TrxG and PcG proteins but not methylated histones remain associated with DNA through replication.

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

Barriers and Facilitators to Harm Reduction for Opioid Use Disorder: A Qualitative Study of People With Lived Experience.

STUDY OBJECTIVE: Although an increasing number of emergency departments (ED) offer opioid agonist treatment, naloxone, and other harm reduction measures, little is known about patient perspectives on harm reduction practices delivered in the ED. The objective of this study was to identify patient-focused barriers and facilitators to harm reduction strategies in the ED. METHODS: We conducted semistructured interviews with a convenience sample of individuals in Massachusetts diagnosed with opioid use disorder. We developed an interview guide, and interviews were recorded, transcribed, and analyzed in an iterative process using reflexive thematic analysis. After initial interviews and coding, we triangulated the results among a focus group of 4 individuals with lived experience. RESULTS: We interviewed 25 participants with opioid use disorder, 6 recruited from 1 ED and 19 recruited from opioid agonist treatment clinics. Key themes included accessibility of harm reduction supplies, lack of self-care resulting from withdrawal and hopelessness, the impact of stigma on the likelihood of using harm reduction practices, habit and knowledge, as well as the need for user-centered harm reduction interventions. CONCLUSION: In this study, people with lived experience discussed the characteristics and need for user-centered harm reduction strategies in the ED that centered on reducing stigma, treatment of withdrawal, and availability of harm reduction materials.

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea.

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day-1 for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69-39.94 km.day-1 for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.

Inshore and offshore marine migration pathways of Atlantic salmon post-smolts from multiple rivers in Scotland, England, Northern Ireland, and Ireland.

The migratory behavior of Atlantic salmon (Salmo salar) post-smolts in coastal waters is poorly understood. In this collaborative study, 1914 smolts, from 25 rivers, in four countries were tagged with acoustic transmitters during a single seasonal migration. In total, 1105 post-smolts entered the marine study areas and 438 (39.6%) were detected on a network of 414 marine acoustic receivers and an autonomous underwater vehicle. Migration pathways (defined as the shortest distance between two detections) of up to 575 km and over 100 days at sea were described for all 25 populations. Post-smolts from different rivers, as well as individuals from the same river, used different pathways in coastal waters. Although difficult to generalize to all rivers, at least during the year of this study, no tagged post-smolts from rivers draining into the Irish Sea were detected entering the areas of sea between the Hebrides and mainland Scotland, which is associated with a high density of finfish aquaculture. An important outcome of this study is that a high proportion of post-smolts crossed through multiple legislative jurisdictions and boundaries during their migration. This study provides the basis for spatially explicit assessment of the impact risk of coastal pressures on salmon during their first migration to sea.

Differences among families in craniofacial shape at early life-stages of Arctic charr (Salvelinus alpinus).

BACKGROUND: Organismal fitness can be determined at early life-stages, but phenotypic variation at early life-stages is rarely considered in studies on evolutionary diversification. The trophic apparatus has been shown to contribute to sympatric resource-mediated divergence in several taxa. However, processes underlying diversification in trophic traits are poorly understood. Using phenotypically variable Icelandic Arctic charr (Salvelinus alpinus), we reared offspring from multiple families under standardized laboratory conditions and tested to what extent family (i.e. direct genetic and maternal effects) contributes to offspring morphology at hatching (H) and first feeding (FF). To understand the underlying mechanisms behind early life-stage variation in morphology, we examined how craniofacial shape varied according to family, offspring size, egg size and candidate gene expression. RESULTS: Craniofacial shape (i.e. the Meckel's cartilage and hyoid arch) was more variable between families than within families both across and within developmental stages. Differences in craniofacial morphology between developmental stages correlated with offspring size, whilst within developmental stages only shape at FF correlated with offspring size, as well as female mean egg size. Larger offspring and offspring from females with larger eggs consistently had a wider hyoid arch and contracted Meckel's cartilage in comparison to smaller offspring. CONCLUSIONS: This study provides evidence for family-level variation in early life-stage trophic morphology, indicating the potential for parental effects to facilitate resource polymorphism.

Gene expression in the phenotypically plastic Arctic charr (Salvelinus alpinus): A focus on growth and ossification at early stages of development.

