A histone H4 lysine 20 methyltransferase couples environmental cues to sensory neuron control of developmental plasticity.

Animals change developmental fates in response to external cues. In the nematode Caenorhabditis elegans, unfavorable environmental conditions induce a state of diapause known as dauer by inhibiting the conserved DAF-2 insulin-like signaling (ILS) pathway through incompletely understood mechanisms. We have previously established a role for the C. elegans dosage compensation protein DPY-21 in the control of dauer arrest and DAF-2 ILS. Here, we show that the histone H4 lysine 20 methyltransferase SET-4, which also influences dosage compensation, promotes dauer arrest in part by repressing the X-linked ins-9 gene, which encodes a new agonist insulin-like peptide (ILP) expressed specifically in the paired ASI sensory neurons that are required for dauer bypass. ins-9 repression in dauer-constitutive mutants requires DPY-21, SET-4 and the FoxO transcription factor DAF-16, which is the main target of DAF-2 ILS. By contrast, autosomal genes encoding major agonist ILPs that promote reproductive development are not repressed by DPY-21, SET-4 or DAF-16/FoxO. Our results implicate SET-4 as a sensory rheostat that reinforces developmental fates in response to environmental cues by modulating autocrine and paracrine DAF-2 ILS.

TATN-1 mutations reveal a novel role for tyrosine as a metabolic signal that influences developmental decisions and longevity in Caenorhabditis elegans.

Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

Whole genome sequencing and the transformation of C. elegans forward genetics.

Forward genetics has been an undeniably powerful approach in Caenorhabditis elegans and other model organisms. However, the trek from mutant isolation to identification of the causative molecular lesion can be time-consuming and fraught with obstacles. This has changed with the advent of whole genome sequencing (WGS). The widespread availability of high-throughput sequencing technology, coupled with the increasing affordability of WGS, has enabled the routine use of WGS in the analysis of forward genetic screens. The noteworthy development of one-step mapping/sequencing approaches has largely eliminated the bottleneck of conventional high-resolution mapping, greatly accelerating the journey from mutagenesis to gene discovery. By enabling the use of increasingly complex and diverse genetic backgrounds as substrates for mutagenesis, WGS is expanding the landscape of biological problems that can be interrogated using forward genetic approaches in C. elegans and other organisms.

Is it time to reduce the length of postgraduate training for physician-scientists in internal medicine?

Physician-scientists play a crucial role in advancing medical knowledge and patient care, yet the long periods of time required to complete training may impede expansion of this workforce. We examined the relationship between postgraduate training and time to receipt of NIH or Veterans Affairs career development awards (CDAs) for physician-scientists in internal medicine. Data from NIH RePORTER were analyzed for internal medicine residency graduates who received specific CDAs (K08, K23, K99, or IK2) in 2022. Additionally, information on degrees and training duration was collected. Internal medicine residency graduates constituted 19% of K awardees and 28% of IK2 awardees. Of MD-PhD internal medicine-trained graduates who received a K award, 92% received a K08 award; of MD-only graduates who received a K award, a majority received a K23 award. The median time from medical school graduation to CDA was 9.6 years for K awardees and 10.2 years for IK2 awardees. The time from medical school graduation to K or IK2 award was shorter for US MD-PhD graduates than US MD-only graduates. We propose that the time from medical school graduation to receipt of CDAs must be shortened to accelerate training and retention of physician-scientists.

Ovarian steroid cell tumor with biallelic adenomatous polyposis coli inactivation in a patient with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant cancer predisposition syndrome that accounts for approximately 0.5-1% of all colorectal cancer cases. It is caused by germline mutations in the gene encoding the adenomatous polyposis coli (APC) tumor suppressor. Somatic APC inactivation due to mutation or loss of heterozygosity (LOH) promotes the development of adenomatous polyps by stabilizing the transcriptional coactivator ß-catenin. Although colorectal cancer is by far the most common malignancy seen in FAP patients, the widespread use of prophylactic colectomy in these patients has increased the clinical importance of extracolonic tumors that are part of the neoplastic spectrum in FAP. Many of these tumors exhibit LOH or somatic APC mutation, strongly supporting a causative role of APC inactivation in their pathogenesis. Here we describe a 47-year-old female FAP patient with clinical manifestations of virilization who was found to have an ovarian steroid cell tumor, a rare neoplasm not known to be associated with FAP. Immunohistochemical analysis of the ovarian tumor demonstrated strong nuclear ß-catenin staining consistent with somatic APC inactivation, and molecular analysis confirmed biallelic APC inactivation in the tumor. Our findings provide the first evidence that ovarian steroid cell tumors may be an extracolonic manifestation of FAP and implicate ß-catenin activation as an oncogenic mechanism in ovarian steroid cell tumorigenesis.

