Biology subjects

Jung, H.

Publications and source records attributed to Jung, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Asymmetric distribution of mitochondrial Ca2+ regulators specifies compartment-specific mitochondrial function and neuronal development

Neuronal polarization is essential for functional compartmentalization, enabling dendritic synaptic integration and axonal action potential generation. While structural differences in mitochondria across compartments have been identified, their functional distinctions remain unclear. Here, we uncovered compartment-specific mitochondrial Ca2+ dynamics and their molecular determinants. In axonal mitochondria, Ca2+ uptake through MCU occurs independently of ER-stored Ca2+ release, with faster matrix Ca2+ clearance than dendritic mitochondria, where Ca2+ uptake predominantly originates from ER Ca2+. The ER-independent mitochondrial Ca2+ uptake in axonal mitochondria is mediated by enriched MCU-regulating proteins, MICU1 and MICU2, while higher NCLX expression facilitates rapid Ca2+ clearance. Moreover, NCLX knockdown, which functionally mimics a mental retardation-associated mutation, caused more significant axonal branching defects compared to dendrites in vivo, aligning with its enrichment in axons. These findings highlight fundamental Ca2+-modulating features and developmental importance of neuronal mitochondria in a compartment-specific manner and reveal the key underlying molecular mechanisms.

neuroscience↗

bioRxiv2025

A telomere to telomere phased genome assembly and annotation for the Australian central bearded dragon Pogona vitticeps

BackgroundThe central bearded dragon (Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon. ResultsGenome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome. This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1, suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon. ConclusionThis high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally. Species TaxonomyEukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona; Pogona vitticeps (Ahl, 1926) (NCBI:txid103695). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/651798v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1a9d5fforg.highwire.dtl.DTLVardef@1206061org.highwire.dtl.DTLVardef@97acf7org.highwire.dtl.DTLVardef@1dc939d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Targeting the ITGA3B1 Complex with A Novel Antibody-Drug Conjugate for Metastatic Bladder Cancer

Advances in antibody (Ab) phage display technologies have enabled the development of targeted therapies with enhanced precision and clinical efficacy. Here, we report a phenotypic screening strategy combining target-unbiased live-cell biopanning with in situ chemical crosslinking and mass spectrometry to identify internalizing antibodies and their cognate antigens--key features for effective antibody-drug conjugate (ADC) development. Using this approach, we identified the 2E7 antibody, which exhibits rapid internalization and high specificity for the integrin 3{beta}1 (ITGA3B1) heterodimer, a complex overexpressed in multiple solid tumors, including bladder cancer. We generated an ITGA3B1-targeted ADC by conjugating monomethyl auristatin E (MMAE) to the 2E7 antibody, enabling selective delivery of cytotoxic payloads to ITGA3B1-positive cells. In preclinical bladder cancer models, this ADC demonstrated potent and dose-dependent antitumor efficacy, with significant tumor regression and improved survival. Our findings establish a framework for target discovery using live-cell phenotypic screening and position ITGA3B1 as a promising therapeutic target for ADC-based treatment of advanced bladder cancer. TeaserLive-cell biopanning uncovers ITGA3B1 as a target for ADC development in metastatic bladder cancer.

synthetic biology↗

bioRxiv2025

STIM1 transmembrane helix dimerization captured by AI-guided transition path sampling

