Biology subjects

Liu, L.

Publications and source records attributed to Liu, L..

At least 667 records · Page 37Linked to original sources

bioRxiv2021

Regeneration of humoral immunity from pluripotent stem cells by defined transcription factors

Regeneration of humoral immunity from pluripotent stem cells (PSCs) is a crucial aim in translational medicine. However, reconstitution of complete, sustained, and functional B lymphopoiesis from PSCs has not yet been developed. Here, we successfully achieved regenerative B lymphopoiesis in B-cell deficient animals transplanted with PSC-derived hematopoietic progenitors (iHPCs) guided by synergistic expression of Runx1, Hoxa9, and Lhx2. Upon transplantation, the iHPCs immediately gave rise to pro/pre-B cells in recipients bone marrow, which were able to further differentiate into the entire B cell lineages, including innate B-1a, B-1b, MZ B cells, as well as adaptive FO B cells. In responding to antigen stimuli, the regenerative B cells produced adaptive humoral immune responses, sustained a prolonged antigen-specific antibody production, and formed immune-memory. Particularly, the regenerative B cells in spleen showed developing patterns of immunoglobulin chain-switch and hyper-mutation via a cross-talk with the host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study provides de novo evidence that B lymphopoiesis can be regenerated from PSCs via a HSC-independent approach, which provides insights into treating B-cell related humoral deficiencies using PSCs as unlimited cell resource.

immunology↗

bioRxiv2021

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in the Southern Hemisphere. The presence of PE401 ablates the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both selfish and altruistic characteristics. This offers an insight into a NE arms race that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed. Author summaryBreeding for durable resistance to fungal diseases in crops is a continual challenge for crop breeders. Fungal pathogens evolve ways to overcome host resistance by masking themselves through effector evolution and evasion of broad-spectrum defense responses. Association studies on mapping populations infected by isolate mixtures are often used by researchers to seek out novel sources of genetic resistance. Disease resistance quantitative trait loci (QTL) are often minor or inconsistent across environments. This is a particular problem with septoria diseases of cereals such as septoria nodorum blotch (SNB) of wheat caused by Parastagonospora nodorum. The fungus uses a suite of necrotrophic effectors (NEs) to cause SNB. We characterised a genetic element, called PE401, in the promoter of the major NE gene Tox1, which is present in some P. nodorum isolates. PE401 functions as a transcriptional repressor of Tox1 and exerts epistatic control on another major SNB resistance QTL in the host. In the context of crop protection, constant surveillance of the pathogen population for the frequency of PE401 in conjunction with NE diversity will enable agronomists to provide the best advice to growers on which wheat varieties can be tailored to provide optimal SNB resistance to regional pathogen population genotypes.

microbiology↗

bioRxiv2021

Evolution of a chordate-specific mechanism for myoblast fusion

The size of an animal is determined by the size of its musculoskeletal system. Myoblast fusion is an innovative mechanism that allows for multinucleated muscle fibers to compound the size and strength of individual mononucleated cells. However, the evolutionary history of the control mechanism underlying this important process is currently unknown. The phylum Chordata hosts closely related groups that span distinct myoblast fusion states: no fusion in cephalochordates, restricted fusion and multinucleation in tunicates, and extensive, obligatory fusion in vertebrates. To elucidate how these differences may have evolved, we studied the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed an unexpectedly complex evolutionary history of myoblast fusion in chordates. A pre-vertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Thus, our findings reveal the evolutionary origins of chordate-specific fusogens and illustrate how new genes can shape the emergence of novel morphogenetic traits and mechanisms.

developmental biology↗

bioRxiv2021

TMT-based proteomic analysis of the antibiotic effects of ShangKeHuangShui against Straphylococcus aureus.