Gene expression during development shapes the phenotypes of individuals. Although embryonic gene expression can have lasting effects on developmental trajectories, few studies consider the role of maternal effects, such as egg size, on gene expression. Using qPCR, we characterize relative expression of 14 growth and/or skeletal promoting genes across embryonic development in Arctic charr (Salvelinus alpinus). We test to what extent their relative expression is correlated with egg size and size at early life-stages within the study population. We predict smaller individuals to have higher expression of growth and skeletal promoting genes, due to less maternal resources (i.e., yolk) and prioritization of energy toward ossification. We found expression levels to vary across developmental stages and only three genes (Mmp9, Star, and Sgk1) correlated with individual size at a given developmental stage. Contrary to our hypothesis, expression of Mmp9 and Star showed a non-linear relationship with size (at post fertilization and hatching, respectively), whilst Sgk1 was higher in larger embryos at hatching. Interestingly, these genes are also associated with craniofacial divergence of Arctic charr morphs. Our results indicate that early life-stage variation in gene expression, concomitant to maternal effects, can influence developmental plasticity and potentially the evolution of resource polymorphism in fishes.

Rare variation in non-coding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior.

Genomic and machine learning-based screening of aquaculture-associated introgression into at-risk wild North American Atlantic salmon (Salmo salar) populations.

The negative genetic impacts of gene flow from domestic to wild populations can be dependent on the degree of domestication and exacerbated by the magnitude of pre-existing genetic differences between wild populations and the domestication source. Recent evidence of European ancestry within North American aquaculture Atlantic salmon (Salmo salar) has elevated the potential impact of escaped farmed salmon on often at-risk wild North American salmon populations. Here, we compare the ability of single nucleotide polymorphism (SNP) and microsatellite (SSR) marker panels of different sizes (7-SSR, 100-SSR and 220K-SNP) to detect introgression of European genetic information into North American wild and aquaculture populations. Linear regression comparing admixture predictions for a set of individuals common to the three datasets showed that the 100-SSR panel and 7-SSR panels replicated the full 220K-SNP-based admixture estimates with low accuracy (r2 of .64 and .49, respectively). Additional tests explored the effects of individual sample size and marker number, which revealed that ~300 randomly selected SNPs could replicate the 220K-SNP admixture predictions with greater than 95% fidelity. We designed a custom SNP panel (301-SNP) for European admixture detection in future monitoring work and then developed and tested a python package, salmoneuadmix (https://github.com/CNuge/SalmonEuAdmix), which uses a deep neural network to make de novo estimates of individuals' European admixture proportion without the need to conduct complete admixture analysis utilizing baseline samples. The results demonstrate the mobilization of targeted SNP panels and machine learning in support of at-risk species conservation and management.

Rare variation in noncoding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of noncoding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of noncoding regions: Human Accelerated Regions (HARs), which show signatures of positive selection in humans; experimentally validated neural Vista Enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole genome analysis of >16,600 samples and >4900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in a VE near SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved noncoding regions in ASD risk and suggest potential mechanisms of how changes in regulatory regions can modulate social behavior.

A conserved molecular template underlies color pattern diversity in estrildid finches.

The color patterns that adorn animals' coats not only exhibit extensive diversity linked to various ecological functions but also display recurrences in geometry, orientation, or body location. How processes of pattern formation shape such phenotypic trends remains a mystery. Here, we surveyed plumage color patterns in passerine finches displaying extreme apparent variation and identified a conserved set of color domains. We linked these domains to putative embryonic skin regions instructed by early developmental tissues and outlined by the combinatory expression of few genetic markers. We found that this embryonic prepattern is largely conserved in birds displaying drastic color differences in the adult, interspecies variation resulting from the masking or display of each domain depending on their coloration. This work showed that a simple molecular landscape serves as common spatial template to extensive color pattern variation in finches, revealing that early conserved landmarks and molecular pathways are a major cause of phenotypic trends.

Variation in egg size and offspring phenotype among and within seven Arctic charr morphs.

