Craniofacial morphogenesis requires precisely coordinated proliferation, migration, and differentiation of cranial neural crest (CNC) cells during development. Although DDB1- and CUL4-associated factor 13 (DCAF13) is kno...Craniofacial morphogenesis requires precisely coordinated proliferation, migration, and differentiation of cranial neural crest (CNC) cells during development. Although DDB1- and CUL4-associated factor 13 (DCAF13) is known to play critical roles in early embryogenesis and tumorigenesis, its function in neural crest development remains unknown. Here, we identify a novel role for DCAF13 in craniofacial development. Conditional knockout of Dcaf13 in neural crest lineages resulted in severe craniofacial malformations characterized by impaired skeletal growth and differentiation. Mechanistically, DCAF13 deficiency in mesenchymal cells led to PTEN accumulation, a key negative regulator of PI3K/AKT signaling, thereby suppressing proliferation and differentiation of CNC-derived cells. Our findings establish DCAF13 as a crucial regulator of craniofacial morphogenesis through its control of the PTEN-PI3K/AKT signaling axis, which orchestrates neural crest cell proliferation and differentiation.
Non-syndromic tooth agenesis (TA) is a rare developmental disorder that impairs oral function, systemic health, psychological well-being, and quality of life. The Wnt signaling pathway plays a central role in TA pathogen...Non-syndromic tooth agenesis (TA) is a rare developmental disorder that impairs oral function, systemic health, psychological well-being, and quality of life. The Wnt signaling pathway plays a central role in TA pathogenesis, and emerging evidence implicates that the low-density lipoprotein receptor-related protein 6 (LRP6) is involved in autosomal-dominant inheritance of TA. In this study, whole-exome sequencing (WES) of a Chinese family uncovered a novel missense mutation (c.692C>T, p.T231M) in LRP6. Sanger sequencing validated this variant, which was identified as a de novo mutation in the proband. Functional assays using qRT-PCR and immunofluorescent staining demonstrated that this mutation impairs LRP6 protein function and disrupts Wnt signaling. Our findings broaden the mutational spectrum of non-syndromic TA and underscore the critical role of LRP6 in TA.
The placenta plays an essential role during pregnancy in mammals, with the placental endocrine trophoblast subtypes secreting many growth factors and cytokines to promote fetal growth and maternal adaptation. These endoc...The placenta plays an essential role during pregnancy in mammals, with the placental endocrine trophoblast subtypes secreting many growth factors and cytokines to promote fetal growth and maternal adaptation. These endocrine cells, including trophoblast giant cells (TGCs), glycogen trophoblast cells (GlyTs) and spongiotrophoblast cells (SpTs), are mainly derived from Tpbpα-positive (Tpbpα) trophoblast cells primarily located in the ectoplacental cone (EPC) and later junctional zone (JZ) of the mouse placenta. However, the mechanism driving Tpbpα trophoblast cell differentiation and the functions of the factors secreted by these endocrine cells remain largely unknown. In the present study, we generated the Tpbpα-iCre-EGFP knock-in mice with codon-improved Cre recombinase (iCre) inserted into the endogenous locus of the Tpbpα gene. To examine the specificity and efficiency of iCre recombinase, we crossed the Tpbpα-iCre-EGFP mice with ROSA26Sor reporter mice. The co-expression of EGFP and Td-tomato was detected obviously in the EPC at E8.5 and E9.5, and in the JZ at E13.5. Meanwhile, employing lineage tracing and in situ hybridization, we demonstrated that Tpbpα trophoblast cells could differentiate into SpTs, GlyTs, maternal blood canal (C)-TGCs, parietal (P)-TGCs, and spiral artery-associated (Spa)-TGCs. In addition, no Tpbpα expression or iCre recombinase activity was detected in other organs examined, indicating the specificity of the iCre activity in placental trophoblast cells. In summary, we successfully generated the Tpbpα-iCre-EGFP knock-in mice with enhanced Cre recombinase for modulating specific genes and investigating their functions during pregnancy.