STIM1 is a Ca2+-sensing protein in the endoplasmic reticulum (ER) membrane. The depletion of ER Ca2+ stores induces a large conformational transition of the cytosolic STIM1 C-terminus, initiated by the dimerization of the transmembrane (TM) domain. We use the AI-guided transition path sampling algorithm aimmd to extensively sample the dimerization of STIM1-TM helices in an ER-mimicking lipid bilayer. In nearly 0.5 milliseconds of all-atom molecular dynamics simulations without bias potentials, we harvest over 170 transition paths, each about 1.2 {micro}s long on average. We find that STIM1 dimerizes into three distinct and coexisting configurations, which reconciles conflicting results from earlier crosslinking studies. The dominant X-shaped bound state centers around contacts supported by the SxxxG TM interfacial motif. Mutating residues in this contact interface allows us to tune the STIM1-dimerization propensity in fluorescence experiments. From the trained model of the committor probability of dimerization, we identify the transition state ensemble for TM-helix dimerization. At the transition state, interhelical contacts in the luminal halves of the two monomers dominate, which likely enables the luminal Ca2+-sensing domain in STIM1 to condition the dimerization of the TM helices. Our work demonstrates the unique power of AI-guided simulations to sample rare and slow molecular transitions, and to produce detailed atomistic insight into the mechanism of STIM1 TM-helix dimerization as a key step in ER Ca2+-sensing. SignificanceSTIM1 is a protein sensor that signals drops in the Ca2+ concentration inside the endoplasmic reticulum (ER) to the cytosol. As Ca2+ levels decrease, the STIM1 transmembrane (TM) helices dimerize. We sample this TM helix dimerization in AI-guided atomistic molecular simulations, revealing two distinct pathways of dimerization. We reconcile conflicting results of earlier crosslinking studies by showing that both reported dimer configurations coexist as the end points of distinct association pathways. The dominance of interhelical contacts on the luminal side at the transition state enables the luminal Ca2+-sensing domain to condition helix dimerization. AI-guided path sampling made it possible to sample rare helix dimerization events far outside the current reach of regular molecular simulations.

biophysics↗

bioRxiv2025

TMEM145 is a key component in stereociliary link structures of outer hair cells

Outer hair cells (OHCs) in the cochlea contain specialized stereociliary structures essential for auditory function. These include horizontal top connectors (HTCs), linking adjacent stereocilia and tectorial membrane-attachment crowns (TM-ACs), anchoring the tallest stereocilia to the tectorial membrane. The known molecular components of these structures, such as stereocilin, otogelin, otogelin-like, and tubby, lack transmembrane domains, suggesting the existence of anchoring proteins. This study identified TMEM145, a transmembrane protein with a Golgi dynamics (GOLD) domain, as a crucial OHC stereocilia component. TMEM145 was expressed in both OHCs and spiral ganglion neurons, with specific localization to TM-ACs and HTCs in OHCs. Tmem145 knockout (KO) mice exhibited profound hearing impairment at three weeks of age, with complete loss of distortion product otoacoustic emissions, indicating OHC dysfunction. Immunostaining and scanning electron microscopy revealed the absence of TM-ACs and HTCs in Tmem145 KO mice. In heterologous cell systems, TMEM145 interacted with stereocilin and tubby, facilitating their extracellular secretion. TMEM145 was undetectable in stereocilin KO and tubby mutant mice, indicating interdependence among these proteins. These findings establish TMEM145 as an essential membrane protein for the structural integrity of OHC stereocilia, providing insights into the molecular architecture of cochlear hair cells and their role in auditory function.

neuroscience↗

bioRxiv2025

A prevalent Krt8-to-Krt5 cellular state transition in skin is co-opted by p63 for enamel organ development

Tooth enamel, the hardest vertebrate tissue, is crucial for mastication and dental protection. Its formation depends on the enamel organ (EO), a specialized epithelial structure derived from oral epithelium. How uniform oral epithelium differentiates into diverse EO cell types remains unclear. While p63, an ectodermal development master regulator, is essential for dental placode formation, its specific roles in EO development have been obscured by early arrest in p63 knockout mice. Using single-cell RNA sequencing from mouse incisors, we show p63 expression across all EO cell types with both shared and distinct functions. Through trajectory reconstruction, we identify p63s role in regulating both amelogenic (AmG) and non-AmG lineage commitment during EO development. Comparative transcriptome analyses reveal that p63 regulates the Krt8-to-Krt5 transition during AmG cell differentiation, paralleling its function in skin development. This parallel is reinforced by comparative motif discovery showing shared transcription factor usage, particularly p63 and AP-2 family members. Chromatin accessibility analyses further illustrate that p63 mediates this transition through chromatin landscape remodeling. These findings demonstrate that p63 co-opts the Krt8-to-Krt5 transition mechanism from skin development for EO formation. Summary statementThis study reveals how p63 repurposes a skin keratinization mechanism for tooth enamel formation, providing novel insights into how specialized dental tissues develop and potential therapeutic targets for enamel disorders.

