An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

Abstract

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.

Germ Granules Prevent Accumulation of Somatic Transcripts in the AdultCaenorhabditis elegansGermline

“…Gene categories were defined using published microarray and serial analysis of gene expression (SAGE) data sets that profiled specific tissues or whole worms which contained or lacked a germline (Baugh et al 2003;Reinke et al 2004;Meissner et al 2009;Wang et al 2009), as described in Rechtsteiner et al (2010) and Knutson et al (2016). Ubiquitous genes (1895 genes) are genes that are expressed (tag .…”

Broad chromosomal domains of histone modification patterns in C. elegans

“…To investigate histone marks on genes expressed in different tissues, we categorized genes based on temporal and spatial expression data (Meissner et al 2009;Wang et al 2009;Rechtsteiner et al 2010) (Methods). ''Ubiquitous genes'' (2580 in total) are expressed across different stages and tissues, whereas ''silent genes'' (415) include serpentine receptors that are not expressed in most stages and tissue types.…”

“…In brief, ''ubiquitous'' or housekeeping genes have transcripts (at least 1 tag) present in muscle, gut, neuron, and adult germline SAGE (Serial Analysis of Gene Expression) data sets (Meissner et al 2009;Wang et al 2009). ''Silent'' serpentine receptor genes are expressed in only a few mature neurons (Kolasinska-Zwierz et al 2009).…”

“…''Silent'' serpentine receptor genes are expressed in only a few mature neurons (Kolasinska-Zwierz et al 2009). ''Germline-specific'' genes are expressed exclusively in the maternal germline, as their transcripts are present in the germline (Reinke et al 2004;Wang et al 2009), maternally loaded into embryos (Baugh et al 2003), and absent from muscle, gut, and neuron SAGE data sets (Meissner et al 2009). ''Soma-specific'' genes are expressed in muscle, gut, and neuron SAGE data sets (Meissner et al 2009) but not in the adult germline (Reinke et al 2004;Wang et al 2009).…”

The Balanced Regulation of Hsc70 by DNJ-13 and UNC-23 Is Required for Muscle Functionality

“…3 In nematodes, unc-23 is linked to motility defects (16 -18). 3 In genome-wide investigations of muscular proteins and their localization, UNC-23 was found to be required for establishing the myosin ultrastructure (18,19). Mutations in unc-23 result in motility dysfunctions in all developmental stages, including a head-bent phenotype and weakness in the attachment structures connecting hypodermis and muscle cells (16,20).…”