Skeletal muscle is the most widespread tissue in mammals and mediates several functions, and whose development is controlled by a coordinated transcriptional hierarchy that regulates the activities of a range of muscle g...Skeletal muscle is the most widespread tissue in mammals and mediates several functions, and whose development is controlled by a coordinated transcriptional hierarchy that regulates the activities of a range of muscle genes. Skeletal muscle comprises various cells that create communication strategies for exchanging biological information. Nevertheless, the features and developmental programs of several of these cell lines remain unknown. We constructed a complete single-cell landscape of prenatal to postnatal developing bovine skeletal muscle and compared its single-cell transcriptomic characteristics with those of humans and mice. This landscape involved cellular heterogeneity, dynamic gene expression profiles, critical regulons during cell fate decisions, extensive networks of intercellular communication, and a gene regulation network. Overall, our results identify a developmental coordinate of the pluripotency spectrum among bovines, humans, and mice. This finding suggests evolutionary conservation and species-specific differences in the skeletal muscle systems, extending to cell types, gene expression, and regulatory elements. These results offer insights into evolutionary conserved and divergent processes during mammalian skeletal muscle development.
Genetic variants of CHD7, encoding a chromatin remodeler, lead to CHARGE syndrome with congenital deficits in multiple organs. One crucial unsolved question is the causal mechanisms of most protein-altering variants of C...Genetic variants of CHD7, encoding a chromatin remodeler, lead to CHARGE syndrome with congenital deficits in multiple organs. One crucial unsolved question is the causal mechanisms of most protein-altering variants of CHD7. One hypothesis is that these variants impair the enzymatic activity of CHD7, that is ATPase and nucleosome remodeling activities. Herein, we compared the phenotype of two new mouse models for CHARGE syndrome in parallel, with the Dppa3-cre/Chd7 line carrying a Chd7 truncation variant and the Chd7 line carrying an enzymatic-deficient missense variant. While the Dppa3-cre/Chd7 line displayed typical disease-relevant phenotypes of CHARGE syndrome as other reported lines, some of these phenotypes, such as body growth and circling behavior, were surprisingly mild in the ATPase-deficient Chd7 mouse line. Thus, our results demonstrated the different contribution of the enzymatic activity of CHD7 in growth and organogenesis.
Chromatin remodeling proteins are evolutionarily conserved factors involved in a wide range of biological processes. In this review, we describe ATRX, a chromatin remodeling protein belonging to the SWI/SNF2 family. Its...Chromatin remodeling proteins are evolutionarily conserved factors involved in a wide range of biological processes. In this review, we describe ATRX, a chromatin remodeling protein belonging to the SWI/SNF2 family. Its association with different protein complexes, and its roles in embryonic development, sexual differentiation, as well as retinal and brain function. We further discuss and integrate current findings on pathologies associated with ATRX dysfunction such as ATR-X syndrome, focusing on its etiology, clinical features, and potential diagnostic tools. Finally, we propose that ATRX may contribute to the progression of certain neurodegenerative diseases and review recent literature supporting this hypothesis.
Vascular endothelial cell senescence is closely associated with cardiovascular disease. The deficiency of natriuretic peptide receptor A (NPRA), encoded by the natriuretic peptide receptor 1 (NPR1) gene, contributes to v...Vascular endothelial cell senescence is closely associated with cardiovascular disease. The deficiency of natriuretic peptide receptor A (NPRA), encoded by the natriuretic peptide receptor 1 (NPR1) gene, contributes to vascular endothelial aging, but the cause of its reduced expression is unclear. In this study, we performed reverse chromatin immunoprecipitation (R-ChIP) to analyze the binding proteins of the NPR1 promoter and found that nucleolar and coiled body phosphoprotein 1 (NOLC1) functions as a key regulator of NPRA transcription in endothelial cells. Similar to NPRA, NOLC1 is also expressed at reduced levels in senescent endothelial cells. Knockdown of NOLC1 decreased both NPRA mRNA and protein expression levels. Furthermore, inhibition of NOLC1 expression triggered cellular senescence hallmarks, including elevated p53/p21 levels, enhanced SA-β-gal activity, ROS accumulation, G0/G1 cell cycle arrest, and impaired migration. NPRA overexpression rescued senescence and dysfunction in NOLC1-deficient cells, restoring proliferative and migratory capacity. In conclusion, our findings demonstrate that nucleolar phosphoprotein NOLC1 is a key regulator of NPRA transcription in endothelial senescence.