Maternal effects have the potential to alter early developmental processes of offspring and contribute to adaptive diversification. Egg size is a major contributor to offspring phenotype, which can influence developmental trajectories and potential resource use. However, to what extent intraspecific variation in egg size facilitates evolution of resource polymorphism is poorly understood. We studied multiple resource morphs of Icelandic Arctic charr, ranging from an anadromous morph-with a phenotype similar to the proposed ancestral phenotype-to sympatric morphs that vary in their degree of phenotypic divergence from the ancestral anadromous morph. We characterized variation in egg size and tested whether egg size influenced offspring phenotype at early life stages (i.e., timing of- and size at- hatching and first feeding [FF]). We predicted that egg size would differ among morphs and be less variable as morphs diverge away from the ancestral anadromous phenotype. We also predicted that egg size would correlate with offspring size and developmental timing. We found morphs had different egg size, developmental timing, and size at hatching and FF. Egg size increased as phenotypic proximity to the ancestral anadromous phenotype decreased, with larger eggs generally giving rise to larger offspring, especially at FF, but egg size had no effect on developmental rate. The interaction between egg size and the environment may have a profound impact on offspring fitness, where the resulting differences in early life-history traits may act to initiate and/or maintain resource morphs diversification.

"Just give them a choice": Patients' perspectives on starting medications for opioid use disorder in the ED.

OBJECTIVES: Medications for opioid use disorder (MOUD) prescribed in the emergency department (ED) have the potential to save lives and help people start and maintain recovery. We sought to explore patient perspectives regarding the initiation of buprenorphine and methadone in the ED with the goal of improving interactions and fostering shared decision making (SDM) around these important treatment options. METHODS: We conducted semistructured interviews with a purposeful sample of people with opioid use disorder (OUD) regarding ED visits and their experiences with MOUD. The interview guide was based on the Ottawa Decision Support Framework, a framework for examining decisional needs and tailoring decisional support, and the research team's experience with MOUD and SDM. Interviews were recorded, transcribed, and analyzed in an iterative process using both the Ottawa Framework and a social-ecological framework. Themes were identified and organized and implications for clinical care were noted and discussed. RESULTS: Twenty-six participants were interviewed, seven in person in the ED and 19 via video conferencing software. The majority had tried both buprenorphine and methadone, and almost all had been in an ED for an issue related to opioid use. Participants reported social, pharmacological, and emotional factors that played into their decision making. Regarding buprenorphine, they noted advantages such as its efficacy and logistical ease and disadvantages such as the need to wait to start it (risk of precipitated withdrawal) and that one could not use other opioids while taking it. Additionally, participants felt that: (1) both buprenorphine and methadone should be offered; (2) because "one person's pro is another person's con," clinicians will need to understand the facets of the options; (3) clinicians will need to have these conversations without appearing judgmental; and (4) many patients may not be "ready" for MOUD, but it should still be offered. CONCLUSIONS: Although participants were supportive of offering buprenorphine in the ED, many felt that methadone should also be offered. They felt that treatment should be tailored to an individual's needs and circumstances and clarified what factors might be important considerations for people with OUD.

Loss of non-motor kinesin KIF26A causes congenital brain malformations via dysregulated neuronal migration and axonal growth as well as apoptosis.

Kinesins are canonical molecular motors but can also function as modulators of intracellular signaling. KIF26A, an unconventional kinesin that lacks motor activity, inhibits growth-factor-receptor-bound protein 2 (GRB2)- and focal adhesion kinase (FAK)-dependent signal transduction, but its functions in the brain have not been characterized. We report a patient cohort with biallelic loss-of-function variants in KIF26A, exhibiting a spectrum of congenital brain malformations. In the developing brain, KIF26A is preferentially expressed during early- and mid-gestation in excitatory neurons. Combining mice and human iPSC-derived organoid models, we discovered that loss of KIF26A causes excitatory neuron-specific defects in radial migration, localization, dendritic and axonal growth, and apoptosis, offering a convincing explanation of the disease etiology in patients. Single-cell RNA sequencing in KIF26A knockout organoids revealed transcriptional changes in MAPK, MYC, and E2F pathways. Our findings illustrate the pathogenesis of KIF26A loss-of-function variants and identify the surprising versatility of this non-motor kinesin.

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint.

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph(p) mutants reveals an acceleration of S phase, showing that ph(p) regulates S phase length. Like ph(p) mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph(p) are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.

