Biology subjects

Bao, H.

Publications and source records attributed to Bao, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Sulfatide deficiency-induced astrogliosis and myelin lipid dyshomeostasis are independent of Trem2-mediated microglial activation

Disrupted lipid homeostasis and neuroinflammation often co-exist in neurodegenerative disorders including Alzheimers disease (AD). However, the intrinsic connection and causal relationship between these deficits remain elusive. Our previous studies show that the loss of sulfatide (ST), a class of myelin-enriched lipids, causes AD-like neuroinflammatory responses, cognitive impairment, bladder enlargement, as well as lipid dyshomeostasis. To better understand the relationship between neuroinflammation and lipid disruption induced by ST deficiency, we established a ST-deficient mouse model with constitutive Trem2 knockout and studied the impact of Trem2 in regulating ST deficiency-induced microglia-mediated neuroinflammation, astrocyte activation and lipid disruption. Our study demonstrates that Trem2 regulates ST deficiency-induced microglia-mediated neuroinflammatory pathways and astrogliosis at the transcriptomic level, but not astrocyte activation at the protein level, suggesting that Trem2 is indispensable for ST deficiency-induced microglia-mediated neuroinflammation but not astrogliosis. Meanwhile, ST loss-induced lipidome disruption and free water retention were consistently observed in the absence of Trem2. Collectively, these results emphasize the essential role of Trem2 in mediating lipid loss-associated microglia-mediated neuroinflammation, but not both astrogliosis and myelin lipid disruption. Moreover, we demonstrated that attenuating neuroinflammation has a limited impact on brain ST loss-induced lipidome alteration or AD-like peripheral disorders. Our findings suggest that preserving lipidome and astrocyte balance may be crucial in decelerating the progression of AD.

neuroscience↗

bioRxiv2024

DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides

Membrane scaffold proteins-based nanodiscs (NDs) have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in NDs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of NDs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional ND technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we created a new class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free NDs (DeFrNDs) with high efficiency. NDs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of DeFrNDs, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.

biophysics↗

bioRxiv2024

Rhizobial effector NopM ubiquitinates Nod factor receptor NFR5 and promotes rhizobial infection in Lotus japonicus

Bacterial pathogens and nitrogen-fixing rhizobia employ type III protein secretion system (T3SS) effectors as potent tools to manipulate plant signaling pathways, thereby facilitating infection and host colonization. However, the molecular mechanisms by which rhizobial effectors affect legume infection remain largely elusive. In this study, we investigated the symbiotic role of T3SS effectors in the interaction between Sinorhizobium fredii NGR234 and Lotus japonicus. Mutants deficient in the T3SS genes Tts1 or NopA showed enhanced rhizobial infection of L. japonicus roots. Further mutant analysis showed that the NopT effector negatively affects infection, while the NopM effector in the absence of NopT promotes infection. Notably, NopM interacts with the Nod factor receptors LjNFR1 and LjNFR5. NopM ubiquitinates LjNFR5 on ten lysine residues as identified using mass spectrometry. Expression of NopM in L. japonicus resulted in an approximately twofold increase in LjNFR5 protein levels and enhanced rhizobial infection. Our findings indicate that NopM directly interferes with the symbiotic signaling pathway through interaction and ubiquitination of Nod factor receptors, which likely benefits rhizobial infection of L. japonicus. Our research contributes to the intricate interplay between the Nod factor signaling pathway and rhizobial T3SS effectors delivered into host cells. Significance StatementThis study unravels intricate mechanisms underlying the symbiotic interaction between Sinorhizobium fredii NGR234 and Lotus japonicus. By focusing on the role of the rhizobial effector NopM, this research sheds light on how bacteria manipulate host symbiotic signaling to promote infection and colonization. NopM interacts with NFRs and ubiquitinates LjNFR5, suggesting a novel strategy employed by rhizobia to enhance symbiotic signaling.

microbiology↗

bioRxiv2024

The Rhizobial effector NopT targets Nod factor receptors to regulate symbiosis in Lotus japonicus