Post-traumatic infection is a serious orthopedic trauma complication commonly caused by Staphylococcus aureus (SAU). ShangKeHuangShui, also known as Yellow Aqueous Concentrate of Traumatology Herbs (YACTH), is prepared from six Chinese herbal medicines, which has been used for decades in our hospital to prevent post-traumatic infection. In the present study, we investigated the in vitro antibacterial effects and underlying mechanism of YACTH against SAU. YACTH exhibited significant antibacterial activity against SAU with the minimum inhibitory concentration (MIC) of 8.625 mg/mL. Proteomic analysis based on Tandem mass tag (TMT) showed different protein expression levels in SAU under the YACTH and control conditions. Compared to the control group, the expression level of 490 proteins of YACTH treated group significantly changed (>1.2 fold, P <0.05). Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes and multiple metabolic pathways. We then selected 26 target proteins to further conduct Parallel reaction monitoring (PRM). The results revealed that the 9 down-regulated proteins were mainly involved in RNA polymerase, oxidative phosphorylation, ABC transporter and glycolysis; whereas, the 17 up-regulated proteins are related to RNA degradation, mismatch repair and ribosome. In conclusion, YACTH, which contains the water-soluble components of six Chinese herbs, is first reported to exhibit antibacterial activity against SAU in vitro. The results provide novel insights into the antibacterial mechanisms of YACTH on protein networks, which may help us find new potential antibiotic targets.

microbiology↗

bioRxiv2021

Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization

Synthetic genome evolution provides a dynamic approach to systematically and straightforwardly explore evolutionary processes. SCRaMbLE is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260,000 rearrangement events after SCRaMbLEing of a novel yeast strain harboring 6 synthetic yeast chromosomes. Remarkably, we find that the rearrangement events exhibit a specific landscape of rearrangement frequency. We further reveal that the landscape is shaped by combinatorial effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. Enormous numbers of rearrangements by SCRaMbLE provide a driving force to potentiate directed genome evolution, and investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.

synthetic biology↗

bioRxiv2021

A novel imaging ligand as a biomarker for mutant huntingtin-lowering in Huntington's disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (PolyQ) tract. While several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, no method is currently available to visualize mHTT levels in the living brain. Here we demonstrate the development of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region-and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum have a functional restorative effect that can be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

neuroscience↗

bioRxiv2021

CAT, AGTR2, L-SIGN and DC-SIGN are potential receptors for the entry of SARS-CoV-2 into human cells

Since December 2019, the COVID-19 caused by SARS-CoV-2 has been widely spread all over the world. It is reported that SARS-CoV-2 infection affects a series of human tissues, including lung, gastrointestinal tract, kidney, etc. ACE2 has been identified as the primary receptor of the SARS-CoV-2 Spike (S) protein. The relatively low expression level of this known receptor in the lungs, which is the predominantly infected organ in COVID-19, indicates that there may be some other co-receptors or alternative receptors of SARS-CoV-2 to work in coordination with ACE2. Here, we identified twenty-one candidate receptors of SARS-CoV-2, including ACE2-interactor proteins and SARS-CoV receptors. Then we investigated the protein expression levels of these twenty-one candidate receptors in different human tissues and found that five of which CAT, MME, L-SIGN, DC-SIGN, and AGTR2 were specifically expressed in SARS-CoV-2 affected tissues. Next, we performed molecular simulations of the above five candidate receptors with SARS-CoV-2 S protein, and found that the binding affinities of CAT, AGTR2, L-SIGN and DC-SIGN to S protein were even higher than ACE2. Interestingly, we also observed that CAT and AGTR2 bound to S protein in different regions with ACE2 conformationally, suggesting that these two proteins are likely capable of the co-receptors of ACE2. Conclusively, we considered that CAT, AGTR2, L-SIGN and DC-SIGN were the potential receptors of SARS-CoV-2. Moreover, AGTR2 and DC-SIGN tend to be highly expressed in the lungs of smokers, which is consistent with clinical phenomena of COVID-19, and further confirmed our conclusion. Besides, we also predicted the binding hot spots for these putative protein-protein interactions, which would help develop drugs against SARS-CoV-2.

