Lions & sea lions & bears, oh my: utilizing museum specimens to study the ossification sequence of carnivoran taxa.

BACKGROUND: Mammalian skeletons are largely formed before birth. Heterochronic changes in skeletal formation can be investigated by comparing the order of ossification for different elements of the skeleton. Due to the challenge of collecting prenatal specimens in viviparous taxa, opportunistically collected museum specimens provide the best material for studying prenatal skeletal development across many mammalian species. Previous studies have investigated ossification sequence in a range of mammalian species, but little is known about the pattern of bone formation in Carnivora. Carnivorans have diverse ecologies, diets, and biomechanical specializations and are well-suited for investigating questions in evolutionary biology. Currently, developmental data on carnivorans is largely limited to domesticated species. To expand available data on carnivoran skeletal development, we used micro-computed tomography (micro-CT) to non-invasively evaluate the degree of ossification in all prenatal carnivoran specimens housed in the Harvard Museum of Comparative Zoology. By coding the presence or absence of bones in each specimen, we constructed ossification sequences for each species. Parsimov-based genetic inference (PGi) was then used to identify heterochronic shifts between carnivoran lineages and reconstruct the ancestral ossification sequence of Carnivora. RESULTS: We used micro-CT to study prenatal ossification sequence in six carnivora species: Eumetopias jubatus (Steller sea lion, n = 6), Herpestes javanicus (small Indian mongoose, n = 1), Panthera leo (lion, n = 1), Urocyon cinereoargenteus (gray fox, n = 1), Ursus arctos arctos (Eurasian brown bear, n = 1), and Viverricula indica (small Indian civet, n = 5). Due to the relatively later stage of collection for the available specimens, few heterochronic shifts were identified. Ossification sequences of feliform species showed complete agreement with the domestic cat. In caniforms, the bear and fox ossification sequences largely matched the dog, but numerous heterochronic shifts were identified in the sea lion. CONCLUSIONS: We use museum specimens to generate cranial and postcranial micro-CT data on six species split between the two major carnivoran clades: Caniformia and Feliformia. Our data suggest that the ossification sequence of domestic dogs and cats are likely good models for terrestrial caniforms and feliforms, respectively, but not pinnipeds.

Survival analysis on subchondral bone length for total knee replacement.

OBJECTIVE: Use subchondral bone length (SBL), a new MRI-derived measure that reflects the extent of cartilage loss and bone flattening, to predict the risk of progression to total knee replacement (TKR). METHODS: We employed baseline MRI data from the Osteoarthritis Initiative (OAI), focusing on 760 men and 1214 women with bone marrow lesions (BMLs) and joint space narrowing (JSN) scores, to predict the progression to TKR. To minimize bias from analyzing both knees of a participant, only the knee with a higher Kellgren-Lawrence (KL) grade was considered, given its greater potential need for TKR. We utilized the Kaplan-Meier survival curves and Cox proportional hazards models, incorporating raw and normalized values of SBL, JSN, and BML as predictors. The study included subgroup analyses for different demographics and clinical characteristics, using models for raw and normalized SBL (merged, femoral, tibial), BML (merged, femoral, tibial), and JSN (medial and lateral compartments). Model performance was evaluated using the time-dependent area under the curve (AUC), Brier score, and Concordance index to gauge accuracy, calibration, and discriminatory power. Knee joint and region-level analyses were conducted to determine the effectiveness of SBL, JSN, and BML in predicting TKR risk. RESULTS: The SBL model, incorporating data from both the femur and tibia, demonstrated a predictive capacity for TKR that closely matched the performance of the BML score and the JSN grade. The Concordance index of the SBL model was 0.764, closely mirroring the BML's 0.759 and slightly below JSN's 0.788. The Brier score for the SBL model stood at 0.069, showing comparability with BML's 0.073 and a minor difference from JSN's 0.067. Regarding the AUC, the SBL model achieved 0.803, nearly identical to BML's 0.802 and slightly lower than JSN's 0.827. CONCLUSION: SBL's capacity to predict the risk of progression to TKR highlights its potential as an effective imaging biomarker for knee osteoarthritis.