developmental biology↗

bioRxiv2025

Protein Synthesis Blockade Prevents Fear Memory Reactivation via Inhibition of Engram Synapse Strengthening

Memory relies on ensembles of engram cells in the brain. While previous studies have established the existence of these cells, the relationship between cellular and synaptic activity remains unclear. To address this, we applied the dual e-GRASP technique in mice to examine synaptic connectivity between the ventral CA1 and the basal amygdala during memory formation. We found that contextual fear conditioning increased engram-to-engram synapse (engram synapse) density and induced structural potentiation, underscoring their importance in associative memory. Additionally, we investigated the role of protein synthesis in memory formation by inducing retrograde amnesia using anisomycin, a protein synthesis inhibitor. Mice injected with anisomycin once (1xANI) showed impaired natural recall but still displayed fear responses upon optogenetic reactivation of engram cells. In contrast, mice that received four anisomycin injections over six hours (4xANI) showed significantly impaired natural recall and failed to exhibit fear responses upon optogenetic reactivation. This behavioural phenotype was correlated with synaptic changes: 1xANI mice exhibited no significant reduction in engram synapse density but had significantly smaller spine sizes, whereas 4xANI mice showed both a significant reduction in engram synapse density and spine size. Our results indicate that protein synthesis inhibition significantly reduces engram synapse density and spine size, changes that correlate with reductions in fear memory during both natural recall and artificial reactivation.

neuroscience↗

bioRxiv2025

Action features dominate cortical representation during natural vision

Cortical resources are allocated into systems specialized for processing ecologically relevant features of the world. These features are typically studied in isolation using controlled experimental stimuli, making it difficult to assess the relative importance of different kinds of features that tend to overlap during natural vision. In the current study, we evaluated the relative contributions of action, agent, and scene features in predicting cortical activity while participants viewed a 1-hour nature documentary. We tested four sets of model features: semantic vectors capturing the observed action, agent, and scene features derived from an annotation of the stimulus, as well as low-level visual motion-energy features. We used banded ridge regression to fit vertex-wise encoding models from the combination of all four sets of features. While each set of features predicted neural activity in the expected areas, the action features predicted more widespread activity. A variance partitioning analysis revealed that the action features captured the most unique variance in neural activity across ten times as many cortical vertices as the agent or scene features. Our findings suggest that cortical activity during dynamic, natural vision is dominated by features for understanding the actions of others.

neuroscience↗

bioRxiv2024

Image Processing in the Acute to Chronic Pain Signatures (A2CPS) Project

The Acute to Chronic Pain Signatures (A2CPS) project is a large-scale, multi-site initiative aimed at identifying biomarkers and biosignatures that predict the transition from acute to chronic pain. The project is collecting multimodal, longitudinal data from over 2,500 individuals at risk for developing chronic pain after surgery. Here we describe the neuroimaging component of A2CPS, including the acquisition protocols, processing pipelines, and contents of the initial data release. The imaging protocol includes structural, diffusion, resting-state and task-based functional magnetic resonance imaging (MRI) data. Data are collected across multiple clinical sites using different scanner manufacturers, with attention to protocol harmonization and quality control. The processing pipeline integrates several established neuroimaging tools to extract potential biomarkers, including measures of brain structure, connectivity, and pain-related neural signatures. The first data release includes pre-surgical imaging data for 595 participants, with high quality ratings across modalities (98.7% of sMRI, 99.8% of dMRI, and 94.6% of fMRI images were rated as acceptable or better). Initial analyses demonstrate expected relationships between brain-derived measures and clinical variables, such as associations between brain age and psychological factors. This dataset represents a valuable resource for both pain research and neuroimaging methods development, with future releases planned to include additional participants and expanded analysis pipelines and processed data derivatives.