bioinformatics↗

bioRxiv2021

Mice and primates use distinct strategies for visual segmentation

The rodent visual system has attracted great interest in recent years due to its experimental tractability, but the fundamental mechanisms used by the mouse to represent the visual world remain unclear. In the primate, researchers have argued from both behavioral and neural evidence that a key step in visual representation is "figure-ground segmentation," the delineation of figures as distinct from backgrounds [1-4]. To determine if mice also show behavioral and neural signatures of figure-ground segmentation, we trained mice on a figure-ground segmentation task where figures were defined by gratings and naturalistic textures moving counterphase to the background. Unlike primates, mice were severely limited in their ability to segment figure from ground using the opponent motion cue, with segmentation behavior strongly dependent on the specific carrier pattern. Remarkably, when mice were forced to localize naturalistic patterns defined by opponent motion, they adopted a strategy of brute force memorization of texture patterns. In contrast, primates, including humans, macaques, and mouse lemurs, could readily segment figures independent of carrier pattern using the opponent motion cue. Consistent with mouse behavior, neural responses to the same stimuli recorded in mouse visual areas V1, RL, and LM also did not support texture-invariant segmentation of figures using opponent motion. Modeling revealed that the texture dependence of both the mouses behavior and neural responses could be explained by a feedforward neural network lacking explicit segmentation capabilities. These findings reveal a fundamental limitation in the ability of mice to segment visual objects compared to primates.

neuroscience↗

bioRxiv2021

Accurately positioning functional residues with robotics-inspired computational protein design

Accurate positioning of functional residues is critical for the design of new protein functions, but has remained difficult because of the prevalence of irregular local geometries in active sites. Here we introduce two computational methods that build local protein geometries from sequence with atomic accuracy: fragment kinematic closure (FKIC) and loophash kinematic closure (LHKIC). FKIC and LHKIC integrate two approaches: robotics-inspired kinematics of protein backbones and insertion of peptide fragments, and show up to 140-fold improvements in native-like predictions over either approach alone. We then integrate these methods into a new design protocol, pull-into-place (PIP), to position functionally important sidechains via design of new structured loop conformations. We validate PIP by remodeling a sizeable active site region in an enzyme and confirming the engineered new conformations of two designs with crystal structures. The described methods can be applied broadly to the design of many new protein geometries and functions.

bioengineering↗

bioRxiv2021

Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 [A] resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD {beta}-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition. HighlightsO_LICryo-EM structure of neutralizing antibody 5-7 in complex with SARS CoV-2 spike C_LIO_LI5-7 recognizes NTD outside of the previously identified antigenic supersite C_LIO_LI5-7 binds to a site known to accommodate numerous hydrophobic ligands C_LIO_LIStructural basis of 5-7 neutralization tolerance to some variants of concern C_LI

immunology↗

bioRxiv2021

DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

cancer biology↗

bioRxiv2021

A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal C) were measured to be 6 nM, 2.5 nM, and 8.9 {micro}M respectively. In addition, we found Sal A has a slightly lower IC50 (3.9 {micro}M) than Sal C. Interesting, simple caffeic acid can also disrupt the formation of 6-HB with sub-mM concentration. A pilot high throughput screening (HTS) a small marine natural product library validates the assay with a Z factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitor and assess their binding affinity.

biochemistry↗

bioRxiv2021

Engineered small extracellular vesicles as a FGL1/PD-L1 dual-targeting delivery system for alleviating immune rejection

