ABSTRACT
Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic ß-catenin acetylation downstream of WNT signalling. As acetylated ß-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.
Subject(s)
Amnion/embryology , Glycolysis , Wnt Proteins/metabolism , Acetylation , Animals , Body Patterning , Chick Embryo , Humans , Hydrogen-Ion Concentration , Lactic Acid/metabolism , Mesoderm/metabolism , beta Catenin/metabolismABSTRACT
Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.
Subject(s)
Cell Differentiation/genetics , Mesoderm/cytology , Muscle Development/genetics , Muscle, Skeletal/cytology , Pluripotent Stem Cells/cytology , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Humans , Immunohistochemistry , Immunophenotyping , In Situ Hybridization , In Vitro Techniques , Mesoderm/metabolism , Mesoderm/physiology , Mice , Muscle Development/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/physiology , Real-Time Polymerase Chain Reaction , Tissue Array Analysis , Wnt Signaling Pathway/geneticsABSTRACT
PURPOSE: To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. METHODS: Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19G28R and CDK19Y32H. RESULTS: We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). CONCLUSION: CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.
Subject(s)
Intellectual Disability , Neurodevelopmental Disorders , Animals , Cyclin-Dependent Kinases/genetics , Gain of Function Mutation , Humans , Infant , Mutation, Missense , Zebrafish/geneticsABSTRACT
In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.
Subject(s)
DNA/genetics , Gene Silencing , Gene Targeting , Germ Cells/metabolism , RNA, Small Interfering/genetics , Animals , Gene Expression Regulation , Genes, Reporter , Genetic Loci , Male , Mice , Mice, Transgenic , RNA Processing, Post-TranscriptionalABSTRACT
Notch signaling exerts multiple roles during different steps of mouse somitogenesis. We have previously shown that segmental boundaries are formed at the interface of the Notch activity boundary, suggesting the importance of the Notch on/off state for boundary formation. However, a recent study has shown that mouse embryos expressing Notch-intracellular domain (NICD) throughout the presomitic mesoderm (PSM) can still form more than ten somites, indicating that the NICD on/off state is dispensable for boundary formation. To clarify this discrepancy in our current study, we created a transgenic mouse lacking NICD boundaries in the anterior PSM but retaining Notch signal oscillation in the posterior PSM by manipulating the expression pattern of a Notch modulator, lunatic fringe. In this mouse, clearly segmented somites are continuously generated, indicating that the NICD on/off state is unnecessary for somite boundary formation. Surprisingly, this mouse also showed a normal rostral-caudal compartment within a somite, conferred by a normal Mesp2 expression pattern with a rostral-caudal gradient. To explore the establishment of normal Mesp2 expression, we performed computer simulations, which revealed that oscillating Notch signaling induces not only the periodic activation of Mesp2 but also a rostral-caudal gradient of Mesp2 in the absence of striped Notch activity in the anterior PSM. In conclusion, we propose a novel function of Notch signaling, in which a progressive oscillating wave of Notch activity is translated into the rostral-caudal polarity of a somite by regulating Mesp2 expression in the anterior PSM. This indicates that the initial somite pattern can be defined as a direct output of the segmentation clock.
Subject(s)
Body Patterning/physiology , Mesoderm/metabolism , Receptors, Notch/metabolism , Somites/embryology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Computer Simulation , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Immunohistochemistry , In Situ Hybridization , Mesoderm/embryology , Mice , Mice, Transgenic , Receptors, Notch/geneticsABSTRACT
In vertebrate embryos, the canonical Wnt ligand primes the formation of dorsal organizers that govern dorsal-ventral patterns by secreting BMP antagonists. In contrast, in Drosophila embryos, Toll-like receptor (Tlr)-mediated NFκB activation initiates dorsal-ventral patterning, wherein Wnt-mediated negative feedback regulation of Tlr/NFκB generates a BMP antagonist-secreting signalling centre to control the dorsal-ventral pattern. Although both Wnt and BMP antagonist are conserved among species, the involvement of Tlr/NFκB and feedback regulation in vertebrate organizer formation remains unclear. By imaging and genetic modification, we reveal that a negative feedback loop between canonical and non-canonical Wnts and Tlr4/NFκB determines the size of zebrafish organizer, and that Tlr/NFκB and Wnts switch initial cue and feedback mediator roles between Drosophila and zebrafish. Here, we show that canonical Wnt signalling stimulates the expression of the non-canonical Wnt5b ligand, activating the Tlr4 receptor to stimulate NFκB-mediated transcription of the Wnt antagonist frzb, restricting Wnt-dependent dorsal organizer formation.
