Discovering Functional ERK Substrates Regulating Caenorhabditis elegans Germline Development

Abstract

The Rat Sarcoma (RAS) GTPAse-mediated extracellular signal-regulated kinase (ERK) pathway regulates multiple biological processes across metazoans. In particular during Caenorhabditis elegans oogenesis, ERK signaling has been shown to regulate over seven distinct biological processes in a temporal and sequential manner. To fully elucidate how ERK signaling cascade orchestrates these different biological processes in vivo, identification of the direct functional substrates of the pathway is critical. This chapter describes the methods that were used to identify ERK substrates in a global manner and study their functions in the germline. These approaches can also be generally applied to study ERK-dependent biological processes in other systems.

Keywords: C. elegans oogenesis; ERK substrates; Functional genomics.

Fig. 1

ERK activation pattern in sensitized genetic backgrounds. (a) Schematic of C. elegans adult hermaphrodite. (b) Schematic of one adult hermaphroditic germline showing different stages of germ cell development, dp MPK-1 pattern and seven MPK-1-dependent processes throughout germline development. DTC marks distal tip cell. TZ marks transition zone. (c) DAPI staining and activation pattern of dpMPK-1 ERK (red) in wild-type, mpk-1 loss-of-function and let-60 gain-of-function alleles at permissive temperature (20 °C). Both mutants have wild-type germline morphology despite decreased or increased/ectopic levels of dpMPK-1 ERK. Asterisks and white lines mark the distal and proximal ends of the germline, respectively [36, 37]. Scale bar: 20 μm

Fig. 2

Flow chart of experimental procedures. (a) Outline of bioinformatics approach based on defined ERK docking site motifs. (b) Rationale of genetic approach using RNAi-based enhancer screen. (c) Outline of biochemical analysis of the functional interactors [21]

Fig. 3

mRNA expression as assayed by in situ hybridization by the Kohara lab, accessible via the Nematode Expression Data Base (NEXTDB). For example, in situ hybridization for drsh-1, an RNase III ribonuclease (a), reveals germline expression, while that for mec-7 beta tubulin (b) does not. Clone 842a08 and 411c2 were used to detect drsh-1 and mec-7 mRNA expression, respectively