neuroscience↗

bioRxiv2024

Common and distinct neural correlates of social interaction perception and theory of mind

Social interaction perception and theory of mind (ToM) frequently co-occur, but their commonalities and distinctions at behavioral and neural levels remain unclear. Participants (N = 231) provided moment-by-moment ratings of four text and four audio narratives on social interactions and ToM engagement, which were reliable (split-half r = .98 and .92, respectively) but only modestly correlated (r = .32). In a second sample (N = 90), we analyzed the co-variation between social interaction and ToM ratings and fMRI activity during text and audio narratives. Social interaction and ToM activity maps generalized across modalities (spatial r = .83 and .57, respectively), both with significant, overlapping clusters in canonical mentalizing regions (FDR q < .01). ToM uniquely engaged the lateral occipitotemporal cortex, left anterior intraparietal sulcus, and right premotor cortex. These results suggest that perceiving social interactions automatically involves mentalizing, and ToM additionally engages brain regions for action understanding and executive functions.

neuroscience↗

bioRxiv2024

Divergent effects of cytomegalovirus and rheumatoid arthritis on senescent CD4+ T cells

CD4+ T cell senescence has been linked to repeated antigen stimulation. However, how different types of chronic antigenic exposures impact T cell differentiation and function remains incompletely understood. Using rheumatoid arthritis (RA) and cytomegalovirus (CMV) as models for persistent stimulation in autoimmunity and chronic viral infection, we performed high-dimensional mass cytometry analyses to examine their effects on CD4+ T cell differentiation. We found that CMV seropositive adults have a significant population of highly differentiated CD27-CD28-CD4+ T cells that exhibited common features of senescence, including CD57 and cytotoxic granule expression. In contrast, CD27-CD28-CD4+ T cells were rare in RA patients and Epstein-Barr virus (EBV) or Herpes simplex virus (HSV) seropositive individuals. The few that were present exhibited a predominantly non-cytotoxic profile, with higher expression of TCF1, CD127, and Ki67. Among CMV seropositive individuals, RA was associated with reduced degranulation of cytotoxic granules and lower cytokine production by senescent CD4+ T cells. We did not find an association with age, sex, clinical characteristics, or medication usage by univariate linear regression analyses. However, in vitro tofacitinib treatment reduced T cell functional activity, suggesting contributions from both the disease and RA treatment. These data uncovered distinct influences from CMV and RA, and their combined impact on senescent CD4+ T cell differentiation and function.

immunology↗

bioRxiv2024

Behaviorally-relevant features of observed actions dominate cortical representational geometry in natural vision

We effortlessly extract behaviorally relevant information from dynamic visual input in order to understand the actions of others. In the current study, we develop and test different classes of models to better understand the neural representational geometries supporting action understanding. Using fMRI, we measured brain activity as participants viewed a diverse set of 90 different video clips depicting social and nonsocial actions in real-world contexts. We developed five behavioral models using arrangement tasks: two models reflecting behavioral judgments of the purpose (transitivity) and the social content (sociality) of the actions depicted in the video stimuli; and three models reflecting behavioral judgments of the visual content (people, objects, and scene) depicted in still frames of the stimuli. We evaluated how well these models predict neural representational geometry and tested them against semantic models based on verb and nonverb embeddings and visual models based on gaze and motion energy. Our results revealed that behavioral models of action meaning/purpose (transitivity, sociality) better reflect neural representational geometry than behavioral models of object, person, and scene features, as well as semantic and visual models throughout much of cortex. The sociality and transitivity models captured a large portion of unique variance throughout the action observation network, extending into regions not typically associated with action perception, like ventral temporal cortex. Overall, these findings expand our understanding of the action observation network and indicate that the social content and purpose of observed actions are predominant in cortical representation.

