The Pax2 transcription factor is activated uniformly in the optic vesicle/cup, but becomes progressively restricted to the forming optic disc and stalk. In the eye, it is not known how Pax2 expression is regulated and pr...The Pax2 transcription factor is activated uniformly in the optic vesicle/cup, but becomes progressively restricted to the forming optic disc and stalk. In the eye, it is not known how Pax2 expression is regulated and progressively restricted, in part because no Pax2 regulatory elements have been identified for this organ. Multiple Pax2-Cre mouse transgenic lines have been produced, but essentially none of these Cre recombinase drivers are active in the visual system. Only Tg(BAC-Pax2-cre) mice have been reported to express Cre in a subset of postnatal retinal astrocytes. We confirm this observation and demonstrate ectopic expression in branchial arches, extraocular muscles, and a subset of GABAergic amacrine cells. Our findings suggest that major eye enhancer(s) for mouse Pax2 reside outside the > 180 kb genomic segment delimited by Pax2 BAC transgenes.
Single-cell RNA sequencing (scRNA-seq) is a rapidly developing and useful technique for elucidating biological mechanisms and characterizing individual cells. Tens of millions of patients worldwide suffer from heart inju...Single-cell RNA sequencing (scRNA-seq) is a rapidly developing and useful technique for elucidating biological mechanisms and characterizing individual cells. Tens of millions of patients worldwide suffer from heart injuries and other types of heart disease. Neonatal mammalian hearts and certain adult vertebrate species, such as zebrafish, can fully regenerate after myocardial injury. However, the adult mammalian heart is unable to regenerate the damaged myocardium. scRNA-seq provides many new insights into pathological and normal hearts and facilitates our understanding of cellular responses to cardiac injury and repair at different stages, which may provide critical clues for effective therapies for adult heart regeneration. In this review, we summarize the application of scRNA-seq in heart development and regeneration and describe how important molecular mechanisms can be harnessed to promote heart regeneration.
Circadian clocks have a fundamental role in many physiological processes. Bmal1 (basic helix-loop-helix ARNT like 1) is a central master circadian clock gene. The global Bmal1 knockout mice were shown to have a loss of c...Circadian clocks have a fundamental role in many physiological processes. Bmal1 (basic helix-loop-helix ARNT like 1) is a central master circadian clock gene. The global Bmal1 knockout mice were shown to have a loss of circadian rhythms, acceleration of aging, and shortened life span. However, global Bmal1 knockout mice did not exactly reflect the Bmal1 function in specific cell or tissue types. To address the importance of circadian rhythms in macrophages, we generated myeloid-specific Bmal1 knockout mice. The efficacy of Bmal1 gene deletion in macrophages was identified at DNA, transcription, protein levels, and function. In contrast to global Bmal1 knockout mice, Bmal1 and Bmal1 mice did not exhibit aging phenotypes. However, the deletion of Bmal1 resulted in a loss of rhythmic expression of the circadian genes in macrophages. RNA-Seq revealed that Bmal1 regulated the expression of cell death-related genes in macrophages. Furthermore, these genes have been identified as clock-controlled genes in rhythmic cell models, and Bmal1 controlled the rhythmic expression of these genes in macrophages. Finally, Bmal1 inhibited RSL3-induced ferroptosis in macrophages through Phgdh. In summary, the model of myeloid-specific Bmal1 knockout mice was successfully constructed, providing a tool for the study of the roles of Bmal1 in macrophages and the peripheral circadian clock. Meanwhile, Bmal1 regulates ferroptosis in macrophages.
Heterotaxy (HTX) is characterized by an abnormality in the organ arrangement along the Left-Right (LR) axis and is caused by the disruption of LR patterning in early development. LR asymmetry is critical for multiple org...Heterotaxy (HTX) is characterized by an abnormality in the organ arrangement along the Left-Right (LR) axis and is caused by the disruption of LR patterning in early development. LR asymmetry is critical for multiple organs. Specifically, proper LR patterning is crucial for cardiac function and is a cause of congenital heart disease (CHD). CACNA1G is a candidate gene identified in patients with CHD and HTX. This gene encodes a T-type, low-voltage-activated calcium channel and is a member of the Cav3.1 channel family. However, its function in cardiac or embryonic development remains unknown. Here, we show that abnormal cacna1g expression in Xenopus tropicalis recapitulates the HTX phenotype found in the patient cohort. By examining early LR patterning markers, including pitx2c and dand5, we discovered that both markers are expressed abnormally, suggesting that LR patterning is disrupted at the earliest stages of the LR signaling cascade. Since cilia have been described as key regulators of LR asymmetry, we checked the process of cilia formation in cacna1g crispants. The LR Organizer (LRO) contained reduced cilia quantity in the cacna1g crispants, which may explain the LR defects. In conclusion, the abnormal expression of cacna1g affects cilia in the LRO, leading to abnormal LR patterning and cardiac looping.
