Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization.

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the "classical" gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina.

Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae.

Mitogen-activated protein kinase (MAPK) pathways control the response to intrinsic and extrinsic stimuli. In the budding yeast Saccharomyces cerevisiae, cells undergo filamentous growth, which is regulated by the fMAPK pathway. To better understand the regulation of the fMAPK pathway, a genetic screen was performed to identify spontaneous mutants with elevated activity of an fMAPK pathway-dependent growth reporter (ste4  FUS1-HIS3). In total, 159 mutants were isolated and analyzed by secondary screens for invasive growth by the plate-washing assay and filament formation by microscopy. Thirty-two mutants were selected for whole-genome sequencing, which identified new alleles in genes encoding known regulators of the fMAPK pathway. These included gain-of-function alleles in STE11, which encodes the MAPKKK, as well as loss-of-function alleles in KSS1, which encodes the MAP kinase, and loss-of-function alleles in RGA1, which encodes a GTPase-activating protein (GAP) for CDC42. New alleles in previously identified pathway modulators were also uncovered in ALY1, AIM44, RCK2, IRA2, REG1, and in genes that regulate protein folding (KAR2), glycosylation (MNN4), and turnover (BLM10). Mutations leading to C-terminal truncations in the transcription factor Ste12p were also uncovered that resulted in elevated reporter activity, identifying an inhibitory domain of the protein from residues 491 to 688. We also find that a diversity of filamentous growth phenotypes can result from combinatorial effects of multiple mutations and by loss of different regulators of the response. The alleles identified here expand the connections surrounding MAPK pathway regulation and reveal new features of proteins that function in the signaling cascade.

Protective Effects of Lipoxin A4 and B4 Signaling on the Inner Retina in a Mouse Model of Experimental Glaucoma.

Glaucoma is a common neurodegenerative disease characterized by progressive degeneration of retinal ganglion cells (RGCs) and the retinal nerve fiber layer (RNFL), resulting in a gradual decline of vision. A recent study by our groups indicated that the levels of lipoxins A4 (LXA4) and B4 (LXB4) in the retina and optic nerve decrease following acute injury, and that restoring their function is neuroprotective. Lipoxins are members of the specialized pro-resolving mediator (SPM) family and play key roles to mitigate and resolve chronic inflammation and tissue damage. Yet, knowledge about lipoxin neuroprotective activity remains limited. Here we investigate the in vivo efficacy of exogenous LXA4 and LXB4 administration on the inner retina in a mouse model of chronic experimental glaucoma. To investigate the contribution of LXA4 signaling we used transgenic knockout (KO) mice lacking the two mouse LXA4 receptors (Fpr2/Fpr3-/-). Functional and structural changes of inner retinal neurons were assessed longitudinally using electroretinogram (ERG) and optical coherence tomography (OCT). At the end of the experiment, retinal samples were harvested for immunohistological assessment. While both lipoxins generated protective trends, only LXB4 treatment was significant, and consistently more efficacious than LXA4 in all endpoints. Both lipoxins also appeared to dramatically reduce Müller glial reactivity following injury. In comparison, Fpr2/Fpr3 deletion significantly worsened inner retinal injury and function, consistent with an essential protective role for endogenous LXA4. Together, these results support further exploration of lipoxin signaling as a treatment for glaucomatous neurodegeneration.

Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma.

The role of glia, particularly astrocytes, in mediating the central nervous system's response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes - collectively referred to as 'reactivity' - in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression - both beneficially and detrimentally - and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic 'gray matter' astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous 'white matter' astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway - those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions - we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.

Ecological inducers of the yeast filamentous growth pathway reveal environment-dependent roles for pathway components.

Signaling modules, such as mitogen-activated protein kinase (MAPK) pathways, are evolutionarily conserved drivers of cell differentiation and stress responses. In many fungal species including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model budding yeast Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was used, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found that induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Combinations of inducers (galactose and ethanol) stimulated the pathway to near-maximal levels, which showed dispensability of several fMAPK pathway components (e.g., mucin sensor, p21-activated kinase), but not others (e.g., adaptor, MAPKKK) and required the Ras2-protein kinase A pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments. IMPORTANCE Filamentous growth is a cell differentiation response and important aspect of fungal biology. In plant and animal fungal pathogens, filamentous growth contributes to virulence. One signaling pathway that regulates filamentous growth is an evolutionarily conserved MAPK pathway. The yeast Saccharomyces cerevisiae is a convenient model to study MAPK-dependent regulation of filamentous growth, although the inducers of the pathway are not clear. Here, we exposed yeast cells to ecologically relevant compounds (e.g., plant compounds), which identified new inducers of the MAPK pathway. In combination, the inducers activated the pathway to near-maximal levels but did not cause detrimental phenotypes associated with previously identified hyperactive alleles. This context allowed us to identify conditional bypass for multiple pathway components. Thus, near-maximal induction of a MAPK pathway by ecologically relevant inducers provides a powerful tool to assess cellular signaling during a fungal differentiation response.