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model.

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

The functional and evolutionary impacts of human-specific deletions in conserved elements.

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5, CPEB4, and PPP2CA. Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.

Epigenetics as a mediator of genetic risk in osteoarthritis: role during development, homeostasis, aging, and disease progression.

The identification of genomic loci that are associated with osteoarthritis (OA) has provided a starting point for understanding how genetic variation activates catabolic processes in the joint. However, genetic variants can only alter gene expression and cellular function when the epigenetic environment is permissive to these effects. In this review, we provide examples of how epigenetic shifts at distinct life stages can alter the risk for OA, which we posit is critical for the proper interpretation of genome-wide association studies (GWAS). During development, intensive work on the growth and differentiation factor 5 (GDF5) locus has revealed the importance of tissue-specific enhancer activity in controlling both joint development and the subsequent risk for OA. During homeostasis in adults, underlying genetic risk factors may help establish beneficial or catabolic "set points" that dictate tissue function, with a strong cumulative effect on OA risk. During aging, methylation changes and the reorganization of chromatin can "unmask" the effects of genetic variants. The destructive function of variants that alter aging would only mediate effects after reproductive competence and thus avoid any evolutionary selection pressure, as consistent with larger frameworks of biological aging and its relationship to disease. A similar "unmasking" may occur during OA progression, which is supported by the finding of distinct expression quantitative trait loci (eQTLs) in chondrocytes depending on the degree of tissue degradation. Finally, we propose that massively parallel reporter assays (MPRAs) will be a valuable tool to test the function of putative OA GWAS variants in chondrocytes from different life stages.

Lineage-specific differences and regulatory networks governing human chondrocyte development.

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology.

Human and African ape myosin heavy chain content and the evolution of hominin skeletal muscle.

Humans are unique among terrestrial mammals in our manner of walking and running, reflecting 7 to 8 Ma of musculoskeletal evolution since diverging with the genus Pan. One component of this is a shift in our skeletal muscle biology towards a predominance of myosin heavy chain (MyHC) I isoforms (i.e. slow fibers) across our pelvis and lower limbs, which distinguishes us from chimpanzees. Here, new MyHC data from 35 pelvis and hind limb muscles of a Western gorilla (Gorilla gorilla) are presented. These data are combined with a similar chimpanzee dataset to assess the MyHC I content of humans in comparison to African apes (chimpanzees and gorillas) and other terrestrial mammals. The responsiveness of human skeletal muscle to behavioral interventions is also compared to the human-African ape differential. Humans are distinct from African apes and among a small group of terrestrial mammals whose pelvis and lower limb muscle is slow fiber dominant, on average. Behavioral interventions, including immobilization, bed rest, spaceflight and exercise, can induce modest decreases and increases in human MyHC I content (i.e. -9.3% to 2.3%, n = 2033 subjects), but these shifts are much smaller than the mean human-African ape differential (i.e. 31%). Taken together, these results indicate muscle fiber content is likely an evolvable trait under selection in the hominin lineage. As such, we highlight potential targets of selection in the genome (e.g. regions that regulate MyHC content) that may play an important role in hominin skeletal muscle evolution.

Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals.

Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus' impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

The developmental impacts of natural selection on human pelvic morphology.

Evolutionary responses to selection for bipedalism and childbirth have shaped the human pelvis, a structure that differs substantially from that in apes. Morphology related to these factors is present by birth, yet the developmental-genetic mechanisms governing pelvic shape remain largely unknown. Here, we pinpoint and characterize a key gestational window when human-specific pelvic morphology becomes recognizable, as the ilium and the entire pelvis acquire traits essential for human walking and birth. We next use functional genomics to molecularly characterize chondrocytes from different pelvic subelements during this window to reveal their developmental-genetic architectures. We then find notable evidence of ancient selection and genetic constraint on regulatory sequences involved in ilium expansion and growth, findings complemented by our phenotypic analyses showing that variation in iliac traits is reduced in humans compared to African apes. Our datasets provide important resources for musculoskeletal biology and begin to elucidate developmental mechanisms that shape human-specific morphology.