Osteoporosis is a metabolic bone disease primarily caused by a decreased bone formation and increased bone resorption. Osteoclasts are a special class of terminally differentiated cells that play an important role in nor...Osteoporosis is a metabolic bone disease primarily caused by a decreased bone formation and increased bone resorption. Osteoclasts are a special class of terminally differentiated cells that play an important role in normal bone remodeling and bone loss in osteoporosis as well as in a variety of osteolytic diseases. Osteoclasts can be differentiated from monocyte-macrophage cells of the hematopoietic system; they are the key cells in bone resorption. Osteoclast formation and differentiation are regulated by various cytokines and transcription factors. In this review, we summarize recent advances in research on the regulation of osteoclast differentiation and function by factors such as M-CSF, RANKL, AP-1, NFATC1, MITF, and PU.1. Understanding these cytokines and transcription factors can not only help identify targets for osteoclast differentiation but also aid in intervening in the treatment of osteoclast-related diseases.
The γ-protocadherins (γ-Pcdhs) comprise 22 homophilic cell adhesion molecule isoforms, expressed from the Pcdhg gene cluster via promoter choice mechanisms that serve many crucial functions during neural development. Eme...The γ-protocadherins (γ-Pcdhs) comprise 22 homophilic cell adhesion molecule isoforms, expressed from the Pcdhg gene cluster via promoter choice mechanisms that serve many crucial functions during neural development. Emerging evidence supports the hypothesis that distinct isoforms have unique functions. The γC4 isoform, which is expressed from the Pcdhgc4 promoter and includes its unique variable exon, is the sole γ-Pcdh isoform essential for the postnatal survival in mice. Here we describe a new mouse line (C4-GFP) in which Pcdhgc4 with a C-terminal GFP tag is expressed from the Rosa26 locus following excision of a lox-Stop-lox cassette by Cre recombinase. We report that restricted expression of this transgene in the nervous system using Nestin-Cre is sufficient to rescue the neonatal lethality of mice mutant for Pcdhgc4. This new line will be a vital tool for dissecting mechanisms underlying the functions of this essential cell adhesion molecule gene, mutations in which have been associated with neurodevelopmental disorders in humans.
Specific removal of a protein is a key to understanding its function. "Trim-Away" utilizes TRIM21, an antibody receptor and ubiquitin ligase, for acute and specific reduction of proteins. When TRIM21 is expressed in cell...Specific removal of a protein is a key to understanding its function. "Trim-Away" utilizes TRIM21, an antibody receptor and ubiquitin ligase, for acute and specific reduction of proteins. When TRIM21 is expressed in cells, introduction of a specific antibody causes rapid degradation of the targeted protein; however, TRIM21 is endogenously expressed in few cell types. We have generated a mouse line using CRISPR to insert a conditional overexpression cassette of TRIM21 into the safe harbor site, Rosa26. These conditionally-expressing mice can be bred to a wide variety of Cre mice to target cell-specific TRIM21 overexpression in different tissues. Zp3 mice expressed TRIM21 protein specifically in oocytes, whereas Hprt mice expressed the protein globally. When TRIM21-overexpressing oocytes were microinjected with specific antibodies targeting either the IP receptor or SNAP23, these proteins were effectively degraded. In addition, cortical neural cells from globally-overexpressing TRIM21 mice showed a dramatic reduction in IP receptor protein within hours after electroporation of a specific antibody. These experiments confirm the effectiveness of the Trim-Away method for protein reduction. These mice should make a valuable addition to the broader research community, as a wide range of proteins and cell types can be studied using this method.
Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in...Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1 line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for β-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1 mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.
