Functional transcriptomics of a migrating cell in Caenorhabditis elegans

Abstract

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi-treated L4 larvae. We observed expression of 8,000-10,000 genes in the linker cell, 22-25% of which were up-or downregulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67-dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.major sperm protein | nematode development | transcriptional profiling | twin pore potassium channels | conserved uncharacterized proteins C ell migration is pervasive in animal development and is also an unwanted part of human cancer. Several different varieties of cell migration, ranging from social protozoa to the human immune system, have been extensively studied (1), but there remains a need for detailed analysis of the different components of migration in a simple cell type that can be exhaustively probed through anatomy, genetics, and genomics. We have undertaken to develop the linker cell (LC) of Caenorhabditis elegans as such a model. The LC is a single cell, attached to the front of a proliferating male gonad, which carries out a stereotypic, long-range migration to generate the shape of the mature gonad (Fig. 1A). During 25 h of the second to fourth larval stages (L2 through L4), the LC moves anteriorly along the ventral body wall, turns dorsally, proceeds posteriorly, switches sides once more to the ventral bodywall, continues posteriorward, reaches the cloaca, and eventually dies (2-4). Throughout this process, the proliferating and lengthening male gonad follows the LC's path to the cloaca. Stage-specific changes occur during the middle to late stages of migration (L3 and L4 larval stages): the LC changes shape from a spheroid to a pointed ellipsoid; it increases its speed of migration; and the netrin receptor gene unc-5 is down-regulated within the LC, which causes it to switch body walls ventrally ( Fig. 1A; ref. 3). These changes between the L3 and L4 stages require the orphan nuclear receptor NHR-67, whose orthologs TAILLESS and TLX...

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“…Max_prot_size: the size of the largest predicted protein product. Housekeeping: a set of genes that we previously observed, by single-cell RNA-seq, to be consistently active both in whole C. elegans larvae and in three different states of migrating C. elegans linker cells (Schwarz et al, 2012). 7TM_GPCRs : a set of genes encoding G-protein coupled receptors (GPCRs), a class of genes of particular biological interest in deciphering AWC function.…”

“…To identify candidate GPCRs expressed in the AWC neurons, we microdissected GFP-labeled AWC on neurons ( str-2 ::GFP) and performed single-cell RNA-seq (Schwarz et al, 2012). Nine individual AWC neurons were dissected, separately amplified for their transcripts with RT-PCR, and equal aliquots from the nine separate purified products were sequenced as a single AWC RT-PCR pool.…”

“…Among most genes (labeled ‘AWC’ and shown as gray dots) are highlighted 32 genes encoding G-protein coupled receptors shown as green triangles and 984 genes encoding housekeeping functions shown as red dots. The housekeeping genes were identified in a previous single-cell RNA-seq analysis (Schwarz et al, 2012). Full identities, annotations, and expression data for all C. elegans genes are provided in Figure 7—source data 1. DOI: http://dx.doi.org/10.7554/eLife.20023.01010.7554/eLife.20023.011Traits of C. elegans genes expressed in AWC, with RNA-seq expression data generated with our full data set and our most recent computational methods.Data columns are as follows.…”

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“…Furthermore, NHR-67 mediates male linker cell migration--the male equivalent cell lineage of the hermaphrodite AC. Interestingly, similar to the AC, nhr-67 also promotes the expression of the MMP zmp-1 (Kato and Sternberg, 2009; Schwarz et al, 2012), but is not required to maintain the linker cell in a non-dividing state. A relationship between vertebrate NHR-67 orthologs and cell cycle regulation does exist in vertebrate development and cancer progression.…”

Nuclear receptors in nematode development: Natural experiments made by a phylum

“…Whole transcriptome expression analysis of a linker cell inhibited for NHR-67 by RNAi identified 22-25% of 10,000 analyzed genes as upor down-regulated (20-fold) [111]. The involvement of genes identified as NHR-67 dependent in linker cell migration was confirmed in 22% (45 of 204) of genes by functional analysis [111].…”