Subject(s)
NF-kappa B , Zebrafish , Animals , Feedback , Ligands , Drosophila , Wnt Signaling PathwayABSTRACT
The African turquoise killifish Nothobranchius furzeri (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the duration of experiments, and we think that these advances will accelerate aging and developmental studies using N. furzeri.
Subject(s)
Cyprinodontiformes , Reverse Genetics , Aging/genetics , Animals , Animals, Genetically Modified , Cyprinodontiformes/genetics , Gene Transfer TechniquesABSTRACT
The rostro-caudal patterning within a somite is periodically established in the presomitic mesoderm (PSM). In the mouse, Mesp2 is required for the rostral property whereas Notch signaling and Ripply2, a Mesp2-induced protein that suppresses Mesp2 transcription, are required for the caudal property. Here, we examined the mechanism behind rostro-caudal patterning by comparing the spatial movement of Notch activity with Mesp2 protein localization in wild-type embryos and those defective in Ripply1 and 2, both of which are expressed in the PSM. Mesp2 protein appears first as a thin band in the middle of the traveling Notch active domain in both wild-type and Ripply1/2-deficient embryos. In wild-type embryos, the Mesp2 band expands anteriorly to the expression front of Tbx6, an activator of Mesp2 transcription. Notch activity becomes localized further anteriorly to this Mesp2 domain, but does not pass over the anterior Mesp2 domain generated in the previous segmentation cycle. As a result, the Notch active domain appears to be restricted between these two Mesp2 domains. In Ripply1/2-deficient embryos, the Mesp2 band becomes more expanded and the Notch domain is finally diminished. Interestingly, Ripply1/2-deficient embryos exhibit anterior expansion of the Tbx6 protein domain, suggesting that Ripply1/2 regulates Mesp2 expression by modulating elimination of Tbx6 proteins. We propose that the rostro-caudal pattern is established by dynamic interaction of Notch activity with two Mesp2 domains, which are defined in successive segmentation cycles by Notch, Tbx6 and Ripply1/2.
Subject(s)
Body Patterning , Embryo, Mammalian/metabolism , Repressor Proteins/metabolism , Somites/metabolism , Animals , Mice , Receptors, Notch/metabolism , Repressor Proteins/genetics , Signal Transduction , Zebrafish/embryologyABSTRACT
Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zebrafish overexpression analyses also showed that the p.G28A and p.N156S alleles were hypomorphic alleles. Importantly, the inhibition of CDK8 kinase activity in zebrafish embryos using a specific chemical inhibitor induced craniofacial and heart defects similar to the patients' phenotype. Taken together, zebrafish studies showed that non-synonymous variants in the kinase domain of CDK8 act as hypomorphic alleles causing human congenital disorder.