neuroscience↗

bioRxiv2024

The Accumulation of Progerin Underlies the Loss of Aortic Smooth Muscle Cells in Hutchinson-Gilford Progeria Syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disorder characterized by multiple aging-like phenotypes, including disease in large arteries. HGPS is caused by an internally truncated prelamin A (progerin) that cannot undergo the ZMPSTE24-mediated processing step that converts farnesyl-prelamin A to mature lamin A; consequently, progerin retains a carboxyl-terminal farnesyl lipid anchor. In cultured cells, progerin and full-length farnesyl-prelamin A (produced in Zmpste24-/- cells) form an abnormal nuclear lamin meshwork accompanied by nuclear membrane ruptures and cell death; however, these proteins differ in their capacity to cause arterial disease. In a mouse model of HGPS (LmnaG609G), progerin causes loss of aortic smooth muscle cells (SMCs) by [~]12 weeks of age. In contrast, farnesyl-prelamin A in Zmpste24-/- mice does not cause SMC loss--even at 21 weeks of age. In young mice, aortic levels of farnesyl-prelamin A in Zmpste24-/- mice and aortic levels of progerin in LmnaG609G/+ mice are the same. However, the levels of progerin and other A-type lamins increase with age in LmnaG609G/+ mice, whereas farnesyl-prelamin A and lamin C levels in Zmpste24-/- mice remain stable. Lmna transcript levels are similar, implying that progerin influences nuclear lamin turnover. We identified a likely mechanism. In cultured SMCs, the phosphorylation of Ser-404 by AKT (which triggers prelamin A degradation) is reduced in progerin. In mice, AKT activity is significantly lower in LmnaG609G/+ aortas than in wild-type or Zmpste24-/- aortas. Our studies identify that the accumulation of progerin in LmnaG609G aortas underlies the hallmark arterial pathology in HGPS. One Sentence SummaryThe age-related accumulation of progerin in smooth muscle cells (SMCs) explains the loss of arterial SMCs in Hutchinson-Gilford progeria syndrome.

cell biology↗

bioRxiv2024

Cancer-independent, second somatic NF1 mutation of normal tissues in neurofibromatosis type 1

INTRODUCTIONCancer predisposition syndromes mediated by recessive cancer genes generate tumours via somatic variants (second hits) in the unaffected allele. Second hits may or may not be sufficient for neoplastic transformation. Here, we performed whole genome and exome sequencing on 479 tissue biopsies from a child with neurofibromatosis type 1, a multi-system cancer-predisposing syndrome mediated by constitutive monoallelic NF1 inactivation. We identified multiple independent NF1 driver variants in histologically normal tissues, but not in 610 biopsies from two non-predisposed children. We corroborated this finding using targeted duplex sequencing, including a further nine adults with the same syndrome. Overall, truncating NF1 mutations were under positive selection in normal tissues from individuals with neurofibromatosis type 1. We demonstrate that normal tissues in neurofibromatosis type 1 commonly harbour second hits in NF1, the extent and pattern of which may underpin the syndromes cancer phenotype.

cancer biology↗

bioRxiv2024

Mature leaves produce a multi-layered wound periderm by integrating phytohormone signaling with ATML1-mediated epidermal specification

The epidermis of plants forms a protective barrier against various stress, but how breaches in the epidermis are repaired is not well understood. Here, we investigated wound healing in the mature leaves of Arabidopsis. We discover a novel type of wound periderm comprising a multi-layered ligno-suberized barrier covered with cuticular wax, which is formed by mesophyll cells that adopt an epidermal fate. Mesophyll cells of protective layer 1 (P1), just beneath the wound, transition into epidermal cells, which seal the wound by depositing cuticle. As P1 undergoes cell death, protective layer 2 (P2), which underlies P1, takes the place of P1 and undergoes ligno-suberization. This multi-layered periderm involves integration of ethylene and jasmonic acid signaling with ATML1, a key transcription factor in epidermal specification, to coordinate cell layer-specific functions. This novel wound periderm also occurs in the leaves of tobacco and Capsella, suggesting it is a widespread phenomenon.

