An emerging complexity of mammalian Ras signal transduction is the assortment of catalytically diverse effectors that may facilitate the elaborate biological activities of Ras in normal and neoplastic cells. The precise role that each effector serves, dynamic regulation of effector utilization, and interplay between effector networks are issues that remain poorly understood. Analysis of C. elegans
vulval development has provided key insights into Ras signaling components and concepts conserved in mammalian cells. The v
ells (VPCs) are a developmental equivalence group of six ventral epithelial cells (P3.p–P8.p) (Sternberg, 2005
) (). The nearby anchor cell (AC) induces VPCs to assume a highly reproducible 3°-3°-2°-1°-2°-3° pattern of fates. The AC-proximal VPC is induced to assume the 1° fate, flanking VPCs assume the 2° fate, and distal uninduced VPCs assume the non-vulval 3° fate.
Along with studies in other systems, analyses of the pro-1° AC inductive signal were instrumental in delineating the first signal transduction pathway connecting the cell surface to the nucleus (Egan and Weinberg, 1993
). The AC secretes LIN-3/EGF (epidermal growth factor), which promotes LET-60/Ras activation of the LIN-45/Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling cascade to regulate the LIN-1 (Ets) and LIN-31 (HNF) transcription factors, thereby inducing 1° fate (Sundaram, 2006
) (). Analogously to human cancers, mutational activation of LET-60/Ras promotes ERK activation, leading to excess vulval induction (), while loss of pathway components results in vulval absence. All constituents of this signaling pathway, particularly LET-60/Ras, are strongly conserved (Figure S1C
). This degree of conservation argued, prematurely, that our understanding of Ras effector signaling was complete. However, subsequent mammalian cell studies characterized additional Ras effectors, with now at least 10 distinct functional classes identified (Repasky et al., 2004
). With many effectors expressed ubiquitously, an unresolved issue is how Ras effector utilization is orchestrated to facilitate the complex biological outputs of Ras.
RAL-1 Antagonizes LET-60-dependent Vulval Induction
Recent analyses have implicated the guanine nucleotide exchange factor for the Ral GTPase (RalGEF) as an effector of importance comparable to Raf in Ras-dependent human oncogenesis (Chien and White, 2003
; Hamad et al., 2002
). Like Ras, Ral functions as a GDP/GTP-regulated switch. Since RalGEF and Ral are conserved in C. elegans
, EGF activation of Ras could involve the RalGEF-Ral pathway in regulation of vulval cell fate.
Two competing models have been proposed to illustrate the mechanisms of vulval fate patterning. The original “morphogen gradient model” posits that a LIN-3/EGF, AC-maximal concentration gradient differentially patterns VPCs dictated by proximity to the AC (Katz et al., 1995
; Katz et al., 1996
; Sternberg and Horvitz, 1986
). This model posits that while strong EGF signal induces 1° fate, diminished EGF signal directly promotes 2° fate for more distal VPCs. Appropriately, an ERK-responsive 1° fate reporter was highly expressed in the presumptive 1° VPC, with transient low expression in presumptive 2° VPCs (Yoo et al., 2004
), but further mechanistic support is lacking.
In contrast, the “sequential induction model” proposes that EGF induces only the most proximal VPC, which becomes 1°. Subsequently this presumptive 1° cell expresses DSL ligands that, via the LIN-12/Notch receptor, laterally induce neighboring VPCs to assume 2° fate (Chen and Greenwald, 2004
). Accordingly, the LET-23/EGF receptor (EGFR) is necessary for 1° but not 2° fate induction (Koga and Ohshima, 1995
; Simske and Kim, 1995
), and pro-1° EGF and pro-2° Notch pathways together are necessary and sufficient to generate initial commitment to the 2°-1°-2° fate pattern (Greenwald, 2005
; Sternberg, 2005
). However, the “sequential induction model” and the “morphogen gradient model” have yet to be mechanistically reconciled.
Importantly, 1° and 2° fates are mutually antagonistic; via “quenching” mechanisms inappropriate pathway activities are reduced to minimize conflicting pro-1° and pro-2° signals in the same cell. Presumptive 1° cells enact programs that antagonize pro-2° signaling (Levitan and Greenwald, 1998
; Yoo and Greenwald, 2005
), and conversely presumptive 2° cells enact programs that antagonize pro-1° signaling (Berset et al., 2001
; Berset et al., 2005
; Yoo et al., 2004
). For example, the LIP-1 ERK protein phosphatase is expressed in presumptive 2° lineages to quench ERK signaling. Thus, the developmental consequences of EGF activation of Ras-Raf signaling in 2° lineages may be minimal, and pro-2° EGF activity mediated through distinct effector pathways.
The most plausible composite model for robust vulval patterning would be one that reconciles these three evidence-based models: graded morphogen signaling, sequential induction, and pathway quenching. Yet nothing is known about the mechanism of the putative pro-2° EGF signal. Despite decades of research into how a single morphogen gradient can induce multiple cell fates, there are few instances in which the mechanism of such differential inductions is understood (Piddini and Vincent, 2009
In this study, we identify a mechanism for EGF pro-2° signaling and thus reconcile the three features of vulval patterning into a unified model. We show that during vulval patterning Ras through Raf transduces a pro-1° signal, then through the RalGEF-Ral pathway transduces a pro-2° signal. Ral signaling antagonizes Raf and regulates the balance of 1° and 2° fates. Ral is necessary and sufficient to drive maximal Notch pro-2° activity, and the RalGEF-Ral pathway is quenched in presumptive 1° cells by restricted Ral expression. In summary, our study establishes that Ras effector utilization is controlled to signal for distinct cellular outcomes. Analogous mechanisms may therefore contribute to the distinct patterns of effector utilization that occur in different settings of mutant Ras-driven human cancers.