Subject(s)
Cyclin-Dependent Kinase 8/genetics , Mutation, Missense , Neurodevelopmental Disorders/genetics , Point Mutation , Abnormalities, Multiple/genetics , Animals , Child , Craniofacial Abnormalities/genetics , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinase 8/deficiency , Cyclin-Dependent Kinase 8/physiology , Cyclin-Dependent Kinases/physiology , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/enzymology , Female , Heart Defects, Congenital/genetics , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Infant , Intellectual Disability/genetics , Loss of Function Mutation , Male , Protein Domains , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Zebrafish/embryology , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/physiologyABSTRACT
The heterochronic genes Lin28a/b and let-7 regulate invertebrate development, but their functions in patterning the mammalian body plan remain unexplored. Here, we describe how Lin28/let-7 influence caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a strikingly increased caudal vertebrae number and tail bud cell proliferation, whereas its knockout did the opposite. Lin28a overexpression downregulated the neural marker Sox2, causing a pro-mesodermal phenotype with a decreased proportion of neural tissue relative to nascent mesoderm. Manipulating Lin28a and let-7 led to opposite effects, and manipulating Lin28a's paralog, LIN28B caused similar yet distinct phenotypes. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan. We propose that the Lin28/let-7 pathway controls the pool of caudal progenitors during tail development, promoting their self-renewal and balancing neural versus mesodermal cell fate decisions.
Subject(s)
MicroRNAs/metabolism , Morphogenesis/physiology , RNA-Binding Proteins/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Proliferation/physiology , Mammals/metabolism , Mice, Transgenic , MicroRNAs/genetics , RNA-Binding Proteins/geneticsABSTRACT
The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.
Subject(s)
Mouse Embryonic Stem Cells/physiology , Proteasome Endopeptidase Complex/metabolism , Repressor Proteins/metabolism , Somites/embryology , Transcription Factors/metabolism , Animals , Cells, Cultured , Mass Spectrometry , Mice , Protein Binding , Proteolysis , T-Box Domain ProteinsABSTRACT
Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatiotemporal regulation of glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of fibroblast growth factor (FGF) signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that glycolysis in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis.
Subject(s)
Amnion/embryology , Amnion/metabolism , Body Patterning , Fibroblast Growth Factors/metabolism , Glycolysis , Vertebrates/embryology , Vertebrates/metabolism , Wnt Signaling Pathway , Animals , Body Patterning/genetics , Cell Movement , Chick Embryo , Extracellular Space/metabolism , Glycolysis/genetics , Hydrogen-Ion Concentration , Mesoderm/embryology , Mesoderm/metabolism , Mice , Phenotype , Tail/embryology , Transcription, Genetic , Vertebrates/genetics , Wnt Signaling Pathway/geneticsABSTRACT
During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.
Subject(s)
Cell Differentiation , Disease Models, Animal , Muscle Fibers, Skeletal/pathology , Muscular Dystrophy, Duchenne/pathology , Pluripotent Stem Cells/pathology , Animals , Cells, Cultured , Mice , Mice, TransgenicABSTRACT
The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific beta-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs' notochordal fate.
Subject(s)
Notochord/embryology , Signal Transduction , Stem Cells/cytology , Wnt Proteins/metabolism , Animals , Apoptosis , Cell Proliferation , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Notochord/metabolism , beta Catenin/metabolismABSTRACT
Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Chromosomes, Artificial, Bacterial , Gene Expression Regulation, Developmental , Mesoderm/metabolism , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Enhancer Elements, Genetic , Mice , Mice, Knockout , Molecular Sequence Data , Phenotype , Recombination, Genetic , Sequence Homology, Nucleic AcidABSTRACT
The metameric structures in vertebrates are based on the periodicity of the somites that are formed one by one from the anterior end of the presomitic mesoderm (PSM). The timing and spacing of somitogenesis are regulated by the segmentation clock, which is characterized by the oscillation of several signaling pathways in mice. The temporal information needs to be translated into a spatial pattern in the so-called determination front, at which cells become responsive to the clock signal. The transcription factor Mesp2 plays a crucial role in this process, regulating segmental border formation and rostro-caudal patterning. However, the mechanisms regulating the spatially restricted and periodic expression of Mesp2 have remained elusive. Using