Cellular progenitors remain poorly characterized in many adult tissues, limited in part by the lack of unbiased techniques to identify progenitors and their progeny. To address this fundamental ...problem, we developed a novel DNA analog-based lineage-tracing technique to detect multiple rounds of cell division in vivo. Here, we apply this technique to determine the adult lineage mechanism of the insulin-secreting β cells of pancreatic islets, an important unresolved question in diabetes research. As expected, gastrointestinal and skin epithelia involve specialized progenitors that repeatedly divide to give rise to postmitotic cells. In contrast, specialized progenitors do not contribute to adult β cells, not even during acute β cell regeneration. Instead, β cells are the products of uniform self-renewal, slowed by a replication refractory period that prevents β cells from immediately redividing. Our approach provides unbiased resolution of previously inaccessible developmental niches and can elucidate lineage mechanisms without candidate markers.
Perhaps the most devastating decline with age is the loss of memory. Therefore, identifying mechanisms to restore memory function with age is critical. Using C. elegans associative learning and ...memory assays, we identified a gain-of-function Gαq signaling pathway mutant that forms a long-term (cAMP response element binding protein CREB-dependent) memory following one conditioned stimulus-unconditioned stimulus (CS-US) pairing, which usually requires seven CS-US pairings. Increased CREB activity in AIM interneurons reduces the threshold for memory consolidation through transcription of a set of previously identified “long-term memory” genes. Enhanced Gαq signaling in the AWC sensory neuron is both necessary and sufficient for improved memory and increased AIM CREB activity, and activation of Gαq specifically in aged animals rescues the ability to form memory. Activation of Gαq in AWC sensory neurons non-cell autonomously induces consolidation after one CS-US pairing, enabling both cognitive function maintenance with age and restoration of memory function in animals with impaired memory performance without decreased longevity.
•Increased Gαq signaling enhances memory and slows age-related cognitive decline•CREB activity underlies enhanced consolidation and memory maintenance with age•The AWC neuron is the site where Gαq signaling regulates memory•Gαq activation in the AWC of aged animals rescues cognitive decline
Arey et al. have found that activating the Gαq signaling pathway in sensory neurons enhances memory in young animals, utilizing the canonical CREB pathway. This pathway also slows age-related memory loss and reverses age-related memory impairment.
•Massed positive associative memory training induces both short-term and intermediate-term memory.•Forgetting is actively regulated and requires protein translation.•C. elegans memory requires ...canonical memory regulators.•C. elegans positive olfactory associative memory is distinct from olfactory adaptation.
While it is thought that short-term memory arises from changes in protein dynamics that increase the strength of synaptic signaling, many of the underlying fundamental molecular mechanisms remain unknown.Our lab developed a Caenorhabditis elegans assay of positive olfactory short-term associative memory (STAM), in which worms learn to associate food with an odor and can remember this association for over 1h. Here we use this massed olfactory associative assay to identify regulators of C. elegans short-term and intermediate-term associative memory (ITAM) processes. We show that there are unique molecular characteristics for different temporal phases of STAM, which include: learning, which is tested immediately after training, short-term memory, tested 30min after training, intermediate-term memory, tested 1h after training, and forgetting, tested 2h after training. We find that, as in higher organisms, C. elegans STAM requires calcium and cAMP signaling, and ITAM requires protein translation. Additionally, we found that STAM and ITAM are distinct from olfactory adaptation, an associative paradigm in which worms learn to disregard an inherently attractive odor after starvation in the presence of that odor. Adaptation mutants show variable responses to short-term associative memory training. Our data distinguish between shorter forms of a positive associative memory in C. elegans that require canonical memory pathways. Study of STAM and ITAM in C. elegans could lead to a more general understanding of the distinctions between these important processes and also to the discovery of novel conserved memory regulators.
Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete ...picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components.
Our understanding of the molecular and genetic regulation of aging and longevity has been greatly augmented through studies using the small model system, C. elegans. It is important to test whether ...mutations that result in a longer life span also extend the health span of the organism, rather than simply prolonging an aged state. C. elegans can learn and remember both associated and non-associated stimuli, and many of these learning and memory paradigms are subject to regulation by longevity pathways. One of the more distressing results of aging is cognitive decline, and while no gross physical defects in C. elegans sensory neurons have been identified, the organism does lose the ability to perform both simple and complex learned behaviors with age. Here we review what is known about the effects of longevity pathways and the decline of these complex learned behaviors with age, and we highlight outstanding questions in the field.
The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and ...chemical odors, and can associate nutritive states with chemical odors, temperature, and the pathogenicity of a food source. Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning. After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (<40 hours), and is dependent on the cAMP Response Element Binding protein (CREB), a transcription factor required for long-term memory across species. Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation. Otsu's method is then used to determine a threshold to separate worms from the background. Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm. We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms. Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.
This spring, students across the globe transitioned from in-person classes to remote learning as a result of the COVID-19 pandemic. This unprecedented change to undergraduate education saw ...institutions adopting multiple online teaching modalities and instructional platforms. We sought to understand students’ experiences with and perspectives on those methods of remote instruction in order to inform pedagogical decisions during the current pandemic and in future development of online courses and virtual learning experiences. Our survey gathered quantitative and qualitative data regarding students’ experiences with synchronous and asynchronous methods of remote learning and specific pedagogical techniques associated with each. A total of 4,789 undergraduate participants representing institutions across 95 countries were recruited via Instagram. We find that most students prefer synchronous online classes, and students whose primary mode of remote instruction has been synchronous report being more engaged and motivated. Our qualitative data show that students miss the social aspects of learning on campus, and it is possible that synchronous learning helps to mitigate some feelings of isolation. Students whose synchronous classes include active-learning techniques (which are inherently more social) report significantly higher levels of engagement, motivation, enjoyment, and satisfaction with instruction. Respondents’ recommendations for changes emphasize increased engagement, interaction, and student participation. We conclude that active-learning methods, which are known to increase motivation, engagement, and learning in traditional classrooms, also have a positive impact in the remote-learning environment. Integrating these elements into online courses will improve the student experience.
The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two ...differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.
Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell ...division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyfluorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.
Non-cognitive skills are essential for success in a variety of settings but are not commonly taught and assessed in undergraduate curricula. The challenge for curricular integration of non-cognitive ...skills stems from varying interpretations of what it means to apply a specific skill effectively. In addition, teaching these skills requires going beyond the foregrounded academic content in traditional coursework. At Minerva University, we articulate core non-cognitive skills precisely and transparently as learning outcomes to guide the design of courses and assessments. We integrate these skills into the academic curriculum and student life alongside traditional learning outcomes. Students are introduced to these outcomes in their first year and continue to apply them throughout their 4 years, receiving feedback and scores across academic, disciplinary, and real-world, experiential learning contexts. We provide examples of the Learning Outcomes, detailing their introduction and application in various contexts across the 4-year arc Students’ college experience. We identify aspects of our approach that can be generalized and implemented in different settings, from individual courses to programs, departments, or entire institutions.
PDF
