No abstract

Animal development is remarkably robust; cell fates are specified with spatial and temporal precision despite physiological and environmental contingencies. Favorable conditions cause Caenorhabditis elegans to develop rapidly through four larval stages (L1-L4) to the reproductive adult. In unfavorable conditions, L2 larvae can enter the developmentally quiescent, stress-resistant dauer larva stage, enabling them to survive for prolonged periods before completing development. A specific progression of cell division and differentiation events occurs with fidelity during the larval stages, regardless of whether an animal undergoes continuous or dauer-interrupted development. The temporal patterning of developmental events is controlled by the heterochronic genes, whose products include microRNAs (miRNAs) and regulatory proteins. One of these proteins, the DAF-12 nuclear hormone receptor, modulates the transcription of certain let-7-family miRNAs, and also mediates the choice between the continuous vs. dauer-interrupted life history. Here, we report a complex feedback loop between DAF-12 and the let-7-family miRNAs involving both the repression of DAF-12 by let-7-family miRNAs and the ligandmodulated transcriptional activation and repression of the let-7-Fam miRNAs by DAF-12. We propose that this feedback loop functions to ensure robustness of cell fate decisions and to coordinate cell fate with developmental arrest.gene regulation ͉ microRNA ͉ nuclear hormone receptor T he complexity of animal development requires the coordination of temporal, spatial, and physiological cues. Successful development is necessary to achieve optimal fitness, and natural selection has favored developmental programs that robustly produce a desired outcome in a range of physiological and environmental conditions. Forward genetics has revealed much of the internal programming of several developmental processes in standardized laboratory conditions. A fundamental question emerging from these studies is how these genetic circuits are affected by conditions that are more similar to natural, variable environments. Caenorhabditis elegans has been an excellent system to uncover developmental mechanisms because of its well-defined cell lineage and tractability to genetic analysis. C. elegans development is robust in a wide range of environmental conditions, providing an excellent opportunity to determine how genetic pathways are modulated in these diverse environments (1).There are two distinct life histories a C. elegans larva may follow: a rapid, continuous life history that occurs in favorable environments and an extended, interrupted life history (2) that occurs in response to increased population density, decreasing food supplies, and elevated temperature (Fig. 1) (3). Within the continuous life history, animals progress through four larval stages (L1-L4) to a reproductively competent adult within Ϸ40-50 h after hatching (4). By contrast, unfavorable environments sensed during L1 stage cause larvae to enter the predauer L2d stage, which is ...

The anchor cell/ventral uterine precursor cell (AC/VU) decision in Caenorhabditis elegans is a canonical example of lin-12/Notch-mediated lateral specification. Two initially equivalent cells interact via the receptor LIN-12 and its ligand LAG-2, so that one becomes the AC and the other a VU. During this interaction, feedback loops amplify a small difference in lin-12 activity, limiting lin-12 transcription to the presumptive VU and lag-2 transcription to the presumptive AC. Here, we find that hlh-2 appears to be required for the VU fate and directly activates lag-2 transcription in the presumptive AC. HLH-2 appears to accumulate selectively in the presumptive AC prior to differential transcription of lin-12 or lag-2, and is therefore the earliest detectable difference between the two cells undergoing the AC/VU decision. The restricted accumulation of HLH-2 to the presumptive AC reflects post-transcriptional down-regulation of HLH-2 in the presumptive VU. Our observations suggest that hlh-2 is regulated as part of the negative feedback that down-regulates lag-2 transcription in the presumptive VU. Finally, we show that the AC/VU decision in an individual hermaphrodite is biased by the relative birth order of the two cells, so that the first-born cell is more likely to become the VU. We propose models to suggest how birth order, HLH-2 accumulation, and transcription of lag-2 may be linked during the AC/VU decision.

Dauer diapause is a stress-resistant, developmentally quiescent, and long-lived larval stage adopted by Caenorhabditis elegans when conditions are unfavorable for growth and reproduction. This chapter contains methods to induce dauer larva formation, to isolate dauer larvae, and to study pre- and post-dauer stages.

G2A is a heptahelical cell surface protein that has recently been described as a potential tumor suppressor, based on its ability to counteract transformation of pre-B cells and ®broblasts by Bcr-Abl, an oncogenic tyrosine kinase. We have isolated cDNAs encoding G2A in the course of screening libraries for clones that cause oncogenic transformation of NIH3T3 ®broblasts. When expressed at high levels in NIH3T3 cells by retroviral transduction, G2A induced a full range of phenotypes characteristic of oncogenic transformation, including loss of contact inhibition, anchorage-independent survival and proliferation, reduced dependence on serum, and tumorigenicity in mice. When expressed by transfection, G2A greatly enhanced the ability of a weakly oncogenic form of Raf-1 to transform NIH3T3 cells. These results demonstrate that G2A is potently oncogenic both on its own and in cooperation with another oncogene. Expression of G2A in ®broblasts and endothelial cells resulted in changes in cell morphology and cytoskeleton structure that were equivalent to those induced by the G protein subunit Ga 13 . Transformation of NIH3T3 cells via G2A expression was completely suppressed by co-expression of LscRGS, a GTPase activating protein that suppresses signaling by Ga 12 and Ga 13 . Hyperactivity of Ga 12 or Ga 13 has previously been shown to result in activation of Rho GTPases. G2A expression resulted in activation of Rho, and transformation via G2A was suppressed by a dominant negative form of RhoA. These results indicate that G2A may be directly coupled to Ga 13 , and that it is the activation of this Rho-activating Ga protein which is responsible for the ability of G2A to transform ®bro-blasts.