high-resolution fluorescent in situ hybridization in conjunction with immunohistochemical analyses, we have found a clear link between Mesp2 transcription and the periodic waves of Notch activity. We also find that Mesp2 transcription is spatially defined by Tbx6: Mesp2 transcription and Tbx6 protein initially share an identical anterior border in the PSM, but once translated, Mesp2 protein leads to the suppression of Tbx6 protein expression post-translationally via rapid degradation mediated by the ubiquitin-proteasome pathway. This reciprocal regulation is the spatial mechanism that successively defines the position of the next anterior border of Mesp2. We further show that FGF signaling provides a spatial cue to position the expression domain of Mesp2. Taken together, we conclude that Mesp2 is the final output signal by which the temporal information from the segmentation clock is translated into segmental patterning during mouse somitogenesis.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Biological Clocks/physiology , Periodicity , Somites/embryology , Somites/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Proteasome Endopeptidase Complex/metabolism , Receptors, Notch/metabolism , Signal Transduction , T-Box Domain Proteins , Transcription Factors/genetics , Transcription, Genetic/genetics , Ubiquitin/metabolism , Wnt Proteins/metabolismABSTRACT
The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. Here we report that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. We also generated a knock-in mouse in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespb enhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Conserved Sequence , Enhancer Elements, Genetic , Evolution, Molecular , Mesoderm/metabolism , Somites/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Gene Targeting , Mesoderm/embryology , Mice , Models, Biological , Molecular Sequence Data , Mutation/genetics , Organ Specificity , Oryzias/genetics , Receptors, Notch/metabolism , Signal Transduction , T-Box Domain Proteins , Transcription, GeneticABSTRACT
The Mesp2 transcription factor plays essential roles in segmental border formation and in the establishment of rostro-caudal patterning within a somite. A possible Mesp2 target gene, Ripply2, was identified by microarray as being downregulated in the Mesp2-null mouse. Ripply2 encodes a putative transcriptional co-repressor containing a WRPW motif. We find that Mesp2 binds to the Ripply2 gene enhancer, indicating that Ripply2 is a direct target of Mesp2. We then examined whether Ripply2 is responsible for the repression of genes under the control of Mesp2 by generating a Ripply2-knockout mouse. Unexpectedly, Ripply2-null embryos show a rostralized phenotype, in contrast to Mesp2-null mice. Gene expression studies together with genetic analyses further revealed that Ripply2 is a negative regulator of Mesp2 and that the loss of the Ripply2 gene results in the prolonged expression of Mesp2, leading to a rostralized phenotype via the suppression of Notch signaling. Our study demonstrates that a Ripply2-Mesp2 negative-feedback loop is essential for the periodic generation of the rostro-caudal polarity within a somite.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Body Patterning , Repressor Proteins/physiology , Somites/physiology , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Developmental , Glycosyltransferases/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Repressor Proteins/geneticsABSTRACT
In the past decades, the function of the Wnt canonical pathway during embryogenesis has been intensively investigated; however, little survey of neonatal and adult tissues has been made, and the role of this pathway remains largely unknown. To investigate its role in mature tissues, we generated two new reporter transgenic mouse lines, ins-TOPEGFP and ins-TOPGAL, that drive EGFP and beta-galactosidase expression under TCF/beta-catenin, respectively. To obtain the accurate expression pattern, we flanked these transgenes with the HS4 insulator to reduce chromosomal positional effects. Analysis of embryos showed that the reporter genes were activated in regions where canonical Wnt activity has been implicated. Furthermore, their expression patterns were consistent in both lines, indicating the accuracy of the reporter signal. In the neonatal brain, the reporter signal was detected in the mesencephalon and hippocampus. In the adult mice, the reporter signal was found in the mature pericenteral hepatocytes in the normal liver. Furthermore, during inflammation the number of T cells expressing the reporter gene increased in the adult spleen. Thus, in this research, we identified two organs, i.e., the liver and spleen, as novel organs in which the Wnt canonical signal is in motion in the adult. These transgenic lines will provide us broader opportunities to investigate the function of the Wnt canonical pathway in vivo.