Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

BACKGROUND: Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. RESULTS: We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. CONCLUSIONS: The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Complementary yet divergent effects of exercise and an exercise mimetic on microbiome in high-fat diet-induced obesity.

Exercise is beneficial for obesity, partially through increased mitochondrial activity and raised nicotinamide adenine dinucleotide (NAD), a coenzyme critical for mitochondrial function and metabolism. Recent work has shown that increasing the availability of NAD through pharmacological means improves metabolic health in rodent models of diet-induced obesity and that the effect of these supplements when administered orally may be modulated by the gut microbiome. The gut microbiome is altered by both diet and exercise and is thought to contribute to some aspects of high-fat diet-induced metabolic dysfunction. We examined the independent and combined effects of treadmill exercise and nicotinamide mononucleotide (NMN) supplementation on the gut microbiome of female C57Bl6/J mice chronically fed a high-fat diet. We showed that 8 wk of treadmill exercise, oral-administered NMN, or combined therapy exert unique effects on gut microbiome composition without changing bacterial species richness. Exercise and NMN exerted additive effects on microbiota composition, and NMN partially or fully restored predicted microbial functions, specifically carbohydrate and lipid metabolism, to control levels. Further research is warranted to better understand the mechanisms underpinning the interactions between exercise and oral NAD+ precursor supplementation on gut microbiome.NEW & NOTEWORTHY Exercise and NAD+ precursor supplementation exerted additive and independent effects on gut microbiota composition and inferred function in female mice with diet-induced obesity. Notably, combining exercise and oral nicotinamide mononucleotide supplementation restored inferred microbial functions to control levels, indicating that this combination may improve high-fat diet-induced alterations to microbial metabolism.

Concurrent betaine administration enhances exercise-induced improvements to glucose handling in obese mice.

BACKGROUND AND AIMS: Betaine supplementation has been shown to enhance hepatic lipid metabolism in obese mice and improve exercise performance in healthy populations. We examined effects of betaine supplementation, alone or in combination with treadmill exercise, on the metabolic consequences of high fat diet (HFD)-induced obesity in mice. METHODS AND RESULTS: Male C57BL/6 J mice were fed chow or HFD. After 15 weeks, HFD mice were split into: HFD, HFD with betaine (1.5% w/v), HFD with treadmill exercise, and HFD with both betaine and exercise (15 m/min for 45min, 6 days/week; n = 12/group) for 10 weeks. Compared to HFD mice, body weight was significantly reduced in exercise and exercise-betaine mice, but not in mice given betaine alone. Similarly, adiposity was reduced by exercise but not by betaine alone. HFD-induced glucose intolerance was slightly improved by exercise, but not with betaine alone. Significantly greater benefits were observed in exercise-betaine mice, compared to exercise alone, such that GTT-outcomes were similar to controls. This was associated with reduced insulin levels during ipGTT, suggesting enhanced insulin sensitivity. Modest benefits were observed in fatty acid metabolism genes in skeletal muscle, whilst limited effects were observed in the liver. HFD-induced increases in hepatic Mpc1 (mitochondrial pyruvate carrier 1) were normalized by all treatments, suggesting potential links to altered glucose metabolism. CONCLUSIONS: Our data show that drinking 1.5% betaine was sufficient to augment metabolic benefits of exercise in obese mice. These processes appear to be facilitated by altered glucose metabolism, with limited effects on hepatic lipid metabolism.

Nutrient Sensing by Histone Marks: Reading the Metabolic Histone Code Using Tracing, Omics, and Modeling.

Several metabolites serve as substrates for histone modifications and communicate changes in the metabolic environment to the epigenome. Technologies such as metabolomics and proteomics have allowed us to reconstruct the interactions between metabolic pathways and histones. These technologies have shed light on how nutrient availability can have a dramatic effect on various histone modifications. This metabolism-epigenome cross talk plays a fundamental role in development, immune function, and diseases like cancer. Yet, major challenges remain in understanding the interactions between cellular metabolism and the epigenome. How the levels and fluxes of various metabolites impact epigenetic marks is still unclear. Discussed herein are recent applications and the potential of systems biology methods such as flux tracing and metabolic modeling to address these challenges and to uncover new metabolic-epigenetic interactions. These systems approaches can ultimately help elucidate how nutrients shape the epigenome of microbes and mammalian cells.

