Predicting mutation outcome from early stochastic variation in genetic interaction partners.

Many mutations, including those that cause disease, only have a detrimental effect in a subset of individuals. The reasons for this are usually unknown, but may include additional genetic variation and environmental risk factors. However, phenotypic discordance remains even in the absence of genetic variation, for example between monozygotic twins, and incomplete penetrance of mutations is frequent in isogenic model organisms in homogeneous environments. Here we propose a model for incomplete penetrance based on genetic interaction networks. Using Caenorhabditis elegans as a model system, we identify two compensation mechanisms that vary among individuals and influence mutation outcome. First, feedback induction of an ancestral gene duplicate differs across individuals, with high expression masking the effects of a mutation. This supports the hypothesis that redundancy is maintained in genomes to buffer stochastic developmental failure. Second, during normal embryonic development we find that there is substantial variation in the induction of molecular chaperones such as Hsp90 (DAF-21). Chaperones act as promiscuous buffers of genetic variation, and embryos with stronger induction of Hsp90 are less likely to be affected by an inherited mutation. Simultaneously quantifying the variation in these two independent responses allows the phenotypic outcome of a mutation to be more accurately predicted in individuals. Our model and methodology provide a framework for dissecting the causes of incomplete penetrance. Further, the results establish that inter-individual variation in both specific and more general buffering systems combine to determine the outcome inherited mutations in each individual.

Niche-associated activation of rac promotes the asymmetric division of Drosophila female germline stem cells.

BACKGROUND: Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division. METHODS AND FINDINGS: Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented. CONCLUSIONS: Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis.

Tyramine Acts Downstream of Neuronal XBP-1s to Coordinate Inter-tissue UPRER Activation and Behavior in C. elegans.

In C. elegans, expression of the UPRER transcription factor xbp-1s in neurons cell non-autonomously activates the UPRER in the intestine, leading to enhanced proteostasis and lifespan. To better understand this signaling pathway, we isolated neurons from animals expressing neuronal xbp-1s for transcriptomic analysis, revealing a striking remodeling of transcripts involved in neuronal signaling. We then identified signaling molecules required for cell non-autonomous intestinal UPRER activation, including the biogenic amine tyramine. Expression of xbp-1s in just two pairs of neurons that synthesize tyramine, the RIM and RIC interneurons, induced intestinal UPRER activation and extended longevity, and exposure to stress led to splicing and activation of xbp-1 in these neurons. In addition, we found that neuronal xbp-1s modulates feeding behavior and reproduction, dependent upon tyramine synthesis. XBP-1s therefore remodels neuronal signaling to coordinately modulate intestinal physiology and stress-responsive behavior, functioning as a global regulator of organismal responses to stress.

Neuronal XBP-1 Activates Intestinal Lysosomes to Improve Proteostasis in C. elegans.

The unfolded protein response of the endoplasmic reticulum (UPRER) is a crucial mediator of secretory pathway homeostasis. Expression of the spliced and active form of the UPRER transcription factor XBP-1, XBP-1s, in the nervous system triggers activation of the UPRER in the intestine of Caenorhabditis elegans (C. elegans) through release of a secreted signal, leading to increased longevity. We find that expression of XBP-1s in the neurons or intestine of the worm strikingly improves proteostasis in multiple tissues, through increased clearance of toxic proteins. To identify the mechanisms behind this enhanced proteostasis, we conducted intestine-specific RNA-seq analysis to identify genes upregulated in the intestine when XBP-1s is expressed in neurons. This revealed that neuronal XBP-1s increases the expression of genes involved in lysosome function. Lysosomes in the intestine of animals expressing neuronal XBP-1s are more acidic, and lysosomal protease activity is higher. Moreover, intestinal lysosome function is necessary for enhanced lifespan and proteostasis. These findings suggest that activation of the UPRER in the intestine through neuronal signaling can increase the activity of lysosomes, leading to extended longevity and improved proteostasis across tissues.

Fitness trade-offs and environmentally induced mutation buffering in isogenic C. elegans.

Mutations often have consequences that vary across individuals. Here, we show that the stimulation of a stress response can reduce mutation penetrance in Caenorhabditis elegans. Moreover, this induced mutation buffering varies across isogenic individuals because of interindividual differences in stress signaling. This variation has important consequences in wild-type animals, producing some individuals with higher stress resistance but lower reproductive fitness and other individuals with lower stress resistance and higher reproductive fitness. This may be beneficial in an unpredictable environment, acting as a "bet-hedging" strategy to diversify risk. These results illustrate how transient environmental stimuli can induce protection against mutations, how environmental responses can underlie variable mutation buffering, and how a fitness trade-off may make variation in stress signaling advantageous.

Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells.

Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis.

Germline stem cell number in the Drosophila ovary is regulated by redundant mechanisms that control Dpp signaling.

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homologue, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. Here, we further examine the role of Dpp in GSC maintenance. In contrast to the above model, we find that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. We demonstrate that one function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur.