ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.
Subject(s)
Ear , Voltage-Dependent Anion Channels , Zebrafish Proteins , Zebrafish , Animals , Ear/embryology , Mutation/genetics , Zebrafish/genetics , Zebrafish/embryology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolismABSTRACT
We developed an ex silico evolutionary-based systematic synteny approach to define and name the duplicated genes in vertebrates. The first convention for the naming of genes relied on historical precedent, the order in the human genome, and mutant phenotypes in model systems. However, total-genome duplication that resulted in teleost genomes required the naming of duplicated orthologous genes (ohnologs) in a specific manner. Unfortunately, as we review here, such naming has no defined criteria, and some ohnologs and their orthologs have suffered from incorrect nomenclature, thus creating confusion in comparative genetics and disease modeling. We sought to overcome this barrier by establishing an ex silico evolutionary-based systematic approach to naming ohnologs in teleosts. We developed software and compared gene synteny in zebrafish using the spotted gar genome as a reference, representing the unduplicated ancestral state. Using new criteria, we identified several hundred potentially misnamed ohnologs and validated the principle manually. Also see the video abstract here: https://youtu.be/UKNLa_TvSgY.
Subject(s)
Evolution, Molecular , Zebrafish , Animals , Biological Evolution , Humans , Phylogeny , Synteny/geneticsABSTRACT
The brain ventricular system (BVS) consists of brain ventricles and channels filled with cerebrospinal fluid (CSF). Disturbance of CSF flow has been linked to scoliosis and neurodegenerative diseases, including hydrocephalus. This could be due to defects of CSF production by the choroid plexus or impaired CSF movement over the ependyma dependent on motile cilia. Most vertebrates have horizontal body posture. They retain additional evolutionary innovations assisting CSF flow, such as the Reissner fiber. The causes of hydrocephalus have been studied using animal models including rodents (mice, rats, hamsters) and zebrafish. However, the horizontal body posture reduces the effect of gravity on CSF flow, which limits the use of mammalian models for scoliosis. In contrast, fish swim against the current and experience a forward-to-backward mechanical force akin to that caused by gravity in humans. This explains the increased popularity of the zebrafish model for studies of scoliosis. "Slit-ventricle" syndrome is another side of the spectrum of BVS anomalies. It develops because of insufficient inflation of the BVS. Recent advances in zebrafish functional genetics have revealed genes that could regulate the development of the BVS and CSF circulation. This review will describe the BVS of zebrafish, a typical teleost, and vertebrates in general, in comparative perspective. It will illustrate the usefulness of the zebrafish model for developmental studies of the choroid plexus (CP), CSF flow and the BVS.
Subject(s)
Hydrocephalus , Scoliosis , Humans , Cricetinae , Animals , Mice , Rats , Zebrafish/physiology , Cerebral Ventricles , Brain , Cerebrospinal Fluid/physiology , MammalsABSTRACT
The circumventricular organs (CVOs) function by mediating chemical communication between blood and brain across the blood-brain barrier. Their origin and developmental mechanisms involved are not understood in enough detail due to a lack of molecular markers common for CVOs. These rather small and inconspicuous organs are found in close vicinity to the third and fourth brain ventricles suggestive of ancient evolutionary origin. Recently, an integrated approach based on analysis of CVOs development in the enhancer-trap transgenic zebrafish led to an idea that almost all of CVOs could be highlighted by GFP expression in this transgenic line. This in turn suggested that an enhancer along with a set of genes it regulates may illustrate the first common element of developmental regulation of CVOs. It seems to be related to a mechanism of suppression of the canonical Wnt/ ß-catenin signaling that functions in development of fenestrated capillaries typical for CVOs. Based on that observation the common molecular elements of the putative developmental mechanism of CVOs will be discussed in this review.
Subject(s)
Biological Evolution , Circumventricular Organs/growth & development , Circumventricular Organs/metabolism , Vertebrates/growth & development , Vertebrates/metabolism , Animals , Blood-Brain Barrier/metabolism , Circumventricular Organs/embryology , Vertebrates/embryologyABSTRACT
The function of the inner ear depends on the maintenance of high concentrations of K+ ions. The slow-inactivating delayed rectifier Kv2.1/KCNB1 channel works in the inner ear in mammals. The kcnb1 gene is expressed in the otic vesicle of developing zebrafish, suggesting its role in development of the inner ear. In the present study, we found that a Kcnb1 loss-of-function mutation affected development of the inner ear at multiple levels, including otic vesicle expansion, otolith formation, and the proliferation and differentiation of mechanosensory cells. This resulted in defects of kinocilia and stereocilia and abnormal function of the inner ear detected by behavioral assays. The quantitative transcriptional analysis of 75 genes demonstrated that the kcnb1 mutation affected the transcription of genes that are involved in K+ metabolism, cell proliferation, cilia development, and intracellular protein trafficking. These results demonstrate a role for Kv2.1/Kcnb1 channels in development of the inner ear in zebrafish.
Subject(s)
Cell Proliferation , Ear, Inner/embryology , Mechanotransduction, Cellular , Potassium Channels, Voltage-Gated/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Cilia/genetics , Cilia/metabolism , Loss of Function Mutation , Potassium Channels, Voltage-Gated/genetics , Protein Transport/genetics , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.
Subject(s)
Genome/genetics , Heart Valves/physiology , Zebrafish/genetics , Animals , Animals, Genetically Modified/genetics , Embryo, Nonmammalian/physiology , Gene Expression Regulation, Developmental/genetics , Genomics/methods , Heart Septal Defects/genetics , Myocardium/pathology , Organogenesis/genetics , Pacemaker, Artificial , Wnt Signaling Pathway/genetics , Zebrafish Proteins/geneticsABSTRACT
BACKGROUND: Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. RESULTS: To characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. CONCLUSION: Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.
Subject(s)
Zebrafish , Animals , Heart Rate , Humans , Sinoatrial Node , Transcriptome , Zebrafish/geneticsABSTRACT
In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.
Subject(s)
Cytoplasm/metabolism , Polyadenylation/physiology , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , Zebrafish Proteins/biosynthesis , Zebrafish/embryology , Zygote/metabolism , Animals , Female , RNA, Messenger/genetics , Zebrafish/genetics , Zebrafish Proteins/genetics , Zygote/cytologyABSTRACT
Development of the brain ventricular system of vertebrates and the molecular mechanisms involved are not fully understood. The developmental genes expressed in the elements of the brain ventricular system such as the ependyma and circumventricular organs act as molecular determinants of cell adhesion critical for the formation of brain ventricular system. They control brain development and function, including the flow of cerebrospinal fluid. Here, we describe the novel distantly related member of the zebrafish L1-CAM family of genes-camel. Whereas its maternal transcripts distributed uniformly, the zygotic transcripts demonstrate clearly defined expression patterns, in particular in the axial structures: floor plate, hypochord, and roof plate. camel expresses in several other cell lineages with access to the brain ventricular system, including the midbrain roof plate, subcommissural organ, organum vasculosum lamina terminalis, median eminence, paraventricular organ, flexural organ, and inter-rhombomeric boundaries. This expression pattern suggests a role of Camel in neural development. Several isoforms of Camel generated by differential splicing of exons encoding the sixth fibronectin type III domain enhance cell adhesion differentially. The antisense oligomer morpholino-mediated loss-of-function of Camel affects cell adhesion and causes hydrocephalus and scoliosis manifested via the tail curled down phenotype. The subcommissural organ's derivative-the Reissner fiber-participates in the flow of cerebrospinal fluid. The Reissner fiber fails to form upon morpholino-mediated Camel loss-of-function. The Camel mRNA-mediated gain-of-function causes the Reissner fiber misdirection. This study revealed a link between Chl1a/Camel and Reissner fiber formation, and this supports the idea that CHL1 is one of the scoliosis factors.
Subject(s)
Cell Adhesion Molecules/metabolism , Cerebral Ventricles/embryology , Cerebral Ventricles/metabolism , Gene Expression Regulation, Developmental , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Animals , Cell Adhesion , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Hydrocephalus/genetics , Hydrocephalus/pathology , Morpholinos/pharmacology , Phenotype , Phylogeny , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Zebrafish/genetics , Animals , Gene Deletion , Heterozygote , Nerve Tissue Proteins/genetics , Nucleic Acid Denaturation , Voltage-Dependent Anion Channels/genetics , Zebrafish Proteins/geneticsABSTRACT
The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.