Intronic regulation of SARS-CoV-2 receptor (ACE2) expression mediated by immune signaling and oxidative stress pathways.

The angiotensin-converting enzyme 2 (ACE2) protein is a key catalytic regulator of the renin-angiotensin system (RAS), involved in fluid homeostasis and blood pressure modulation. ACE2 also serves as a cell-surface receptor for some coronaviruses such as SARS-CoV and SARS-CoV-2. Improved characterization of ACE2 regulation may help us understand the effects of pre-existing conditions on COVID-19 incidence, as well as pathogenic dysregulation following viral infection. Here, we perform bioinformatic analyses to hypothesize on ACE2 gene regulation in two different physiological contexts, identifying putative regulatory elements of ACE2 expression. We perform functional validation of our computational predictions via targeted CRISPR-Cas9 deletions of these elements in vitro, finding them responsive to immune signaling and oxidative-stress pathways. This contributes to our understanding of ACE2 gene regulation at baseline and immune challenge. Our work supports pursuit of these putative mechanisms in our understanding of infection/disease caused by current, and future, SARS-related viruses such as SARS-CoV-2.

Detection of Neanderthal Adaptively Introgressed Genetic Variants That Modulate Reporter Gene Expression in Human Immune Cells.

Although some variation introgressed from Neanderthals has undergone selective sweeps, little is known about its functional significance. We used a Massively Parallel Reporter Assay (MPRA) to assay 5,353 high-frequency introgressed variants for their ability to modulate the gene expression within 170 bp of endogenous sequence. We identified 2,548 variants in active putative cis-regulatory elements (CREs) and 292 expression-modulating variants (emVars). These emVars are predicted to alter the binding motifs of important immune transcription factors, are enriched for associations with neutrophil and white blood cell count, and are associated with the expression of genes that function in innate immune pathways including inflammatory response and antiviral defense. We combined the MPRA data with other data sets to identify strong candidates to be driver variants of positive selection including an emVar that may contribute to protection against severe COVID-19 response. We endogenously deleted two CREs containing expression-modulation variants linked to immune function, rs11624425 and rs80317430, identifying their primary genic targets as ELMSAN1, and PAN2 and STAT2, respectively, three genes differentially expressed during influenza infection. Overall, we present the first database of experimentally identified expression-modulating Neanderthal-introgressed alleles contributing to potential immune response in modern humans.

Interspecies transcriptomics identify genes that underlie disproportionate foot growth in jerboas.

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has "mouse-like" arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as "nodes" for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

Identification of IGF2BP1-related lncRNA-miRNA-mRNA network in goat skeletal muscle satellite cells.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) plays essential roles in the proliferation of skeletal muscle satellite cells (MuSCs). Increasing evidence has shown that IGF2BP1 regulates the expression of noncoding RNAs and mRNAs. However, the related molecular network remains to be fully understood. Therefore, we performed RNA sequencing and analyzed the microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and mRNAs differentially expressed in goat MuSCs treated with IGF2BP1 overexpressing and empty vectors. A total of 36 miRNAs, 59 lncRNAs, and 44 mRNAs were differentially expressed caused by IGF2BP1. Expectedly, they were enriched in muscle development-related Rap1, PI3K-AKT, and FoxO signaling pathways. Finally, we constructed a lncRNA-miRNA-mRNA interaction network containing 30 lncRNAs, 15 miRNAs, and 34 mRNAs, in which several miRNAs, including miR-133a-3p, miR-204-5p, miR-125a-3p, miR-145-3p, and miR-423-5p, relate with cell growth and participate in muscle development. Overall, we constructed an IGF2BP1-related network, which provides new insight into the myogenic proliferation of goat.

Joint disease-specificity at the regulatory base-pair level.