Structural insights into the career path between pre- and postgraduate physician-scientist training programs.

The growing complexities of clinical medicine and biomedical research have clouded the career path for physician-scientists. In this perspective piece, we address one of the most opaque career stage transitions along the physician-scientist career path, the transition from medical school to research-focused internal medicine residency programs, or physician-scientist training programs (PSTPs). We present the perspectives of medical scientist training program (MSTP) and PSTP directors on critical features of PSTPs that can help trainees proactively align their clinical and scientific training for successful career development. We aim to provide both trainees and MSTP directors with a conceptual framework to better understand and navigate PSTPs. We also offer interview-specific questions to help trainees gather data and make informed decisions in choosing a residency program that best supports their career.

Pearls of wisdom for aspiring physician-scientist residency applicants and program directors.

Postgraduate physician-scientist training programs (PSTPs) enhance the experiences of physician-scientist trainees following medical school graduation. PSTPs usually span residency and fellowship training, but this varies widely by institution. Applicant competitiveness for these programs would be enhanced, and unnecessary trainee anxiety relieved, by a clear understanding of what factors define a successful PSTP matriculant. Such information would also be invaluable to PSTP directors and would allow benchmarking of their admissions processes with peer programs. We conducted a survey of PSTP directors across the US to understand the importance they placed on components of PSTP applications. Of 41 survey respondents, most were from internal medicine and pediatrics residency programs. Of all components in the application, two elements were considered very important by a majority of PSTP directors: (a) having one or more first-author publications and (b) the thesis advisor's letter. Less weight was consistently placed on factors often considered more relevant for non-physician-scientist postgraduate applicants - such as US Medical Licensing Examination scores, awards, and leadership activities. The data presented here highlight important metrics for PSTP applicants and directors and suggest that indicators of scientific productivity and commitment to research outweigh traditional quantitative measures of medical school performance.

Functional divergence of dafachronic acid pathways in the control of C. elegans development and lifespan.

Steroid hormone and insulin/insulin-like growth factor signaling (IIS) pathways control development and lifespan in the nematode Caenorhabditis elegans by regulating the activity of the nuclear receptor DAF-12 and the FoxO transcription factor DAF-16, respectively. The DAF-12 ligands Delta(4)- and Delta(7)-dafachronic acid (DA) promote bypass of the dauer diapause and proper gonadal migration during larval development; in adults, DAs influence lifespan. Whether Delta(4)- and Delta(7)-DA have unique biological functions is not known. We identified the 3-beta-hydroxysteroid dehydrogenase (3betaHSD) family member HSD-1, which participates in Delta(4)-DA biosynthesis, as an inhibitor of DAF-16/FoxO activity. Whereas IIS promotes the cytoplasmic sequestration of DAF-16/FoxO, HSD-1 inhibits nuclear DAF-16/FoxO activity without affecting DAF-16/FoxO subcellular localization. Thus, HSD-1 and IIS inhibit DAF-16/FoxO activity via distinct and complementary mechanisms. In adults, HSD-1 was required for full lifespan extension in IIS mutants, indicating that HSD-1 interactions with IIS are context-dependent. In contrast to the Delta(7)-DA biosynthetic enzyme DAF-36, HSD-1 is dispensable for proper gonadal migration and lifespan extension induced by germline ablation. These findings provide insights into the molecular interface between DA and IIS pathways and suggest that Delta(4)- and Delta(7)-DA pathways have unique as well as overlapping biological functions in the control of development and lifespan.

Microbiome: Insulin signaling shapes gut community composition.

In a C. elegans model of host-microbiome interactions, interrogation of a genetically diverse panel of host strains with a defined, complex bacterial community reveals an important role for a conserved insulin-like signaling pathway in shaping the phylogenetic composition of the gut microbiome.

Starvation Responses Throughout the Caenorhabditiselegans Life Cycle.