Exercise-induced benefits on glucose handling in a model of diet-induced obesity are reduced by concurrent nicotinamide mononucleotide.

Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity and subsequent increases in nicotinamide adenine dinucleotide (NAD+), an important metabolic cofactor. Recent studies have shown that increasing NAD+ levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high-fat-diet (HFD)-fed mice. However, the effects of combined exercise and NMN supplementation are unknown. Thus, here we examined the combined effects of NMN and treadmill exercise in female mice with established obesity after 10 wk of diet. Five-week-old female C57BL/6J mice were exposed to a control diet (n = 16) or HFD. Mice fed a HFD were either untreated (HFD; n = 16), received NMN in drinking water (400 mg/kg; HNMN; n = 16), were exposed to treadmill exercise 6 days/wk (HEx; n = 16), or were exposed to exercise combined with NMN (HNEx; n = 16). Although some metabolic benefits of NMN have been described, at this dose, NMN administration impaired several aspects of exercise-induced benefits in obese mice, including glucose tolerance, glucose-stimulated insulin secretion from islets, and hepatic triglyceride accumulation. HNEx mice also exhibited increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN cotreatment. Our data show that NMN treatment impedes the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism.NEW & NOTEWORTHY NMN dampened exercise-induced benefits on glucose handling in diet-induced obesity. NMN administration alongside treadmill exercise enhanced the ratio of antioxidants to prooxidants. We suggest that NMN administration may not be beneficial when NAD+ levels are replete.

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased ß-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson's Disease.

Analysis of experience-regulated transcriptome and imprintome during critical periods of mouse visual system development reveals spatiotemporal dynamics.

Visual system development is light-experience dependent, which strongly implicates epigenetic mechanisms in light-regulated maturation. Among many epigenetic processes, genomic imprinting is an epigenetic mechanism through which monoallelic gene expression occurs in a parent-of-origin-specific manner. It is unknown if genomic imprinting contributes to visual system development. We profiled the transcriptome and imprintome during critical periods of mouse visual system development under normal- and dark-rearing conditions using B6/CAST F1 hybrid mice. We identified experience-regulated, isoform-specific and brain-region-specific imprinted genes. We also found imprinted microRNAs were predominantly clustered into the Dlk1-Dio3 imprinted locus with light experience affecting some imprinted miRNA expression. Our findings provide the first comprehensive analysis of light-experience regulation of the transcriptome and imprintome during critical periods of visual system development. Our results may contribute to therapeutic strategies for visual impairments and circadian rhythm disorders resulting from a dysfunctional imprintome.

Impacts of obesity, maternal obesity and nicotinamide mononucleotide supplementation on sperm quality in mice.

Male fertility and sperm quality are negatively impacted by obesity. Furthermore, recent evidence has shown that male offspring from obese rat mothers also have reduced sperm quality and fertility. Here, we extend work in this area by comparing the effects of both maternal obesity and offspring post-weaning diet-induced obesity, as well as their combination, on sperm quality in mice. We additionally tested whether administration of the NAD+-booster nicotinamide mononucleotide (NMN) can ameliorate the negative effects of obesity and maternal obesity on sperm quality. We previously showed that intraperitoneal (i.p.) injection of NMN can reduce the metabolic deficits induced by maternal obesity or post-weaning dietary obesity in mice. In this study, female mice were fed a high-fat diet (HFD) for 6 weeks until they were 18% heavier than a control diet group. Thereafter, HFD and control female mice were mated with control diet males, and male offspring were weaned into groups receiving control or HFD. At 30 weeks of age, mice received 500 mg/kg body weight NMN or vehicle PBS i.p. for 21 days. As expected, adiposity was increased by both maternal and post-weaning HFD but reduced by NMN supplementation. Post-weaning HFD reduced sperm count and motility, while maternal HFD increased offspring sperm DNA fragmentation and levels of aberrant sperm chromatin. There was no evidence that the combination of post-weaning and maternal HFD exacerbated the impacts in sperm quality suggesting that they impact spermatogenesis through different mechanisms. Surprisingly NMN reduced sperm count, vitality and increased sperm oxidative DNA damage, which was associated with increased NAD+ in testes. A subsequent experiment using oral NMN at 400 mg/kg body weight was not associated with reduced sperm viability, oxidative stress, mitochondrial dysfunction or increased NAD+ in testes, suggesting that the negative impacts on sperm could be dependent on dose or mode of administration.