Subject(s)
Evolution, Molecular , Genome/genetics , Sharks/genetics , Animals , Calcium/metabolism , Cell Lineage/immunology , Fish Proteins/classification , Fish Proteins/genetics , Gene Deletion , Genomics , Immunity, Cellular/genetics , Molecular Sequence Annotation , Molecular Sequence Data , Osteogenesis/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phylogeny , Protein Structure, Tertiary/genetics , Sharks/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Time Factors , Vertebrates/classification , Vertebrates/genetics , Zebrafish/genetics , Zebrafish/growth & developmentABSTRACT
Kv2.1 voltage-gated potassium channels consist of two types of α-subunits: (a) electrically-active Kcnb1 α-subunits and (b) silent or modulatory α-subunits plus ß-subunits that, similar to silent α-subunits, also regulate electrically-active subunits. Voltage-gated potassium channels were traditionally viewed, mainly by electrophysiologists, as regulators of the electrical activity of the plasma membrane in excitable cells, a role that is performed by transmembrane protein domains of α-subunits that form the electric pore. Genetic studies revealed a role for this region of α-subunits of voltage-gated potassium channels in human neurodevelopmental disorders, such as epileptic encephalopathy. The N- and C-terminal domains of α-subunits interact to form the cytoplasmic subunit of heterotetrameric potassium channels that regulate electric pores. Subsequent animal studies revealed the developmental functions of Kcnb1-containing voltage-gated potassium channels and illustrated their role during brain development and reproduction. These functions of potassium channels are discussed in this review in the context of regulatory interactions between electrically-active and regulatory subunits.
Subject(s)
Growth and Development/genetics , Shab Potassium Channels/physiology , Animals , Brain/embryology , Brain/growth & development , Humans , Neurodevelopmental Disorders/genetics , Reproduction/geneticsABSTRACT
BACKGROUND: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. RESULTS: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. CONCLUSION: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Nervous System/metabolism , Repressor Proteins/genetics , Transcription Factors/genetics , Zebrafish Proteins/genetics , Animals , Animals, Genetically Modified , Conserved Sequence , Embryonic Development/genetics , Green Fluorescent Proteins/metabolism , Habenula/embryology , Habenula/growth & development , Humans , ZebrafishABSTRACT
The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit Kv6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit Kv2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.
Subject(s)
Brain/embryology , Cerebral Ventricles/embryology , Organogenesis , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Potassium Channels, Voltage-Gated/physiology , Voltage-Dependent Anion Channels/genetics , Zebrafish Proteins/genetics , Animals , Animals, Genetically Modified , Brain/metabolism , Cell Proliferation/genetics , Cerebral Ventricles/metabolism , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Hydrocephalus/embryology , Hydrocephalus/genetics , Neuroepithelial Cells/metabolism , Neuroepithelial Cells/physiology , Organogenesis/genetics , Potassium Channels, Voltage-Gated/genetics , Protein Subunits/antagonists & inhibitors , Protein Subunits/physiology , Shab Potassium Channels/antagonists & inhibitors , Shab Potassium Channels/physiology , ZebrafishABSTRACT
The brain ventricular system (BVS) consists of brain ventricles and channels connecting ventricles filled with cerebrospinal fluid (CSF). The disturbance of CSF flow has been linked to neurodegenerative disease including hydrocephalus, which manifests itself as an abnormal expansion of BVS. This relatively common developmental disorder has been observed in human and domesticated animals and linked to functional deficiency of various cells lineages facing BVS, including the choroid plexus or ependymal cells that generate CSF or the ciliated cells that cilia beating generates CSF flow. To understand the underlying causes of hydrocephalus, several animal models were developed, including rodents (mice, rat, and hamster) and zebrafish. At another side of a spectrum of BVS anomalies there is the "slit-ventricle" syndrome, which develops due to insufficient inflation of BVS. Recent advances in functional genetics of zebrafish brought to light novel genetic elements involved in development of BVS and circulation of CSF. This review aims to reveal common elements of morphologically different BVS of zebrafish as a typical representative of teleosts and other vertebrates and illustrate useful features of the zebrafish model for studies of BVS. Along this line, recent analyses of the two novel zebrafish mutants affecting different subunits of the potassium voltage-gated channels allowed to emphasize an important functional convergence of the evolutionarily conserved elements of protein transport essential for BVS development, which were revealed by the zebrafish and mouse studies.