Organisms from the five kingdoms of life use minerals to harden their tissues and make teeth, shells and skeletons, in the process of biomineralization. The sea urchin larval skeleton is an excellent system to study the...Organisms from the five kingdoms of life use minerals to harden their tissues and make teeth, shells and skeletons, in the process of biomineralization. The sea urchin larval skeleton is an excellent system to study the biological regulation of biomineralization and its evolution. The gene regulatory network (GRN) that controls sea urchin skeletogenesis is known in great details and shows similarity to the GRN that controls vertebrates' vascularization while it is quite distinct from the GRN that drives vertebrates' bone formation. Yet, transforming growth factor beta (TGF-β) signaling regulates both sea urchin and vertebrates' skeletogenesis. Here, we study the upstream regulation and identify transcriptional targets of TGF-β in the Mediterranean Sea urchin species, Paracentrotus lividus. TGF-βRII is transiently active in the skeletogenic cells downstream of vascular endothelial growth factor (VEGF) signaling, in P. lividus. Continuous perturbation of TGF-βRII activity significantly impairs skeletal elongation and the expression of key skeletogenic genes. Perturbation of TGF-βRII after skeletal initiation leads to a delay in skeletal elongation and minor changes in gene expression. TGF-β targets are distinct from its transcriptional targets during vertebrates' bone formation, suggesting that the role of TGF-β in biomineralization in these two phyla results from convergent evolution.
The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeolog...The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeologs of sox21, namely sox21.S and sox21.L. Previous studies focused on Sox21.S, but its amino acid sequence is divergent, lacking conserved poly-A stretches and bearing more similarity with ancestral homologs. In contrast, Sox21.L shares higher sequence similarity with mouse and chick Sox21. To determine if Sox21.S and Sox21.L have distinct functions, we conducted gain and loss-of-function studies in Xenopus embryos. Our studies revealed that Sox21.S and Sox21.L are functionally redundant, but Sox21.L is more effective at driving changes than Sox21.S. These results also support our earlier findings in ectodermal explants, demonstrating that Sox21 function is dose-dependent. While Sox21 is necessary for primary neuron formation, high levels prevent their formation. Strikingly, these proteins autoregulate, with high levels of Sox21.L reducing sox21.S and sox21.L mRNA levels, and decreased Sox21.S promoting increased expression of sox21.L. Our findings shed light on the intricate concentration-dependent roles of Sox21 homeologs in Xenopus neurogenesis.
Olfactory sensory neurons (OSNs) are one of a few neuron types that are generated continuously throughout life in mammals. The persistence of olfactory sensory neurogenesis beyond early development has long been thought...Olfactory sensory neurons (OSNs) are one of a few neuron types that are generated continuously throughout life in mammals. The persistence of olfactory sensory neurogenesis beyond early development has long been thought to function simply to replace neurons that are lost or damaged through exposure to environmental insults. The possibility that olfactory sensory neurogenesis may also serve an adaptive function has received relatively little consideration, largely due to the assumption that the generation of new OSNs is stochastic with respect to OSN subtype, as defined by the single odorant receptor gene that each neural precursor stochastically chooses for expression out of hundreds of possibilities. Accordingly, the relative birthrates of different OSN subtypes are predicted to be constant and impervious to olfactory experience. This assumption has been called into question, however, by evidence that the birthrates of specific OSN subtypes can be selectively altered by manipulating olfactory experience through olfactory deprivation, enrichment, and conditioning paradigms. Moreover, studies of recovery of the OSN population following injury provide further evidence that olfactory sensory neurogenesis may not be strictly stochastic with respect to subtype. Here we review this evidence and consider mechanistic and functional implications of the prospect that specific olfactory experiences can regulate olfactory sensory neurogenesis rates in a subtype-selective manner.
The organization of the olfactory glomerular map involves the convergence of olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into glomeruli in the olfactory bulb (OB). A remarkable feature of t...The organization of the olfactory glomerular map involves the convergence of olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into glomeruli in the olfactory bulb (OB). A remarkable feature of the olfactory glomerular map formation is that the identity of OR instructs the topography of the bulb, resulting in thousands of discrete glomeruli in mice. Several lines of evidence indicate that ORs control the expression levels of various kinds of transmembrane proteins to form glomeruli at appropriate regions of the OB. In this review, we will discuss how the OR identity is decoded by OSNs into gene expression through intracellular regulatory mechanisms.