Caenorhabditis elegans survives on ephemeral food sources in the wild, and the species has a variety of adaptive responses to starvation. These features of its life history make the worm a powerful model for studying developmental, behavioral, and metabolic starvation responses. Starvation resistance is fundamental to life in the wild, and it is relevant to aging and common diseases such as cancer and diabetes. Worms respond to acute starvation at different times in the life cycle by arresting development and altering gene expression and metabolism. They also anticipate starvation during early larval development, engaging an alternative developmental program resulting in dauer diapause. By arresting development, these responses postpone growth and reproduction until feeding resumes. A common set of signaling pathways mediates systemic regulation of development in each context but with important distinctions. Several aspects of behavior, including feeding, foraging, taxis, egg laying, sleep, and associative learning, are also affected by starvation. A variety of conserved signaling, gene regulatory, and metabolic mechanisms support adaptation to starvation. Early life starvation can have persistent effects on adults and their descendants. With its short generation time, C. elegans is an ideal model for studying maternal provisioning, transgenerational epigenetic inheritance, and developmental origins of adult health and disease in humans. This review provides a comprehensive overview of starvation responses throughout the C. elegans life cycle.

Modular metabolite assembly in Caenorhabditis elegans depends on carboxylesterases and formation of lysosome-related organelles.

Signaling molecules derived from attachment of diverse metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, many aspects of their biogenesis remain unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of most modular ascarosides as well as previously undescribed modular glucosides. Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism, suggesting that modular glucosides, like the ascarosides, may serve signaling functions. We further show that carboxylesterases that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides via ester and amide linkages. Further exploration of LRO function and carboxylesterase homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms.

Caenorhabditis elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity.

Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals.

Two membrane-associated tyrosine phosphatase homologs potentiate C. elegans AKT-1/PKB signaling.

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues.

Requirement for translocon-associated protein (TRAP) α in insulin biogenesis.

The mechanistic basis for the biogenesis of peptide hormones and growth factors is poorly understood. Here, we show that the conserved endoplasmic reticulum membrane translocon-associated protein α (TRAPα), also known as signal sequence receptor 1, plays a critical role in the biosynthesis of insulin. Genetic analysis in the nematode Caenorhabditis elegans and biochemical studies in pancreatic ß cells reveal that TRAPα deletion impairs preproinsulin translocation while unexpectedly disrupting distal steps in insulin biogenesis including proinsulin processing and secretion. The association of common intronic single-nucleotide variants in the human TRAPα gene with susceptibility to type 2 diabetes and pancreatic ß cell dysfunction suggests that impairment of preproinsulin translocation and proinsulin trafficking may contribute to the pathogenesis of type 2 diabetes.

Training the physician-scientist: views from program directors and aspiring young investigators.

There is growing concern that the physician-scientist is endangered due to a leaky training pipeline and prolonged time to scientific independence (1). The NIH Physician-Scientist Workforce Working Group has concluded that as many as 1,000 individuals will need to enter the pipeline each year to sustain the workforce (2). Moreover, surveys of postgraduate training programs document considerable variability in disposition and infrastructure (3). Programs can be broadly grouped into two classes: physician-scientist training programs (PSTPs) that span residency and fellowship training, and research-in-residency programs (RiRs), which are limited to residency but trainees are able to match into PSTPs upon transitioning to fellowship (Figure 1). Funding sources for RiRs and PSTPs are varied and include NIH KL2 and T32 awards, charitable foundations, philanthropy, and institutional support. Furthermore, standards for research training and tools for evaluating programmatic success are lacking. Here, we share consensus generated from iterative workshops hosted by the Alliance of Academic Internal Medicine (AAIM) and the student-led American Physician Scientists Association (APSA).

N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.

Chromoanasynthetic Genomic Rearrangement Identified in a N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen in Caenorhabditis elegans.

Chromoanasynthesis is a recently discovered phenomenon in humans with congenital diseases that is characterized by complex genomic rearrangements (CGRs) resulting from aberrant repair of catastrophic chromosomal damage. How these CGRs are induced is not known. Here, we describe the structure and function of dpDp667, a causative CGR that emerged from a Caenorhabditis elegans dauer suppressor screen in which animals were treated with the point mutagen N-ethyl-N-nitrosourea (ENU). dpDp667 comprises nearly 3 Mb of sequence on the right arm of the X chromosome, contains three duplications and one triplication, and is devoid of deletions. Sequences from three out of the four breakpoint junctions in dpDp667 reveal microhomologies that are hallmarks of chromoanasynthetic CGRs. Our findings suggest that environmental insults and physiological processes that cause point mutations may give rise to chromoanasynthetic rearrangements associated with congenital disease. The relatively subtle phenotype of animals harboring dpDp667 suggests that the prevalence of CGRs in the genomes of mutant and/or phenotypically unremarkable animals may be grossly underestimated.

Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans.

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental principles underlying aging in animals.