Academic journal article Genetics

Spatiotemporal Gene Expression Analysis of the Caenorhabditis Elegans Germline Uncovers a Syncytial Expression Switch

Academic journal article Genetics

Spatiotemporal Gene Expression Analysis of the Caenorhabditis Elegans Germline Uncovers a Syncytial Expression Switch

Article excerpt

AT the core of metazoan sexual reproduction lies the development of haploid gametes, namely the sperm and the egg produced from diploid germline stem cells (LHernault 1997; Yoshida 2010; Sánchez and Smitz 2012; Chu and Shakes 2013; Ellis and Stanfield 2014; Tanaka 2014). This developmental process includes well-defined cellular steps, but the genetic program that drives them is largely unknown. During gametogenesis, the mitotically cycling stem cells must produce diploid cells that lose their stem cell identity, accumulate proteins required for entry into meiosis, and then proceed onto two consecutive rounds of meiotic cell divisions (meiosis I and II). A critical phase within gametogenesis occurs before the first meiotic division during prophase I. Specifically, homologous chromosomes find each other, pair, synapse, and exchange segments via homologous recombination in preparation for their segregation away from each other toward opposite poles of the meiosis I spindle. During prophase I of oogenesis in many organisms, the oocytes also accumulate transcripts required for early stages of embryogenesis [reviewed in Sánchez and Smitz (2012) and Kim et al. (2013)]. Yet, despite decades of studies providing descriptions of the gametogenic processes, we know very little about what drives and controls the development of sperm and oocytes. This stands in contrast to studies in yeast in which the availability of large quantities of synchronized meiotic cells have allowed transcriptome and ribosome profiling analysis of the budding yeast sporulation process (Chu et al. 1998; Primig et al. 2000; Brar et al. 2012).

The Caenorhabditis elegans gonad offers a unique opportunity for the study of progression from the undifferentiated proliferative germ cells through spermatogenesis or oogenesis (LHernault 1997; Dernburg 2001; Couteau et al. 2004; Greenstein 2005; Zetka 2009; Schvarzstein et al. 2010; Chu and Shakes 2013; Lui and Colaiácovo 2013; Nousch and Eckmann 2013; Ellis and Stanfield 2014). Nuclei in both the adult male and hermaphrodite tube-like gonads are arranged along the distal-proximal axis in a spatiotemporal fashion from the germ stem cell mitotic proliferative zone through the various stages of meiotic prophase, and then differentiate into mature sperm or oocytes (Kimble and Crittenden 2005; Shakes et al. 2009; Pazdernik and Schedl 2013; Ellis and Stanfield 2014) (Figure 1A and Figure 6A). Important contributions to our understanding of the genes required for germ cell progression were achieved through RNA-sequencing (RNA-seq), DNA microarray, and SAGE (serial analysis of gene expression) analyses of the entire C. elegans gonad (Reinke et al. 2000, 2004; Wang et al. 2009; Ortiz et al. 2014). However, due to the gonad's syncytial nature, in which germ "cells" do not become fully cellularized until late diakinesis, and instead maintain a bridge or connection to a common core referred to as the rachis to which they contribute their cytoplasmic contents that flow into developing oocytes, it has so far been impossible to analyze the dynamics of the transcriptome through this process.

Recent advancements in high-throughput sequencing have enabled the analysis of mRNA abundances from small biological samples as small as individual cells (Tang et al. 2011; Hashimshony et al. 2012; Avital et al. 2014; Islam et al. 2014; Macaulay and Voet 2014). Among the available methods, CEL-Seq (cell expression by linear amplification and sequencing) is a multiplexed method that uses in vitro transcription for single-cell transcriptome analysis (Hashimshony et al. 2012). However, these methods have only recently started to be used to analyze transcriptome dynamics through an entire organ or microscopically defined syncytial tissue (Jan 2017).

Here, we report the development of a novel approach for transcriptome analysis of syncytial tissues by combining ARCTURUS Laser Capture Microscopy (LCM) for sample extraction and CEL-Seq analysis. …

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