BACKGROUND: Increasing evidence suggests that circular RNA (circRNA) plays a regulatory role in the progression of renal cell carcinoma (RCC). However, the precise function and underlying mechanism of circSCNN1A in RCC p...BACKGROUND: Increasing evidence suggests that circular RNA (circRNA) plays a regulatory role in the progression of renal cell carcinoma (RCC). However, the precise function and underlying mechanism of circSCNN1A in RCC progression still remain unclear. METHODS: The expression levels of circSCNN1A, microRNA-590-5p (miR-590-5p), claudin 8 (CLDN8), cyclin D1, matrix metalloprotein 2 (MMP2), MMP9, E-cadherin, N-cadherin and vimentin were detected by a quantitative real-time polymerase chain reaction and Western blotting analysis. Immunohistochemistry assay was performed to analyze the positive expression rate of CLDN8. Cell proliferation was investigated by cell colony formation, 5-Ethynyl-2'-deoxyuridine and DNA content quantitation assays. Cell migration and invasion were assessed by wound-healing and transwell invasion assays. Interactions among circSCNN1A, miR-590-5p and CLDN8 were identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft mouse model assay was conducted to verify the effect of circSCNN1A on tumor formation in vivo. RESULTS: CircSCNN1A and CLDN8 expression were significantly downregulated, while miR-590-5p was upregulated in both RCC tissues and cells. CircSCNN1A overexpression inhibited RCC cell proliferation, migration and invasion, accompanied by decreases of cyclin D1, MMP2, MMP9, N-cadherin and vimentin expression and an increase of E-cadherin expression. CircSCNN1A acted as a miR-590-5p sponge and regulated RCC cell processes by binding to miR-590-5p. CLDN8, a target gene of miR-590-5p, was involved in the regulation of the biological behaviors of RCC cells by miR-590-5p. In addition, circSCNN1A induced CLDN8 production by interacting with miR-590-5p. Further, circSCNN1A suppressed tumor formation in vivo. CONCLUSION: CircSCNN1A inhibited RCC cell proliferation, migration and invasion by regulating the miR-590-5p/CLDN8 pathway.
The vomeronasal organ (VNO) is a specialized chemoreceptive structure in many vertebrates that detects chemical stimuli, mostly pheromones, which often elicit innate behaviors such as mating and aggression. Previous stud...The vomeronasal organ (VNO) is a specialized chemoreceptive structure in many vertebrates that detects chemical stimuli, mostly pheromones, which often elicit innate behaviors such as mating and aggression. Previous studies in rodents have demonstrated that chemical stimuli are actively transported to the VNO via a blood vessel-based pumping mechanism, and this pumping mechanism is necessary for vomeronasal stimulation in behaving animals. However, the molecular mechanisms that regulate the vomeronasal pump remain mostly unknown. In this study, we observed a high level of expression of phosphodiesterase 5A (PDE5A) in the vomeronasal blood vessel of mice. We provided evidence to support the potential role of PDE5A in vomeronasal pump regulation. Local application of PDE5A inhibitors-sildenafil or tadalafil-to the vomeronasal organ (VNO) reduced stimulus delivery into the VNO, decreased the pheromone-induced activity of vomeronasal sensory neurons, and attenuated male-male aggressive behaviors. PDE5A is well known to play a role in regulating blood vessel tone in several organs. Our study advances our understanding of the molecular regulation of the vomeronasal pump.
Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have bee...Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.
Cilia play a key role in the regulation of signaling pathways required for embryonic development, including the proper formation of the neural tube, the precursor to the brain and spinal cord. Forward genetic screens wer...Cilia play a key role in the regulation of signaling pathways required for embryonic development, including the proper formation of the neural tube, the precursor to the brain and spinal cord. Forward genetic screens were used to generate mouse lines that display neural tube defects (NTD) and secondary phenotypes useful in interrogating function. We describe here the L3P mutant line that displays phenotypes of disrupted Sonic hedgehog signaling and affects the initiation of cilia formation. A point mutation was mapped in the L3P line to the gene Rsg1, which encodes a GTPase-like protein. The mutation lies within the GTP-binding pocket and disrupts the highly conserved G1 domain. The mutant protein and other centrosomal and IFT proteins still localize appropriately to the basal body of cilia, suggesting that RSG1 GTPase activity is not required for basal body maturation but is needed for a downstream step in axonemal elongation.
HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have gen...HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2 homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2 embryos is indistinguishable from wild-type Hand2 expression, and Hand efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2 will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.
Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previousl...Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.
The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares sever...The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.