It is well-documented that type-III effectors are required by Gram-negative pathogens to directly target different host cellular pathways to promote bacterial infection. However, in the context of legume-rhizobium symbiosis, the role of rhizobial effectors in regulating plant symbiotic pathways remains largely unexplored. Here, we show that NopT, a YopT-type cysteine protease of Sinorhizobium fredii NGR234 directly targets the plants symbiotic signaling pathway by associating with two Nod factor receptors (NFR1 and NFR5 of Lotus japonicus). NopT inhibits cell death triggered by co-expression of NFR1/NFR5 in Nicotiana benthamiana. Full-length NopT physically interacts with NFR1 and NFR5. NopT proteolytically cleaves NFR5 both in vitro and in vivo, but can be inactivated by NFR1 as a result of phosphorylation. NopT plays an essential role in mediating rhizobial infection in L. japonicus. Autocleaved NopT retains the ability to cleave NFR5 but no longer interacts with NFR1. Interestingly, genomes of certain Sinorhizobium species only harbor nopT genes encoding truncated proteins without the autocleavage site. These results reveal an intricate interplay between rhizobia and legumes, in which a rhizobial effector protease targets NFR5 to suppress symbiotic signaling. NFR1 appears to counteract this process by phosphorylating the effector. This discovery highlights the role of a bacterial effector in regulating a signaling pathway in plants and opens up the perspective of developing kinase-interacting proteases to fine-tune cellular signaling processes in general. One-sentence summaryThe rhizobial effector NopT and two Nod factor receptors of Lotus japonicus, NFR1 and NFR5, mutually interact to precisely regulate rhizobial symbiosis.

plant biology↗

bioRxiv2023

AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationship between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3 cells and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor exerts its influence on various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, fostering their growth and development, suggesting a broad impact of AhR on intestinal health. In conclusion, our study demonstrates the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.

immunology↗

bioRxiv2023

Evolutionarily Conserved CKI1-Mediated Two-Component Signaling is Required for Female Germline Specification in Marchantia polymorpha

In land plants, gametes derive from a small number of dedicated haploid cells. 1 In angiosperms, one central cell and one egg cell are differentiated in the embryo sac as female gametes for double fertilization, while in non-flowering plants, only one egg cell is generated in the female sexual organ, called the archegonium. 2,3 The central cell specification of Arabidopsis thaliana is controlled by the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1), which is a two-component signaling (TCS) activator sharing downstream regulatory components with the cytokinin signaling pathway. 4-7 Our phylogenetic analysis suggested that CKI1 orthologs broadly exist in land plants. However, the role of CKI1 in the archegonium-bearing non-flowering plants remains unclear. Here we found that the sole CKI1 ortholog in the liverwort Marchantia polymorpha, MpCKI1, which functions through conserved downstream TCS components, regulates the female germline specification for egg cell development in the archegonium. In M. polymorpha, the archegonium develops three-dimensionally from a single cell, accumulating MpBONOBO (MpBNB), a master regulator for germline initiation and differentiation. 8 We visualized female germline specification by capturing the distribution pattern of MpBNB in the discrete stages of early archegonium development, and found that the MpBNB accumulation is restricted to female germline cells. MpCKI1 is required for the proper MpBNB accumulation in the female germline, and is critical for the asymmetric cell divisions that specify the female germline cells. These results suggest that CKI1-mediated TCS originated during early land plant evolution and participates in female germ cell specification in deeply-diverged plant lineages.

plant biology↗

bioRxiv2023

Substrate-Specific Function of PNPLA3 Facilitates Hepatic VLDL-Triglyceride Secretion During Stimulated Lipogenesis

The I148M variant of PNPLA3 is strongly linked to hepatic steatosis. Evidence suggests a gain-of-function role for the I148M mutant as an ATGL inhibitor, leaving the physiological relevance of wild-type PNPLA3 undefined. Here we show that PNPLA3 selectively degrades triglycerides (TGs) enriched in polyunsaturated fatty acids (PUFAs) independently of ATGL in cultured cells and mice. Lipidomics and metabolite tracing analyses demonstrated that PNPLA3 mobilizes PUFAs from intracellular TGs for phospholipid desaturation, supporting hepatic secretion of TG-rich lipoproteins. Consequently, mice with liver-specific knockout or acute knockdown of PNPLA3 both exhibited aggravated liver steatosis and concomitant decreases in plasma VLDL-TG, phenotypes that manifest only under lipogenic conditions. I148M-knockin mice similarly displayed impaired hepatic TG secretion during lipogenic stimulation. Our results highlight a specific context whereby PNPLA3 facilitates the balance between hepatic TG storage and secretion and suggest the potential contributions of I148M variant loss-of-function to the development of hepatic steatosis in humans. Summary StatementWe define the physiological role of wild type PNPLA3 in maintaining hepatic VLDL-TG secretion.

physiology↗

bioRxiv2022

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Root system is critical for plant growth and development. To develop plant growth regulators functioning in root development, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that mimicked the effects of auxin on root development. Genetic, pharmacological and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

plant biology↗

bioRxiv2022

Reconstruction of the cell pseudo-space from single-cell RNA sequencing data with scSpace