Given the pleiotropic nature of coding sequences and that many loci exhibit multiple disease associations, it is within non-coding sequence that disease-specificity likely exists. Here, we focus on joint disorders, finding among replicated loci, that GDF5 exhibits over twenty distinct associations, and we identify causal variants for two of its strongest associations, hip dysplasia and knee osteoarthritis. By mapping regulatory regions in joint chondrocytes, we pinpoint two variants (rs4911178; rs6060369), on the same risk haplotype, which reside in anatomical site-specific enhancers. We show that both variants have clinical relevance, impacting disease by altering morphology. By modeling each variant in humanized mice, we observe joint-specific response, correlating with GDF5 expression. Thus, we uncouple separate regulatory variants on a common risk haplotype that cause joint-specific disease. By broadening our perspective, we finally find that patterns of modularity at GDF5 are also found at over three-quarters of loci with multiple GWAS disease associations.

Shifting epigenetic contexts influence regulatory variation and disease risk.

Epigenetic shifts are a hallmark of aging that impact transcriptional networks at regulatory level. These shifts may modify the effects of genetic regulatory variants during aging and contribute to disease pathomechanism. However, these shifts occur on the backdrop of epigenetic changes experienced throughout an individual's development into adulthood; thus, the phenotypic, and ultimately fitness, effects of regulatory variants subject to developmental- versus aging-related epigenetic shifts may differ considerably. Natural selection therefore may act differently on variants depending on their changing epigenetic context, which we propose as a novel lens through which to consider regulatory sequence evolution and phenotypic effects. Here, we define genomic regions subjected to altered chromatin accessibility as tissues transition from their fetal to adult forms, and subsequently from early to late adulthood. Based on these epigenomic datasets, we examine patterns of evolutionary constraint and potential functional impacts of sequence variation (e.g., genetic disease risk associations). We find that while the signals observed with developmental epigenetic changes are consistent with stronger fitness consequences (i.e., negative selection pressures), they tend to have weaker effects on genetic risk associations for aging-related diseases. Conversely, we see stronger effects of variants with increased local accessibility in adult tissues, strongest in young adult when compared to old. We propose a model for how epigenetic status of a region may influence the effects of evolutionary relevant sequence variation, and suggest that such a perspective on gene regulatory networks may elucidate our understanding of aging biology.

Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses.

Type I interferons (IFNs) are our first line of defense against virus infection. Recent studies have suggested the ability of SARS-CoV-2 proteins to inhibit IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wild-type SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are scarce. Here we demonstrate that SARS-CoV-2 infection induces a type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Furthermore, we show that physiological levels of IFNα detected in patients with moderate COVID-19 is sufficient to suppress SARS-CoV-2 replication in human airway cells.

Subchondral Bone Length in Knee Osteoarthritis: A Deep Learning-Derived Imaging Measure and Its Association With Radiographic and Clinical Outcomes.

OBJECTIVE: To develop a bone shape measure that reflects the extent of cartilage loss and bone flattening in knee osteoarthritis (OA) and test it against estimates of disease severity. METHODS: A fast region-based convolutional neural network was trained to crop the knee joints in sagittal dual-echo steady-state magnetic resonance imaging sequences obtained from the Osteoarthritis Initiative (OAI). Publicly available annotations of the cartilage and menisci were used as references to annotate the tibia and the femur in 61 knees. Another deep neural network (U-Net) was developed to learn these annotations. Model predictions were compared to radiologist-driven annotations on an independent test set (27 knees). The U-Net was applied to automatically extract the knee joint structures on the larger OAI data set (n = 9,434 knees). We defined subchondral bone length (SBL), a novel shape measure characterizing the extent of overlying cartilage and bone flattening, and examined its relationship with radiographic joint space narrowing (JSN), concurrent pain and disability (according to the Western Ontario and McMaster Universities Osteoarthritis Index), as well as subsequent partial or total knee replacement. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for each outcome were estimated using relative changes in SBL from the OAI data set stratified into quartiles. RESULTS: The mean SBL values for knees with JSN were consistently different from knees without JSN. Greater changes of SBL from baseline were associated with greater pain and disability. For knees with medial or lateral JSN, the ORs for future knee replacement between the lowest and highest quartiles corresponding to SBL changes were 5.68 (95% CI 3.90-8.27) and 7.19 (95% CI 3.71-13.95), respectively. CONCLUSION: SBL quantified OA status based on JSN severity and shows promise as an imaging marker in predicting clinical and structural OA outcomes.