There is an urgent need for developing new immunosuppressive agents due to the toxicity of long-term use of broad immunosuppressive agents post organ transplantation. Comprehensive sample analysis revealed dysregulation of FGL1/LAG-3 and PD-L1/PD-1 immune checkpoints in allogeneic heart transplantation mice and clinical kidney transplant patients. In order to enhance these two immunosuppressive signal axes, we developed a bioengineering strategy to simultaneously display FGL1/PD-L1 (FP) on the surface of small extracellular vesicles (sEVs). Among various cell sources, FP sEVs derived from mesenchymal stem cells (MSCs) not only enriched FGL1/PD-L1 expression but also maintained the immunomodulatory properties of unmodified MSC sEVs. Next, we confirmed that FGL1 and PD-L1 on sEVs were specifically bound to their receptors LAG-3 and PD-1 on target cells. Importantly, FP sEVs significantly inhibited T cell activation and proliferation in vitro and a heart allograft model. Furthermore, FP sEVs encapsulated with low-dose FK506 (FP sEVs@FK506) exerted stronger effects on inhibiting T cell proliferation, reducing CD8+ T cell density and cytokine production in the spleens and heart grafts, inducing regulatory T cells in lymph nodes, and extending graft survival. Taken together, dual-targeting sEVs have the potential to boost the immune inhibitory signalings in synergy and slow down transplant rejection.

bioengineering↗

bioRxiv2021

Engineered AIM-based Selective Autophagy to Degrade Proteins and Organelles

Techniques for disrupting of protein function are essential for biological researches and therapeutics development. Though well-established, genetic perturbation strategies may have off-target effects and/or trigger compensatory mechanisms, and cannot efficiently eliminate existing protein variants or aggregates1, 2. Therefore, precise and direct protein-targeting methods are highly desired. Here we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescent-tagged proteins and peroxisome organelle, using a tobacco-based transient expression system, and by degrading endogenous proteins in transgenic Arabidopsis expressing engineered receptors. With the wide substrate scope and specificity of selective autophagy, our method provides a convenient and robust strategy for eliminating proteins and aggregates, and may enable developing new treatments for protein-related disorders.

cell biology↗

bioRxiv2021

Single-cell RNA sequencing uncovers the excitatory/inhibitory synaptic unbalance in the retrosplenial cortex after peripheral nerve injury

Nerve injury in the somatosensory pathway may induce maladaptive changes at the transcriptional or protein level, contributing to the development and maintenance of neuropathic pain. In contrast to the retrosplenial cortex (RSC), which processes nociceptive information and exhibits structural and molecular changes after nerve injury, detailed transcriptional changes in the RSC are not yet known. Here we confirm the involvement of the RSC in regulating pain sensation and observe that the same peripheral stimulation activates more retrosplenial neurons after nerve injury; reducing the activities of CaMKII+ splenial cells relieves peripheral pain hypersensitivity after nerve injury. Using a single-cell RNA sequencing (scRNA-seq) approach, we identified cell-type-specific gene expression changes after nerve injury, and the gene set enrichment analysis results revealed suppressed ion homeostasis in CaMKII+ neurons. Furthermore, examination of the expression of genes encoding ligand-gated ion channels showed a decrease in Gabar1a but an increase in Gria1 in CaMKII+ neurons; consistently, we confirmed the unbalanced excitatory/inhibitory synaptic transmission by using the electrophysiological recording approach. Moreover, micro-infusion of 1-Naphthyl acetyl spermine in the RSC to reduce excitatory synaptic transmission alleviated peripheral pain hypersensitivity. Our data confirm the involvement of the RSC in pain regulation and provide information on cell type-dependent transcriptomic changes after nerve injury, which will contribute to the understanding of the mechanisms mediating neuropathic pain.

neuroscience↗

bioRxiv2021

Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.

cell biology↗

bioRxiv2021

COVID-19 Mortality is Associated with Impaired Innate Immunity in Pre-existing Health Conditions