plant biology↗

bioRxiv2024

Convergent evolution of somatic escape variants in SERPINA1 in the liver in alpha-1 anti-trypsin deficiency

Somatic variants accumulate in non-malignant tissues with age1,2. Functional variants leading to clonal advantage of hepatocytes accumulate in the liver from patients with acquired chronic liver disease (CLD)3-5. Whether these somatic variants are common to CLD from differing aetiologies is unknown. We analysed somatic variants in the liver from patients with genetic CLD from alpha-1 anti-trypsin (A1AT) deficiency or haemochromatosis. We show that somatic variants in SERPINA1, the gene encoding A1AT, are strongly selected for in A1AT deficiency, with evidence of convergent evolution. Acquired variants cluster at the 3 end of SERPINA1 leading to C-terminal missense or truncation variants of A1AT. In vitro, C-terminal truncation variants abrogate disease-associated A1AT polymerisation and retention in the endoplasmic reticulum, supporting the C-terminal domain swap mechanism. Therefore, somatic escape variants from a deleterious germline variant are selected for in A1AT deficiency, suggesting adaptive functional somatic variants are disease-specific in CLD and point to disease-associated mechanisms.

genomics↗

bioRxiv2024

Mll4 regulates tooth enamel development

Amelogenesis, the process of enamel formation, is tightly regulated and essential for producing the tooth enamel that protects teeth from decay and wear. Disruptions in amelogenesis can result in amelogenesis imperfecta, a group of genetic conditions characterized by defective enamel, including enamel hypoplasia, marked by thin or underdeveloped enamel. Mutations in the KMT2D (MLL4) gene, which encodes a histone H3-lysine 4-methyltransferase, are associated with Kabuki syndrome, a developmental disorder that can involve dental anomalies such as enamel hypoplasia. However, the specific role of KMT2D in amelogenesis remains poorly understood. To address this gap, we generated a conditional knockout mouse model with ectoderm-specific deletion of Kmt2d (Krt14-Cre;Kmt2dfl/fl, or Kmt2d-cKO) and characterized the resulting enamel defects using gross, radiographic, histological, cellular, and molecular analyses. Micro-computed tomography and scanning electron microscopy revealed that adult Kmt2d-cKO mice exhibited 100% penetrant amelogenesis imperfecta, characterized by hypoplastic and hypomineralized enamel, partially phenocopying human Kabuki syndrome. Additionally, Kmt2d-cKO neonates developed molar tooth germs with subtle cusp shape alterations and mild delays in ameloblast differentiation at birth. RNA-seq analysis of the first molar tooth germ at birth revealed that 33.7% of known amelogenesis-related genes were significantly downregulated in the Kmt2d-cKO teeth. Integration with KMT2D CUT&RUN-seq results identified 8 overlapping genes directly targeted by KMT2D. Re-analysis of a single-cell RNA-seq dataset in the developing mouse incisors revealed distinct roles for these genes in KMT2D-regulated differentiation across various cell subtypes within the dental epithelium. Among these genes, Satb1 and Sp6 are likely direct targets involved in the differentiation of pre-ameloblasts into ameloblasts. Taken together, we propose that KMT2D plays a crucial role in amelogenesis by directly activating key genes involved in ameloblast differentiation, offering insights into the molecular basis of enamel development and related dental pathologies.

developmental biology↗

bioRxiv2024

Mll4 regulates postnatal palate growth and midpalatal suture development

MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the MLL4 gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the Mll4-cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in Mll4-cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in Mll4-cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the Mll4-cKO mice emerged as early as one day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as Col2a1 and Acan, at birth. Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the Mll4-cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in Mll4-cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.

developmental biology↗