Nydam ML, Gamble D, Gordon T
… +10 more, Hiebert LS, Hutchings B, Liberti A, Mondal J, Moody K, Olhasso M, Popsuj S, Sato A, Oğul Ünal FN, Di Gregorio A
Syrian hamsters (Mesocricetus auratus) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristic...Syrian hamsters (Mesocricetus auratus) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristics. Although embryo manipulation techniques such as embryo collection, pronuclear microinjection, and embryo transfer have been established, gene knock-in (KI) hamsters have not yet been reported. Here, we report the successful generation of gene KI Syrian hamsters by microinjecting CRISPR/Cas9 components and plasmid DNA into pronuclear-stage zygotes. Targeted insertion of a DNA cassette up to 8 kb was achieved at the ROSA26 orthologous locus and other genomic sites. Importantly, we confirmed functional expression of a reporter cassette inserted at the ROSA26 site, providing evidence of transcriptional activity at this locus in Syrian hamsters. Furthermore, we demonstrated that frozen-thawed KI embryos could give rise to live offspring using a simplified freezing and thawing protocol originally developed for mice and rats. These results confirm the feasibility and applicability of advanced genome editing technologies in Syrian hamsters. These technological advancements enable the development of versatile KI models for applications such as gene expression monitoring and conditional mutagenesis, thereby expanding the utility of Syrian hamsters as model organisms, comparable to mice and rats.
Astrocytes are a major glial cell type, playing multiple roles in the development, function, and pathogenesis of the brain. Accordingly, neuronal-astrocyte communication is an important research area. However, because th...Astrocytes are a major glial cell type, playing multiple roles in the development, function, and pathogenesis of the brain. Accordingly, neuronal-astrocyte communication is an important research area. However, because these cell types share the same developmental origin, selective manipulation of each cell type is needed for precise mechanistic understanding. Here, we generated two new Cre driver lines for selective gene manipulation in astrocytes: Slc7a10-IRES-Cre and Aldh1l1-IRES-Cre. An internal ribosome entry site (IRES)-Cre cassette was knocked-in to the 3'-untranslated region of the solute carrier family 7 member 10 (Slc7a10) or aldehyde dehydrogenase 1 family member L1 (Aldh1l1) locus without disrupting gene function. The Slc7a10-IRES-Cre line underwent highly selective recombination in astrocytes of the brain, apart from choroid plexus epithelial cells. The onset of recombination began after completion of differentiation in the astrocyte lineage. By contrast, the Aldh1l1-IRES-Cre line began recombination during astrocyte differentiation at early postnatal stages. Some leaky expression was observed in the oligodendrocyte lineage, probably due to early onset of Cre expression in an uncommitted glial progenitor state. Together, the combination of the two deleter lines with distinct temporal Cre expression patterns serves as valuable tools to understand the development and function of astrocytes.
The mammalian uterus contains glands in the endometrium that develop only or primarily after birth. In the mouse, endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantat...The mammalian uterus contains glands in the endometrium that develop only or primarily after birth. In the mouse, endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful to study endometrial gland development and function. Utilizing CRISPR-Cas9 genome editing, improved Cre recombinase (iCre) was inserted into the endogenous C-X-C motif chemokine ligand 15 (Cxcl15) gene. Cxcl15 mRNA, Cxcl15 protein, and Cxcl15-iCre recombinase activity were specific to the developing GE of the uterus. Cxcl15-iCre mice were crossed with floxed Foxa2 mice to conditionally delete Foxa2 specifically in the glands of the neonatal mouse uterus. This conditional deletion of Foxa2 in the developing neonatal uterus resulted in adult mice that lacked Foxa2 in the GE of the uterus, and the adult mice were infertile. The studies described here establish that Cxcl15-iCre mice are a valuable resource to elucidate and explore mechanisms regulating the development and function of glands in the uterus.
Secreted Frizzled-related proteins (SFRPs) are a small, ancient family of extracellular signaling pathway agonists and antagonists. In this study, we examined their expression patterns during regeneration in the holothur...Secreted Frizzled-related proteins (SFRPs) are a small, ancient family of extracellular signaling pathway agonists and antagonists. In this study, we examined their expression patterns during regeneration in the holothurian Eupentacta fraudatrix using whole-mount in situ hybridization. Our results suggest that both genes are involved in proper water-vascular system formation and coelomic epithelium homeostasis in holothurians.