Dietary Macronutrient Management to Treat Mitochondrial Dysfunction in Parkinson's Disease.

Mitochondrial dysfunction has been demonstrated to play an important role in the pathogenesis of Parkinson's disease (PD). The products of several PD-associated genes, including alpha-synuclein, parkin, pink1, protein deglycase DJ-1, and leucine rich repeat kinase 2, have important roles in mitochondrial biology. Thus, modifying mitochondrial function could be a potential therapeutic strategy for PD. Dietary management can alter mitochondrial function as shifts in dietary macronutrients and their ratios in food can alter mitochondrial energy metabolism, morphology and dynamics. Our studies have established that a low protein to carbohydrate (P:C) ratio can increase lifespan, motor ability and mitochondrial function in a parkin mutant Drosophila model of PD. In this review, we describe mitochondrial dysfunction in PD patients and models, and dietary macronutrient management strategies to reverse it. We focus on the effects of protein, carbohydrate, fatty acids, and their dietary ratios. In addition, we propose potential mechanisms that can improve mitochondrial function and thus reverse or delay the onset of PD.

Reintroducing domesticated wild mice to sociality induces adaptive transgenerational effects on MUP expression.

When brought into captivity, wild animals can adapt to domestication within 10 generations. Such adaptations may decrease fitness in natural conditions. Many selective pressures are disrupted in captivity, including social behavioral networks. Although lack of sociality in captivity appears to mediate domestication, the underlying mechanisms are not well understood. Additionally, determining the contribution of genetic inheritance vs. transgenerational effects during relaxed selection may provide insight into the flexibility of adaptation. When wild-derived mice kept under laboratory conditions for eight generations were reintroduced to sociality and promiscuity (free mate choice), they adapted within two generations. Fitness assessments between this promiscuous lineage and a monogamous laboratory lineage revealed male-specific effects. Promiscuous-line males had deficits in viability, but a striking advantage in attracting mates, and their scent marks were also more attractive to females. Here, we investigate mechanistic details underlying this olfactory signal and identify a role of major urinary protein (MUP) pheromones. Promiscuous-line males inherit higher MUP expression than monogamous-line males through transgenerational inheritance. Sociality-driven maternal and paternal effects reveal intriguing conflicts among parents and offspring over pheromone expression. MUP up-regulation is not driven by hormone-driven transduction pathways, but rather is associated with reduction in DNA methylation of a CpG dinucleotide in the promoter. This reduction in methylation could enhance transcription by promoting the binding of transcription factor USF1 (upstream stimulatory factor 1). Finally, we experimentally demonstrate that increased MUP expression is a female attractant. These results identify molecular mechanisms guiding domestication and adaptive responses to fluctuating sociality.

Modifiers of epigenetic reprogramming show paternal effects in the mouse.

There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.

Paternal high-fat diet consumption induces common changes in the transcriptomes of retroperitoneal adipose and pancreatic islet tissues in female rat offspring.

We previously showed that paternal high-fat diet (HFD) consumption programs ß-cell dysfunction in female rat offspring, together with transcriptome alterations in islets. Here we investigated the retroperitoneal white adipose tissue (RpWAT) transcriptome using gene and pathway enrichment and pathway analysis to determine whether commonly affected network topologies exist between these two metabolically related tissues. In RpWAT, 5108 genes were differentially expressed due to a paternal HFD; the top 5 significantly enriched networks identified by pathway analysis in offspring of HFD fathers compared with those of fathers fed control diet were: mitochondrial and cellular response to stress, telomerase signaling, cell death and survival, cell cycle, cellular growth and proliferation, and cancer. A total of 187 adipose olfactory receptor genes were down-regulated. Interrogation against the islet transcriptome identified specific gene networks and pathways, including olfactory receptor genes that were similarly affected in both tissues (411 common genes, P<0.05). In particular, we highlight a common molecular network, cell cycle and cancer, with the same hub gene, Myc, suggesting early onset developmental changes that persist, shared responses to programmed systemic factors, or crosstalk between tissues. Thus, paternal HFD consumption triggers unique gene signatures, consistent with premature aging and chronic degenerative disorders, in both RpWAT and pancreatic islets of daughters.