Turnover and bypass of p21-activated kinase during Cdc42-dependent MAPK signaling in yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems.

Rapid isolation of intact retinal astrocytes: a novel approach.

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3, or Scd6; whereas most of the remaining transcripts utilize nonsense-mediated mRNA decay factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are upregulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

Chaperone-Dependent Degradation of Cdc42 Promotes Cell Polarity and Shields the Protein from Aggregation.

Rho GTPases are global regulators of cell polarity and signaling. By exploring the turnover regulation of the yeast Rho GTPase Cdc42p, we identified new regulatory features surrounding the stability of the protein. We specifically show that Cdc42p is degraded at 37 °C by chaperones through lysine residues located in the C-terminus of the protein. Cdc42p turnover at 37 °C occurred by the 26S proteasome in an ESCRT-dependent manner in the lysosome/vacuole. By analyzing versions of Cdc42p that were defective for turnover, we show that turnover at 37 °C promoted cell polarity but was defective for sensitivity to mating pheromone, presumably mediated through a Cdc42p-dependent MAP kinase pathway. We also identified one residue (K16) in the P-loop of the protein that was critical for Cdc42p stability. Accumulation of Cdc42pK16R in some contexts led to the formation of protein aggregates, which were enriched in aging mother cells and cells undergoing proteostatic stress. Our study uncovers new aspects of protein turnover regulation of a Rho-type GTPase that may extend to other systems. Moreover, residues identified here that mediate Cdc42p turnover correlate with several human diseases, which may suggest that turnover regulation of Cdc42p is important to aspects of human health.

New Features Surrounding the Cdc42-Ste20 Module that Regulates MAP Kinase Signaling in Yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular responses, including the response to stress and cell differentiation, and are highly conserved across eukaryotes from yeast to humans. In yeast, the canonical activation of several MAPK pathways includes the interaction of the small GTPase Cdc42p with the p21-activated kinase (PAK) Ste20p. We recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is turned over by the 26S proteasome. Ste20p was stabilized when bound to Cdc42p, presumably to sustain MAPK pathway signaling. Ste20p is a major conduit by which signals flow through the fMAPK pathway; however, by genetic approaches we also identified a Ste20p-independent branch of the fMAPK pathway. Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p, while Ste20p-independent signaling required the fMAPK pathway adaptor and Cdc42p-interacting protein, Bem4p. Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p in the fMAPK pathway, Rga1p, which also dampened basal but not active fMAPK pathway activity. Finally, the polarity adaptor and Cdc42p-interacting protein, Bem1p, which also regulates the fMAPK pathway, interacts with the tetra-span protein Sho1p, connecting a sensor at the plasma membrane to a protein that regulates the GTPase module. Collectively, these data reveal new regulatory features surrounding a Rho-PAK module that may extend to other pathways that control cell differentiation.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2 Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2 Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2 Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2 Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated by dcp2 Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

The case for change: aviation worker wellbeing during the COVID 19 pandemic, and the need for an integrated health and safety culture.

The workplace is an important setting for health protection, health promotion and disease prevention. Currently, health and wellbeing approaches at an aviation organisational level are not addressing both human and safety needs. This issue has been intensified since the COVID 19 pandemic. This paper reports on the findings of a survey pertaining to aviation worker wellbeing and organisational approaches to managing wellbeing and mental health. The survey was administered at two different time periods during the COVID 19 pandemic (2020 and 2021). Collectively, feedback was obtained from over 3000 aviation workers. Survey feedback indicates that aviation workers are experiencing considerable challenges in relation to their health and wellbeing. These challenges are not being adequately addressed at an organisational level, which creates risk both from an individual and flight safety perspective. The descriptive findings of both surveys along with a regression analysis is used to make a principled case for augmenting the existing approach to managing aviation worker wellbeing (including mental health), at both an organisational and regulatory level. It is argued that aviation organisations, with the support of the regulator should implement a preventative, ethical and evidence-based strategy to managing wellbeing and mental health risk. Critically, aviation organisations need to advance and integrated health, wellbeing, and safety culture. This necessitates an alignment of human, business, and safety objectives, as articulated in concepts of corporate social responsibility (CSR) and responsible work. Critically, this approach depends on trust and the specification of appropriate protections, so that aviation workers feel safe to routinely report wellbeing levels and challenges, and their impact on operational safety.

Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae.

Mitogen-activated protein kinase (MAPK) pathways control the response to intrinsic and extrinsic stimuli. In the budding yeast Saccharomyces cerevisiae, cells undergo filamentous growth, which is regulated by the fMAPK pathway. To better understand the regulation of the fMAPK pathway, a genetic screen was performed to identify spontaneous mutants with elevated activity of an fMAPK-pathway dependent growth reporter (ste4 FUS1-HIS3). In total, 159 mutants were isolated and analyzed by secondary screens for invasive growth by the plate-washing assay, and filament formation by microscopy. Thirty-two mutants were selected for whole-genome sequencing, which identified new alleles in genes encoding known regulators of the fMAPK pathway. These included gain-of-function alleles in STE11, which encodes the MAPKKK, as well as loss-of-function alleles in KSS1, which encodes the MAP kinase, and RGA1, which encodes a GTPase activating protein (GAP) for CDC42. New alleles in previously identified pathway modulators were also uncovered in ALY1, AIM44, RCK2, IRA2, REG1 and in genes that regulate protein folding (KAR2), glycosylation (MNN4), and turnover (BLM10). C-terminal truncations in the transcription factor Ste12p were also uncovered that resulted in elevated reporter activity, presumably identifying an inhibitory domain in the C-terminus of the protein. We also show that a wide variety of filamentous growth phenotypes result from mutations in different regulators of the response. The alleles identified here expand the connections surrounding MAPK pathway regulation and reveal new features of proteins that function in the signaling cascade.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Regulation of Cdc42 protein turnover modulates the filamentous growth MAPK pathway.

Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children.

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >106 copies/ml and >103 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (≥2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities.

Traumatic avulsion of the tricuspid valve: an unusual cardiac complication of an equine accident.

A stablehand in his 20s presented with increasing dyspnoea on exertion and chest pain months after being kicked directly in the chest by a horse. Further investigations revealed severe isolated, primary tricuspid regurgitation due to partial avulsion of the anterior leaflet. Open surgical repair was successfully performed from which he recovered uneventfully with resolution of his symptoms and minimal residual tricuspid regurgitation. Although uncommon, blunt force trauma to the chest is becoming increasingly recognised as a rare cause of tricuspid regurgitation. Symptoms may be insidious, and a high index of suspicion is required to avoid missing the diagnosis.

Gene by Environment Interactions reveal new regulatory aspects of signaling network plasticity.

Phenotypes can change during exposure to different environments through the regulation of signaling pathways that operate in integrated networks. How signaling networks produce different phenotypes in different settings is not fully understood. Here, Gene by Environment Interactions (GEIs) were used to explore the regulatory network that controls filamentous/invasive growth in the yeast Saccharomyces cerevisiae. GEI analysis revealed that the regulation of invasive growth is decentralized and varies extensively across environments. Different regulatory pathways were critical or dispensable depending on the environment, microenvironment, or time point tested, and the pathway that made the strongest contribution changed depending on the environment. Some regulators even showed conditional role reversals. Ranking pathways' roles across environments revealed an under-appreciated pathway (OPI1) as the single strongest regulator among the major pathways tested (RAS, RIM101, and MAPK). One mechanism that may explain the high degree of regulatory plasticity observed was conditional pathway interactions, such as conditional redundancy and conditional cross-pathway regulation. Another mechanism was that different pathways conditionally and differentially regulated gene expression, such as target genes that control separate cell adhesion mechanisms (FLO11 and SFG1). An exception to decentralized regulation of invasive growth was that morphogenetic changes (cell elongation and budding pattern) were primarily regulated by one pathway (MAPK). GEI analysis also uncovered a round-cell invasion phenotype. Our work suggests that GEI analysis is a simple and powerful approach to define the regulatory basis of complex phenotypes and may be applicable to many systems.

Spatiotemporal control of pathway sensors and cross-pathway feedback regulate a differentiation MAPK pathway in yeast.

Mitogen-activated protein kinase (MAPK) pathways control cell differentiation and the response to stress. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the pathway that regulates the response to osmotic stress (HOG). Here, we show that the two pathways exhibit different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding mucin sensors that regulate the pathways. Cross-pathway regulation from the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. We also show that the shared tetraspan protein Sho1p, which has a dynamic localization pattern throughout the cell cycle, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway regulation, control a MAPK pathway that regulates cell differentiation in yeast.