Periodontal diseases, including periodontitis and gingivitis, constitute a major global health burden, affecting over 1 billion people worldwide. These conditions typically initiate in adulthood and progress chronically,...Periodontal diseases, including periodontitis and gingivitis, constitute a major global health burden, affecting over 1 billion people worldwide. These conditions typically initiate in adulthood and progress chronically, often exhibiting severe manifestations. The socioeconomic impact is particularly acute in low- and middle-income countries, where limited healthcare access exacerbates disease outcomes. Although conventional treatments provide symptomatic relief, they often fail to achieve complete tissue regeneration due to the complex pathophysiology of periodontal destruction. Mesenchymal stem cells (MSCs) have emerged as a transformative therapeutic strategy, demonstrating unique capabilities for immunomodulation, anti-inflammatory effects, and multipotent differentiation. Preclinical studies have documented MSC-mediated regeneration of periodontal ligaments, alveolar bone, and cementum through paracrine signaling and direct tissue integration. Clinical trials further substantiate their potential to improve key outcomes, including clinical attachment levels and probing depth reduction. However, five critical challenges require resolution for successful translation: (1) cellular source standardization, (2) mechanistic understanding of long-term efficacy, (3) safety and immunological profiling, (4) ethical and economic barriers, and (5) clinical translation barriers. This review systematically evaluates current evidence on MSC-based periodontal regeneration, analyzes these translational challenges, and provides strategic guidance for future research. By integrating fundamental science with clinical perspectives, this work advances the development of reliable MSC therapies for periodontal regeneration.
Tissue-specific gene manipulation using Cre/loxP or Flp/frt recombination systems is a cornerstone of genetically engineered mouse models. In this study, we aim to develop a novel hepatocyte-specific, tamoxifen-inducible...Tissue-specific gene manipulation using Cre/loxP or Flp/frt recombination systems is a cornerstone of genetically engineered mouse models. In this study, we aim to develop a novel hepatocyte-specific, tamoxifen-inducible Flp mouse line. BAC (bacterial artificial chromosome)-Alb(albumin)-FlpER2(estrogen receptor ligan binding domain) was developed by inserting IRES-FlpER2 cDNA between the translation stop codon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome. Upon tamoxifen induction in mice crossed with reporter lines, western blotting, immunohistochemistry, immunofluorescence staining, and X-gal staining (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside Staining) were used to verify the recombination efficiency and specificity of this mouse model. Recombination was highly efficient and specific in hepatocytes, with no recombination detected in intrahepatic cholangiocytes or other organs in this mouse model. We generated a new tamoxifen-induced hepatocyte-specific mouse model with highly efficient recombination specifically in hepatocytes, and this model can be used to generate tumor model lines.
Neuronal migration during embryonic development is a fundamental process. In the developing nose of rodents, neurons that form during early neurogenic waves in the olfactory placode leave this structure to migrate toward...Neuronal migration during embryonic development is a fundamental process. In the developing nose of rodents, neurons that form during early neurogenic waves in the olfactory placode leave this structure to migrate toward or into the developing brain as part of the migratory mass. This mass includes gonadotropin-releasing hormone-1 (GnRH-1) neurons, pioneer/terminal nerve (TN) neurons, as well as neural crest-derived olfactory glial cells called olfactory ensheathing cells. There have been a limited number of molecular markers available to effectively trace and functionally manipulate the early migratory neurons that originate in the olfactory region. Contactin-2 (Cntn2), also known as transiently expressed axonal surface glycoprotein-1 (TAG-1), has been used to label various developing neuronal populations, including the commissural neurons of the spinal cord, motor neurons, and TN neurons. Previous single-cell RNA sequencing analyses of the developing olfactory system have identified Cntn2 expression in the TN, suggesting that Cntn2 is a suitable molecular marker for studying nasal migratory neurons. To trace Cntn2 expression in the developing olfactory system, we generated an inducible Cntn2CreERT2 mouse line. In this study, we outline how this mouse line can serve as an effective tool for time-controlled chimeric manipulation of specific neuronal populations of interest.
Genes encoding C2H2-type zinc finger (ZF) proteins constitute one of the largest gene families in animal genomes. Many C2H2-type ZF domains are used for DNA-binding, and others are used for protein-protein interactions a...Genes encoding C2H2-type zinc finger (ZF) proteins constitute one of the largest gene families in animal genomes. Many C2H2-type ZF domains are used for DNA-binding, and others are used for protein-protein interactions and RNA-binding. In ascidian embryos, a gene regulatory network controlling cell fate decision has been studied extensively. However, because ZF proteins have not been well annotated genome-widely, it has not been clear which ZF proteins are possible regulators for cell fate decision in this animal. Therefore, we tried to identify and classify all genes encoding C2H2-type ZF proteins in the genome of an ascidian in the present study. Because the number of ZF domains often varies among possible orthologous proteins, and because ZF domains are not long enough for molecular phylogenetic analyses in many cases, we extensively utilized other bioinformatics tools for their classification. In addition, we found that many ascidian ZF proteins contain fewer ZF domains than their human orthologs; this finding might be related to the compactness of the ascidian genome.
Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles th...Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1 allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.
In the early zebrafish neural plate, proneural cluster domains are defined by surrounding neural progenitor pools (NPPs), generating primary neurogenesis patterns. In each NPP, several Notch-independent Hes/her-type gene...In the early zebrafish neural plate, proneural cluster domains are defined by surrounding neural progenitor pools (NPPs), generating primary neurogenesis patterns. In each NPP, several Notch-independent Hes/her-type genes are expressed in distinct manners. Previous knockdown (KD) experiments induced ectopic neurogenesis in NPPs where only the targeted her genes were expressed, with other her genes absent, suggesting cooperative functions of Notch-independent her genes. In this study, to overcome the inherent limitations in KD approaches, we knocked out (KO) three her genes, her3, her5, and her11, using genome editing techniques. The resulting mutants exhibited ectopic neurogenesis patterns at the end of gastrulation, similar to those observed in KD experiments. KOs of her5 and her11 induced ectopic neurogenesis around the midbrain-hindbrain boundary, whereas her3 KO led to ectopic neurogenesis in rhombomere 1/2 and r4. In these cases, the expression of other Notch-independent her genes was not affected, except for her11, whose expression depended on her5. Analyses of compound mutants revealed that their phenotypes were essentially the sum of those of individual her mutants, indicating independent suppression of neurogenesis by Notch-independent her genes. In conclusion, different Notch-independent her genes collectively define the characteristic pattern of primary neurogenesis in the neural plate.
This review provides a comprehensive overview of Complex I during mammalian oocyte maturation. Complex I (NADH:ubiquinone oxidoreductase) is a crucial member of the electron transport chain and serves two principal funct...This review provides a comprehensive overview of Complex I during mammalian oocyte maturation. Complex I (NADH:ubiquinone oxidoreductase) is a crucial member of the electron transport chain and serves two principal functions during oxidative phosphorylation: NADH oxidation and proton pumping. It is located at the inner mitochondrial membrane and consists of 14 core and 31 accessory subunits that are necessary for its function and assembly. Moreover, Complex I is the primary site of reactive oxygen species (ROS) production among the different tissues. In light of the literature, it has been demonstrated that ROS and oxidative stress are significantly important among the various factors that can affect oocyte maturation. Factors such as malnutrition, alcohol use, obesity, PCOS, aging, and smoking are some of the common causes of infertility. Each one of them causes disruption in the equilibrium of the body's redox system and related with oxidative stress. During oocyte maturation, excessive ROS levels are associated with chromosomal errors and developmental insufficiency. In addition, excess oxidative stress adversely affects embryo growth and development and may cause fetal embryopathies with damage to macromolecules in the cytoskeleton. At this particular juncture, Complex I plays a key role in determining ROS production and the success of the oocyte maturation. This review evaluates mitochondrial Complex I's function, structure, and its crucial role during oocyte maturation.
To identify novel Six1-interacting proteins, we previously screened the fly interactome for Sine oculis-binding partners whose orthologues are also expressed in Xenopus embryos. We identified a zinc-finger MYM-containing...To identify novel Six1-interacting proteins, we previously screened the fly interactome for Sine oculis-binding partners whose orthologues are also expressed in Xenopus embryos. We identified a zinc-finger MYM-containing protein-Zmym2-based on its sequence similarity in a few domains also found in the Drosophila and vertebrate Sine oculis-binding proteins (Sobp). Because recent studies established Zmym2 as a transcriptional repressor that interacts with Six4 during renal development, herein we assessed whether it interacts with Six1, can modify Six1's transcriptional activity, and is involved in cranial neural crest or placode gene expression. Although during early development Zmym2 is expressed in many of the same tissues as Six1 and contains several domains also found in Sobp, we did not detect any interaction by co-immunoprecipitation and did not detect any effect on Six1 + Eya1 transcriptional activity in cultured cells. Nonetheless, increasing the level of Zmym2 in embryos resulted in broader expression domains of neural border, neural tube and neural crest genes, and smaller placode gene domains. These results suggest that although Zmym2 is unlikely to be a bone fide Six1 interacting protein, it appears to indirectly antagonize Six1 function during cranial placode development, promoting neural plate and neural crest gene expression.