Tissues are highly complicated with spatial heterogeneity in gene expression. However, the cutting-edge single-cell RNA-seq technology eliminates the spatial information of individual cells, which contributes to the characterization of cell identities. Herein, we propose single-cell spatial position associated co-embeddings (scSpace), an integrative algorithm to distinguish spatially variable cell subclusters by reconstructing cells onto a pseudo-space with spatial transcriptome references (Visium, STARmap, Slide-seq, etc.). We demonstrated that scSpace can define biologically meaningful cell subpopulations neglected by single-cell RNA-seq or spatially resolved transcriptomics. The use of scSpace to uncover the spatial association within single-cell data, reproduced, the hierarchical distribution of cells in the brain cortex and liver lobules, and the regional variation of cells in heart ventricles and the intestinal villus. scSpace identified cell subclusters in intratelencephalic neurons, which were confirmed by their biomarkers. The application of scSpace in melanoma and Covid-19 exhibited a broad prospect in the discovery of spatial therapeutic markers.

bioinformatics↗

bioRxiv2021

NASP maintains histone H3-H4 homeostasis through two distinct H3 binding modes

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3-H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3-H4 supply. We determine the structures of a sNASP dimer, a complex of sNASP with an H3 3 peptide, and the sNASP-H3-H4-ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 3 helix and the H3 N region, respectively. Functional studies demonstrate the H3 N-interaction represents the major binding mode of NASP in cells and shielding of the H3 N region by NASP is essential in maintaining the H3-H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3-H4 chaperone in guarding histone homeostasis.

biochemistry↗

bioRxiv2021

Catalase protects against non-enzymatic decarboxylations during photorespiration

Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H2O2 resulting from glycolate oxidation into O2 and water. We hypothesize that catalase in the peroxisome also protects against non-enzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO2 release from photorespiration, specifically an increase in the CO2 compensation point, a photorespiratory-dependent decrease in the quantum efficiency of CO2 assimilation, increase in the 12CO2 released in a 13CO2 background and an increase in the post-illumination CO2 burst. Further metabolic evidence suggests this excess CO2 release occurred via the non-enzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these non-enzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted. One Sentence SummaryCatalase guards against additional carbon loss from photorespiration arising from non-enzymatic decarboxylations of photorespiratory intermediates.

plant biology↗

bioRxiv2021

SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization

The establishment of receptive endometrium in human necessitates appropriate decidualization of stromal cells, which involves steroids regulated periodic transformation of endometrial stromal cells during menstrual cycle. Insufficient decidualization of endometrium contributes to not only the failure of embryo implantation and unexplained infertility, but also the occurrence of recurrent spontaneous abortion, intrauterine growth retardation, preeclampsia, and other clinical gynecological diseases. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of decidualization in humans is yet to be fully elucidated. In this investigation, we document that SOX4 is a key regulator of human endometrial stromal cells (hESCs) decidualization by directly regulating PRL and FOXO1 expression as revealed by whole genomic binding of SOX4 assay and RNA-Seq. Besides, our immunoprecipitation and mass spectrometry results unravel that SOX4 modulates progesterone receptor (PGR) stability through repressing E3 ubiquitin ligase HERC4 mediated degradation. More importantly, we provide evidence that dysregulated SOX4-HERC4-PGR axis is a potential cause of defective decidualization and recurrent implantation failure (RIF) in IVF patients. In summary, this study evidences that SOX4 is a new and critical regulator for human endometrial decidualization, and provides insightful information for the pathology of decidualization-related infertility and will pave the way for pregnancy improvement.

physiology↗

bioRxiv2020

Neonicotinoids stimulate H2-limited methane emission in Periplaneta americana through the regulation of gut bacterium community

Methane emitted by insects is considered to be an important source of atmospheric methane. Here we report the stimulation of methane emission in Periplaneta americana, an insect species with abundant methanogens, by neonicotinoids, insecticides widely used to control insect pests. The application of cycloxaprid (CYC) and imidacloprid (IMI) caused foregut expansion in P. americana, and increased the methane production and emission. Antibiotics could mostly eliminate the stimulatory effects. In P. americana gut, hydrogen levels increased and pH values decreased, which could be significantly explained by the gut bacterium community change. The proportion of several bacterium genera increased in guts following CYC treatment, and four genera from five with increased proportions could generate hydrogen at anaerobic conditions. Hydrogen is a central intermediate in methanogenesis. Gut methanogens could use the increased hydrogen to produce more methane, especially at acidic conditions. Following neonicotinoid applications, all increased methanogens in both foregut and hindgut used hydrogen as electron donor to produce methane. Besides, the up-regulation of mcrA, encoding the enzyme that catalyzes the final step of methanogenesis, suggested an enhanced methane production ability in present methanogens. In the termite Coptotermes chaohuensis, another methanogen-abundant insect species, hydrogen levels in gut and methane emission significantly increased after neonicotinoid treatment, which was similar to the results in P. americana. In summary, neonicotinoids changed bacterium community in P. americana gut to generate more hydrogen, which then stimulate gut methanogens to produce and emit more methane. The finding raised a new concern over neonicotinoid applications, and might be a potential environmental risk associated with global warming.