Subject(s)
Cerebral Ventricles/embryology , Animals , Cerebral Ventricles/growth & development , Cricetinae , Humans , Mice , Rats , Vertebrates/embryology , ZebrafishABSTRACT
The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development.
Subject(s)
Drosophila Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Cerebellum/growth & development , Cerebellum/metabolism , Drosophila Proteins/genetics , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Proto-Oncogene Proteins/genetics , Wnt Proteins/genetics , Wnt Signaling Pathway , Zebrafish/genetics , Zebrafish/growth & developmentABSTRACT
The introduction of genomics into the field of developmental biology led to a vast expansion of knowledge about developmental genes and signaling mechanisms they are involved in. Unlike mammals, the zebrafish features seven Zic genes. This provides an interesting insight into Zic gene evolution. In addition, an unprecedented bioimaging capability of semitransparent zebrafish embryos turns to be a crucial factor in medium- to large-scale analysis of the activity of potential regulatory elements. The Zic family of zinc finger proteins plays an important, relatively well-established, role in the regulation of stem cells and neural development and, in particular, during neural fate commitment and determination. At the same time, some Zic genes are expressed in mesodermal lineages, and their deficiency causes a number of developmental defects in axis formation, establishing body symmetry and cardiac morphogenesis. In stem cells, Zic genes are required to maintain pluripotency by binding to the proximal promoters of pluripotency genes (Oct4, Nanog, Sox2, etc.). During embryogenesis, the dynamic nature of Zic transcriptional regulation is manifested by the interaction of these factors with distal enhancers and other regulatory elements associated with the control of gene transcription and, in particular, with the Nodal and Wnt signaling pathways that play a role in establishing basic organization of the vertebrate body. Zic transcription factors may regulate development through acting alone as well as in combination with other transcription factors. This is achieved due to Zic binding to sites adjacent to the binding sites of other transcription factors, including Gli. This probably leads to the formation of multi-transcription factor complexes associated with enhancers.
Subject(s)
Embryonic Development/physiology , Transcription Factors , Wnt Signaling Pathway/physiology , Zebrafish Proteins , Zebrafish , Zinc Fingers/physiology , Animals , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Responsive nanomaterials have emerged as promising candidates as drug delivery vehicles in order to address biomedical diseases such as cancer. In this work, polymer-based responsive nanoparticles prepared by a supramolecular approach are loaded with doxorubicin (DOX) for the cancer therapy. The nanoparticles contain disulfide bonds within the polymer network, allowing the release of the DOX payload in a reducing environment within the endoplasm of cancer cells. In addition, the loaded drug can also be released under acidic environment. In vitro anticancer studies using redox and pH dual responsive nanoparticles show excellent performance in inducing cell death and apoptosis. Zebrafish larvae treated with DOX-loaded nanoparticles exhibit an improved viability as compared with the cases treated with free DOX by the end of a 3 d treatment. Confocal imaging is utilized to provide the daily assessment of tumor size on zebrafish larva models treated with DOX-loaded nanoparticles, presenting sustainable reduction of tumor. This work demonstrates the development of functional nanoparticles with dual responsive properties for both in vitro and in vivo drug delivery in the cancer therapy.
Subject(s)
Drug Delivery Systems , Nanoparticles/chemistry , Polymers/chemistry , Acids/chemistry , Animals , Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Endocytosis/drug effects , HeLa Cells , Humans , Hydrogen-Ion Concentration , Larva/drug effects , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Nanoparticles/ultrastructure , Oxidation-Reduction , Spectroscopy, Fourier Transform Infrared , Zebrafish/metabolismABSTRACT
The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3' untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response.