Alteration of connexin expression is an early signal for chronic kidney disease.

Chronic kidney disease is promoted by a variety of factors that induce chronic inflammation and fibrosis. Inflammation and excessive scaring have been recently associated with disruptions of the gap junction-mediated intercellular communication. Nevertheless, little is known about alterations of the expression of gap junction proteins such as connexin (Cx) 43 and 37 in chronic renal disease. In this study, we investigated the expression of these two Cxs in the hypertensive RenTg mice, the anti-glomerular basement membrane glomerulonephritis, and the unilateral ureteral obstruction models, all leading to the development of chronic kidney disease in mice. Expression of Cx43 was almost negligible in the renal cortex of control mice. In contrast, Cx43 was markedly increased in the endothelium of peritubular and glomerular capillaries of the 3-mo-old RenTg mice, in the glomeruli of mice suffering from glomerulonephritis, and in the tubules after obstructive nephropathy. The Cx43 expression pattern was paralleled closely by that of the adhesion markers such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 as well as the inflammatory biomarker monocyte chemoattractant protein-1. In contrast, Cx37 that was abundantly expressed in the renal cortex of healthy mice was markedly decreased in the three experimental models. Interestingly, Cx43+/- mice showed restricted expression of VCAM-1 after 2 wk of obstructive nephropathy. These findings suggest the importance of Cxs as markers of chronic renal disease and indicate that these proteins may participate in the inflammatory process during the development of this pathology.

The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein.

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. We report here a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. We show that the Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hh(Mrt) without impairing cell division. Our results provide a framework to further investigate how cohesin and associated components might regulate transcription.

Variation in parasite resistance of Arctic charr, Salvelinus alpinus, between and within sympatric morphs.

Genetic variation in resistance against parasite infections is a predominant feature in host-parasite systems. However, mechanisms maintaining genetic polymorphism in resistance in natural host populations are generally poorly known. We explored whether differences in natural infection pressure between resource-based morphs of Arctic charr (Salvelinus alpinus) have resulted in differentiation in resistance profiles. We experimentally exposed offspring of two morphs from Lake Þingvallavatn (Iceland), the pelagic planktivorous charr ("murta") and the large benthivorous charr ("kuðungableikja"), to their common parasite, eye fluke Diplostomum baeri, infecting the eye humor. We found that there were no differences in resistance between the morphs, but clear differences among families within each morph. Moreover, we found suggestive evidence of resistance of offspring within families being positively correlated with the parasite load of the father, but not with that of the mother. Our results suggest that the inherited basis of parasite resistance in this system is likely to be related to variation among host individuals within each morph rather than ecological factors driving divergent resistance profiles at morph level. Overall, this may have implications for evolution of resistance through processes such as sexual selection.

Maintenance of Hox gene expression patterns.

Once established, homeotic gene (Hox) expression is maintained in the original pattern by Polycomb-group (PcG) and trithorax-group (trxG) proteins therefore named maintenance proteins (MPs). PcG and trxG proteins maintain silencing and activation of Hox and many other genes, respectively. We provide here a brief overview of genetics and molecular biology of these proteins and of a third class of proteins termed Enhancers of Trithorax and Polycomb (ETP) that are required for both maintenance of silencing and activation of Hox genes. We examine the recruitment of MPs onto maintenance elements (MEs), their role in the regulation of transcription and the epigenetic marks that could provide maintenance. Lastly, we discuss two important roles of PcG proteins in replication of DNA and stem cell renewal and maintenance.

The periodic coloration in birds forms through a prepattern of somite origin.

The periodic stripes and spots that often adorn animals' coats have been largely viewed as self-organizing patterns, forming through dynamics such as Turing's reaction-diffusion within the developing skin. Whether preexisting positional information also contributes to the periodicity and orientation of these patterns has, however, remained unclear. We used natural variation in colored stripes of juvenile galliform birds to show that stripes form in a two-step process. Autonomous signaling from the somite sets stripe position by forming a composite prepattern marked by the expression profile of agouti Subsequently, agouti regulates stripe width through dose-dependent control of local pigment production. These results reveal that early developmental landmarks can shape periodic patterns upstream of late local dynamics, and thus constrain their evolution.