BackgroundCOVID-19 poses a life-threatening endangerment to individuals with chronic diseases. However, not all comorbidities affect COVID-19 prognosis equally. Some increase the risk of COVID-19 related death by more than six folds while others show little to no impact. To prevent severe outcomes, it is critical that we comprehend pre-existing molecular abnormalities in common health conditions that predispose patients to poor prognoses. In this study, we aim to discover some of these molecular risk factors by associating gene expression dysregulations in common health conditions with COVID-19 mortality rates in different cohorts. MethodsWe focused on fourteen pre-existing health conditions, for which age-and-sex-adjusted hazard ratios of COVID-19 mortality have been documented. For each health condition, we analyzed existing transcriptomics data to identify differentially expressed genes (DEGs) between affected individuals and unaffected individuals. We then tested if fold changes of any DEG in these pre-existing conditions were correlated with hazard ratios of COVID-19 mortality to discover molecular risk factors. We performed gene set enrichment analysis to identify functional groups overrepresented in these risk factor genes and examined their relationships with the COVID-19 disease pathway. ResultsWe found that upregulated expression of 70 genes and downregulated expression of 181 genes in pre-existing health conditions were correlated with increased risk of COVID-19 related death. These genes were significantly enriched with endoplasmic reticulum (ER) function, proinflammatory reaction, and interferon production that participate in viral transcription and immune responses to viral infections. ConclusionsImpaired innate immunity in pre-existing health conditions are associated with increased hazard of COVID-19 mortality. The discovered molecular risk factors are potential prognostic biomarkers and targets for therapeutic interventions.

bioinformatics↗

bioRxiv2021

Knocking-out the human face genes TBX15 and PAX1 in mice alters facial and other physical morphology

DNA variants in or closed to the human TBX15 and PAX1 genes have been repeatedly associated with facial morphology in independent genome-wide association studies, while their functional roles in determining facial morphology remains to be understood. We generated Tbx15 knockout (Tbx15-/-) and Pax1 knockout (Pax1-/-) mice by applying the one-step CRISPR/Cas9 method. A total of 75 adult mice were used for subsequent phenotype analysis, including 38 Tbx15 mice (10 homozygous Tbx15-/-, 18 heterozygous Tbx15+/-, 10 wild-type WT) and 37 Pax1 mice (12 homozygous Pax1-/-, 15 heterozygous Pax1+/-, 10 WT mice). Facial and other physical morphological phenotypes were obtained from three-dimensional (3D) images acquired with the HandySCAN BLACK scanner. Compared to WT mice, the Tbx15-/- mutant mice had significantly shorter faces (P=1.08E-8, R2=0.61) and their ears were in a significantly lower position (P=3.54E-8, R2=0.62) manifesting an "ear dropping" characteristic. Besides these face alternations, Tbx15-/- mutant mice displayed significantly lower weight as well as shorter body and limb length. Pax1-/- mutant mice showed significantly longer noses (P=1.14E-5, R2=0.46) relative to WT mice, but otherwise displayed less obvious morphological alterations than Tbx15-/- mutant mice did. Because the Tbx15 and Pax1 effects on facial morphology we revealed here in mice are largely consistent with previously reported TBX15 and PAX1 face associations in humans, we suggest that the functional role these two genes play on determining the face of mice is similar to the functional impact their human homologues have on the face of humans. Author SummarySeveral independent genome-wide association studies (GWASs) on human facial morphology highlighted DNA variants in or closed to TBX15 and PAX1 with genome-wide significant association with human facial phenotypes. However, direct evidence on the functional involvement if these genes in the development and determination of facial morphology has not been established as of yet. In the current study, our in vivo gene editing experiments in mice for two well-replicated human face TBX15 and PAX1 genes provide novel evidence on the functional involvement of these two genes in facial and other physical morphology in mice, at least. Tbx15-/- mice showed a shortened facial length and manifesting an ear dropping characteristic, Pax1-/- mice showed an increased nose length. Our geometric morphometrics analysis further indicate that there are significant facial morphology differences between groups (Tbx15-/-and Tbx15+/-, Tbx15-/- and Tbx15+/+, Tbx15+/- and Tbx15+/+, Pax1-/- and Pax1+/+). We provide the first direct functional evidence that two well-known and replicated human face genes, Tbx15 and Pax1, impact facial and other body morphology in mice. The general agreement between our findings in knock-out mice with those from previous GWASs suggests that the functional evidence we established here in mice may also be relevant in humans.

genetics↗