Parental programming: how can we improve study design to discern the molecular mechanisms?

The contribution of inherited non-genetic factors to complex diseases is of great current interest. The ways in which mothers and fathers can affect their offspring's health clearly differ as a result of the intimate interactions between mother and offspring during pre- and postnatal life. There is, however, potential for some overlap in mechanisms, particularly epigenetic mechanisms. A small number of epidemiological studies and animal models have investigated the non-genetic contribution of the parents to offspring health. Discovering new mechanisms of disease inheritance is technically difficult, especially in genetically, socially and environmentally heterogeneous human populations. Therefore, rigorous experimental design, appropriate sample numbers and the use of high-throughput technologies are necessary to provide convincing evidence. Based on recent examples from the literature, here we propose several ways to improve human studies that aim to identify the underlying mechanisms of transgenerational inheritance of metabolic disease.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations.

Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.

Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene.

MicroRNAs (miRNAs) are an abundant class of RNAs that are approximately 21-25 nucleotides (nt) long, interact with mRNAs and trigger either translation repression or RNA cleavage (RNA interference, RNAi) depending on the degree of complementarity with their targets. Here we show that the imprinted mouse distal chromosome 12 locus encodes two miRNA genes expressed from the maternally inherited chromosome and antisense to a retrotransposon-like gene (Rtl1) expressed only from the paternal allele.

Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12.

Genomic imprinting causes parental origin-specific gene expression. Cis-acting regulatory elements that control imprinting are not fully understood but involve regions that become differentially methylated on the two parental chromosomes during male and female gametogenesis. Understanding properties of maternally and paternally inherited imprints provides insight into the mechanisms and evolution of genomic imprinting. Previously we identified an intergenic germline-derived differentially methylated region (IG-DMR) that is a candidate control element for an imprinted domain on distal mouse chromosome 12 (ref. 5). The 1-Mb cluster contains the paternally expressed protein-coding genes Dlk1 (refs. 6,7) and Dio3 (ref. 8,9) and several maternally expressed non-coding RNAs, including Gtl2 (refs. 6,7,10) and C/D snoRNAs. A retrotransposon-like gene (Rtl1) is expressed from the paternal chromosome and has an antisense transcript expressed from the maternal chromosome containing two microRNAs with full complementarity to Rtl1 (ref. 12). Here we show that deletion of the IG-DMR from the maternally inherited chromosome causes bidirectional loss of imprinting of all genes in the cluster. When the deletion is transmitted from the father, imprinting is unaltered. These results prove that the IG-DMR is a control element for all imprinted genes on the maternal chromosome only and indicate that the two parental chromosomes control allele-specific gene expression differently.

Epigenetic control and genomic imprinting dynamics of the Dlk1-Dio3 domain.

Genomic imprinting is an epigenetic process whereby genes are monoallelically expressed in a parent-of-origin-specific manner. Imprinted genes are frequently found clustered in the genome, likely illustrating their need for both shared regulatory control and functional inter-dependence. The Dlk1-Dio3 domain is one of the largest imprinted clusters. Genes in this region are involved in development, behavior, and postnatal metabolism: failure to correctly regulate the domain leads to Kagami-Ogata or Temple syndromes in humans. The region contains many of the hallmarks of other imprinted domains, such as long non-coding RNAs and parental origin-specific CTCF binding. Recent studies have shown that the Dlk1-Dio3 domain is exquisitely regulated via a bipartite imprinting control region (ICR) which functions differently on the two parental chromosomes to establish monoallelic expression. Furthermore, the Dlk1 gene displays a selective absence of imprinting in the neurogenic niche, illustrating the need for precise dosage modulation of this domain in different tissues. Here, we discuss the following: how differential epigenetic marks laid down in the gametes cause a cascade of events that leads to imprinting in the region, how this mechanism is selectively switched off in the neurogenic niche, and why studying this imprinted region has added a layer of sophistication to how we think about the hierarchical epigenetic control of genome function.

Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women.

Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10-10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10-7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR-Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.