ecology↗

bioRxiv2020

Coordination of two enhancers drives expression of olfactory trace amine-associated receptors

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.

neuroscience↗

bioRxiv2020

Dysregulation of hippocampal adult-born immature neurons disrupts a brain-wide network for spatial memory

Mounting evidence suggests that cognitive deficits associated with various neurological disorders may arise in part from a small population of dysregulated adult-born neurons in the dentate gyrus (DG). How these dysregulated adult-born neurons contribute to brain-wide network maladaptation and subsequent cognitive deficits remains unknown. Using an established mouse model with a small number of time-stamped dysregulated adult-born immature neurons and spatial memory deficits, we performed resting state functional magnetic resonance imaging and found that approximately 500 deficient immature neurons (<0.1% of total DG granule neurons) are sufficient to induce a significant decrease in the functional connectivity between DG and insular cortex (IC), two brain regions without direct anatomical connections. Furthermore, using a combination of rabies-based retrograde tracing and in vivo fiber photometry recording, we demonstrated that dysregulated adult-born neurons induce aberrant activity and synchrony in local hippocampal CA3 and CA1 regions, as well as distal medial-dorsal thalamus and IC regions during a spatial memory process. These results suggest that a few hundred dysregulated adult-born immature neurons can impact brain-wide network dynamics across several anatomically discrete regions and collectively contribute to impaired cognitive functions.

neuroscience↗

bioRxiv2020

Single-cell screening of SARS-CoV-2 target cells in pets, livestock, poultry and wildlife

A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.

cell biology↗

bioRxiv2019

Genome sequencing and neurotoxin diversity of a wandering spider Pardosa pseudoannulata (pond wolf spider)

Spiders constitute an extensive and diverse branch of the phylum Arthropoda. Whereas the genomes of four web-weaver spider species and a single cave-living spider have been determined, similar studies have not been reported previously for a wandering spider. The pond wolf spider, Pardosa pseudoannulata, is a wandering hunter that immobilizes prey using venom rather than a web. It is also an important predator against a range of agriculturally important insect pests. The increasing interest in its wandering lifestyle and in the potential of spider venom as a tool for pest control have prompted a detailed study on this wandering spider species. We have generated a high-quality genome sequence of P. pseudoannulata and analysed genes associated with the production of silk and venom toxins. Sequencing reveals that P. pseudoannulata has a large genome of 4.26 Gb. The presence of only 16 spidroin genes and four types of silk glands is consistent with the moderate use of silk and the lack of a prey-catching web. A large number of genes encode neurotoxins and there is evidence that the majority are highly selective for invertebrates. Comparison between spider species reveals a correlation between spider neurotoxin selectivity for target organisms and spider prosoma size, suggesting a possible coevolution of these two features. The genome data provides valuable insights into the biology of P. pseudoannulata and its potential role as a natural enemy in pest control.

genomics↗

bioRxiv2019

Müllerian-inhibiting substance (MIS) is both necessary and sufficient for testicular differentiation in Chinese soft-shelled turtle Pelodiscus sinensis

Mullerian-inhibiting substance (Mis, or anti-mullerian hormone, Amh), a member of TGF-{beta} superfamily, as initiator or key regulator in sexual development has been well documented in some vertebrates, especially in fish. However, its functional role has not been identified yet in reptiles. Here we characterized the Mis gene in Chinese soft-shelled turtle Pelodiscus sinensis (P. sinensis), a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The mRNA of Mis was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited male-specific expression pattern throughout embryogenesis. Moreover, Mis was rapidly up-regulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Mis loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker Sox9, and upregulation of the ovarian regulator Cyp19a1. Conversely, overexpression of Mis in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of testis structure, ectopic activation of Sox9, and a remarkable decline in Cyp19a1. Collectively, these findings provide the first solid evidence that Mis is both necessary and sufficient to drive testicular development in a reptilian species, P. sinensis, highlighting the significance of the TGF-{beta} pathway in reptilian sex determination.

developmental biology↗