce support cells of the adult reproductive
   system, including distal tip cells (DTC), sheath, spermatheca, and
   uterus (grey). Each SGP produces one of the two gonadal arms: Z1 makes
   the anterior arm and Z4 makes the posterior arm. (b) Cell lineage
   leading to SGPs and hmcs. Precursor cells (not shown) divide
   asymmetrically to generate one SGP and one hmc. The hmcR cell dies by
   programmed cell death prior to the L1 stage. (c) Merged confocal
   differential interference and fluorescence microscopy image of an L1
   stage worm with reporters expressed in the SGPs (ehn-3::tdTomato, red)
   and the hmc (bgal-1::GFP, green). Inset shows fluorescence images for
   each cell type. (d) Cell dissociates from L1 stage larvae showing
   individual cells expressing ehn-3::tdTomato (D, SGPs) and bgal-1::GFP
   (D’, hmcs). (e) FACS profile of dissociated cells from L1 larvae. GFP
   positive (green) and tdTomato positive cells (red) are outlined with
   boxes

   We previously reported that hnd-1 and the SWI/SNF (SWItching
   defective/Sucrose Non-Fermenting) chromatin remodeling complex play
   roles in the SGP/hmc cell fate decision [[44]6]. hnd-1 encodes a bHLH
   transcription factor and the SWI/SNF chromatin remodeling complex
   regulates gene expression by altering chromatin structure. In animals
   carrying mutations in either of these transcriptional regulators, the
   SGPs usually express SGP-characteristic markers and migrate to form the
   gonadal primordium, but they can also express markers of the hmc cell
   fate and sometimes fail to develop into the tissues of the reproductive
   system [[45]6]; this suggests that SGPs are often partially transformed
   into hmcs in these mutants. The incompletely penetrant phenotype of the
   mutations indicates that there are additional regulators of the SGP/hmc
   cell fate decision.

   Here, we perform transcriptional profiling of isolated SGP and hmc
   cells to identify the gene expression differences underlying their
   distinctive cell fates. We find that the differentiated hmc cell
   expresses genes characteristic of neurons, suggesting that it has
   neuronal properties. In contrast, the SGP cells express genes involved
   in transcription and translation, which is consistent with the fact
   that they are poised to proliferate to generate the tissues of the
   somatic gonad.

Methods

Strains

   C. elegans strains were cultured as described previously [[46]7,
   [47]8]. All strains were grown at 20 °C unless otherwise specified and
   were derived from the Bristol strain N2. Strains were obtained from the
   Caenorhabditis Genetics Center or were generated as described below.
   The following alleles were used in this study and are described in C.
   elegans II [[48]9], cited references, or this work: