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1.
Cell Rep ; 31(7): 107655, 2020 05 19.
Article in English | MEDLINE | ID: mdl-32433964

ABSTRACT

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.


Subject(s)
Human Embryonic Stem Cells/metabolism , Transcription Factors/metabolism , Cell Differentiation/physiology , Cell Line , Human Embryonic Stem Cells/cytology , Humans , Single-Cell Analysis/methods
2.
Elife ; 4: e07178, 2015 Sep 11.
Article in English | MEDLINE | ID: mdl-26359635

ABSTRACT

Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblast stem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs differentiating into mEpiSCs, indicative of its requirement in the shift between these states. However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chick show primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among amniotes.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Finches/embryology , Germ Layers/cytology , Germ Layers/physiology , Animals , Biomarkers/analysis , Finches/growth & development
3.
Genesis ; 53(11): 669-77, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26385755

ABSTRACT

The domesticated zebra finch (Taeniopygia guttata) is a well-established animal model for studying vocal learning. It is also a tractable model for developmental analyses. The finch genome has been sequenced and methods for its transgenesis have been reported. Hatching and sexual maturation in this species takes only two weeks and three months, respectively. Finch colonies can be established relatively easily and its eggs are laid at a stage earlier than in other common avian experimental models, facilitating the analysis of very early avian development. Representing the Neoaves to which 95% of all bird species belong, the finch can potentially complement two existing, Galloanserae developmental models, the chick, and quail. Here, we provide a step-by-step guide for how to set up a finch colony in a conventional laboratory environment. Technical tips are offered to optimize hens' productivity and ensure a constant supply of fertilized finch eggs. Methods of handling finch eggs and embryos for subsequent embryological, cellular, or molecular analyses are also discussed. We conclude by emphasizing scientific values and cost effectiveness of maintaining a finch colony for avian developmental studies. genesis 53:669-677, 2015. © 2015 Wiley Periodicals, Inc.


Subject(s)
Developmental Biology/methods , Finches/growth & development , Models, Biological , Animal Husbandry , Animals , Female , Housing, Animal , Male , Photoperiod , Reproduction , Sex Determination Analysis
4.
PLoS Genet ; 10(1): e1004118, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24465223

ABSTRACT

Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27(Kip1), Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Ear, Inner/growth & development , Hair Cells, Auditory, Inner/cytology , Membrane Proteins/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle , Cell Differentiation/genetics , Cochlea/growth & development , Cochlea/metabolism , Ear, Inner/cytology , Epithelium/growth & development , Epithelium/metabolism , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Protein Binding , Receptor, Fibroblast Growth Factor, Type 1/metabolism , SOXB1 Transcription Factors/genetics , Signal Transduction
5.
Dev Biol ; 385(2): 380-95, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24262986

ABSTRACT

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.


Subject(s)
Cell Cycle/genetics , Evolution, Molecular , Face , Osteogenesis/genetics , Skull/growth & development , Animals , Base Sequence , Blood Vessels/growth & development , Blotting, Western , Coturnix , DNA Primers , Ducks , Species Specificity
6.
Neural Dev ; 8: 13, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23829703

ABSTRACT

BACKGROUND: In order to fulfill their chemosensory function, olfactory neurons are in direct contact with the external environment and are therefore exposed to environmental aggressive factors. Olfaction is maintained through life because, unlike for other sensory neuroepithelia, olfactory neurons have a unique capacity to regenerate after trauma. The mechanisms that control the ontogenesis and regenerative ability of these neurons are not fully understood. Here, we used various experimental approaches in two model systems (chick and mouse) to assess the contribution of retinoic acid signaling in the induction of the olfactory epithelium, the generation and maintenance of progenitor populations, and the ontogenesis and differentiation of olfactory neurons. RESULTS: We show that retinoic acid signaling, although dispensable for initial induction of the olfactory placode, plays a key role in neurogenesis within this neuroepithelium. Retinoic acid depletion in the olfactory epithelium, both in chick and mouse models, results in a failure of progenitor cell maintenance and, consequently, differentiation of olfactory neurons is not sustained. Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid. CONCLUSIONS: Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells. Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.


Subject(s)
Cell Differentiation/drug effects , Cell Lineage/drug effects , Neurogenesis/drug effects , Olfactory Bulb/drug effects , Olfactory Receptor Neurons/drug effects , Stem Cells/drug effects , Tretinoin/pharmacology , Animals , Cell Differentiation/physiology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Mice , Neurogenesis/physiology , Olfactory Bulb/cytology , Olfactory Mucosa/cytology , Olfactory Mucosa/drug effects , Olfactory Receptor Neurons/cytology , Signal Transduction/drug effects , Signal Transduction/physiology , Stem Cells/cytology
7.
Nat Commun ; 3: 1041, 2012.
Article in English | MEDLINE | ID: mdl-22948823

ABSTRACT

The paratympanic organ, a mechanosensory hair cell-containing pouch in the amniote middle ear, was first described 100 years ago, yet its origins remain unresolved. Homology with the anamniote spiracular organ is supported by association with homologous skeletal elements and similar central targets of afferent neurons, suggesting it might be a remnant of the water-dependent lateral line system, otherwise lost during the amniote transition to terrestrial life. However, this is incompatible with studies suggesting that it arises from the first epibranchial (geniculate) placode. Here we show that a previously undiscovered Sox2-positive placode, immediately dorsal to the geniculate placode, forms the paratympanic organ and its afferent neurons, which are molecularly and morphologically distinct from geniculate neurons. These data remove the only obstacle to accepting the homology of the paratympanic organ and spiracular organ. We hypothesize that the paratympanic organ/spiracular organ represents an ancient head ectoderm module, developmentally and evolutionarily independent of both lateral line and epibranchial placodes.


Subject(s)
Ear, Middle/embryology , Ectoderm/embryology , Hair Cells, Auditory/cytology , Vertebrates/embryology , Animals , Biological Evolution , Chick Embryo , Chickens/metabolism , Ear, Middle/cytology , Ear, Middle/metabolism , Ectoderm/cytology , Ectoderm/metabolism , Hair Cells, Auditory/metabolism , Neurons, Afferent/cytology , Neurons, Afferent/metabolism , Phylogeny , Quail/embryology , Quail/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Sense Organs/embryology , Sense Organs/metabolism , Sharks/embryology , Sharks/metabolism , Vertebrates/classification , Vertebrates/genetics , Vertebrates/metabolism
8.
Dev Dyn ; 241(6): 1104-10, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22473893

ABSTRACT

BACKGROUND: The auditory complex of the chick, like that of humans, is made of intimate and highly ordered connections between the inner ear, the middle ear, and the outer ear. Unlike mammals, the middle ear of chick has only one ossicle, known as the columella. The independent lineages of the two suggest that some mechanism must exist that ensures the connectivity between the inner ear and the columella; however, the basis of integration is not known. RESULTS: Using quail-chick chimeras, we demonstrate that columella development depends on signaling interactions. Specifically, both pharyngeal endoderm and cranial paraxial mesoderm can alter the morphology of the columella. Only a discrete region of pharyngeal endoderm exerts this patterning activity, and this region is specified by the overlying paraxial mesoderm. CONCLUSIONS: Paraxial mesoderm is also used in the induction of the inner ear, thus we propose that this overlapping source of signalling cues in both middle and inner ear development may underlie the integration of these structures.


Subject(s)
Ear Ossicles/embryology , Ear, Inner/embryology , Embryonic Induction/physiology , Endoderm/physiology , Mesoderm/physiology , Morphogenesis/physiology , Signal Transduction/physiology , Alcian Blue , Animals , Chick Embryo , Chimera/embryology , Immunohistochemistry , Quail
9.
Dev Biol ; 352(2): 382-92, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21320481

ABSTRACT

The spatial regulation of combinatorial expression of Hox genes is critical for determining hindbrain rhombomere (r) identities. To address the cross-regulatory relationship between Hox genes in hindbrain neuronal specification, we have generated a gain-of-function transgenic mouse mutant Hoxb3(Tg) using the Hoxb2 r4-specific enhancer element. Interestingly, in r4 of the Hoxb3(Tg) mutant where Hoxb3 was ectopically expressed, the expression of Hoxb1 was specifically abolished. The hindbrain neuronal defects of the Hoxb3(Tg) mutant mice were similar to those of Hoxb1(-/-) mutants. Therefore, we hypothesized that Hoxb3 could directly suppress Hoxb1 expression. We first identified a novel Hoxb3 binding site S3 on the Hoxb1 locus and confirmed protein binding to this site by EMSA, and by in vivo ChIP analysis using P19 cells and hindbrain tissues from the Hoxb3(Tg) mutant. We further showed that Hoxb3 could suppress Hoxb1 transcriptional activity by chick in ovo luciferase reporter assay. Moreover, in E10.5 wildtype caudal hindbrain, where Hoxb1 is not expressed, we showed by in vivo ChIP that Hoxb3 was consistently bound to the S3 site on the Hoxb1 gene. This study reveals a novel negative regulatory mechanism by which Hoxb3 as a posterior gene serves to restrict Hoxb1 expression in r4 by direct transcriptional repression to maintain the rhombomere identity.


Subject(s)
Homeodomain Proteins/metabolism , Rhombencephalon/embryology , Rhombencephalon/metabolism , Animals , Animals, Genetically Modified , Avian Proteins/genetics , Avian Proteins/metabolism , Base Sequence , Binding Sites/genetics , Body Patterning , Chick Embryo , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , DNA Primers/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Mice , Mice, Mutant Strains , Mice, Transgenic , Models, Neurological , Neurogenesis/genetics , Neurogenesis/physiology
10.
Dev Dyn ; 240(1): 162-75, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21181941

ABSTRACT

The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time-lapse imaging and molecular techniques. Emu embryos in general take 2-3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50-56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27- to 30-somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed.


Subject(s)
Dromaiidae/embryology , Embryonic Development/physiology , Animals , Cell Size , Chick Embryo , Cloning, Molecular , Dromaiidae/genetics , Embryo, Nonmammalian , Embryonic Development/genetics , Fetal Proteins/genetics , Gene Expression Regulation, Developmental , Genes, Developmental , Glycoproteins/genetics , Hedgehog Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Somites/embryology , Somites/growth & development , T-Box Domain Proteins/genetics , Time-Lapse Imaging , Zygote/cytology , Zygote/growth & development
11.
Development ; 135(20): 3415-24, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18799542

ABSTRACT

The development of the vertebrate inner ear is an emergent process. Its progression from a relatively simple disk of thickened epithelium within head ectoderm into a complex organ capable of sensing sound and balance is controlled by sequential molecular and cellular interactions. Fibroblast growth factor (FGF) and Wnt signals emanating from mesoderm and neural ectoderm have been shown to direct inner ear fate. However, the role of these multiple signals during inner ear induction is unclear. We demonstrate that the action of the FGFs and Wnts is sequential, and that their roles support a model of hierarchical fate decisions that progressively restrict the developmental potential of the ectoderm until otic commitment. We show that signalling by Fgf3 and Fgf19 is required to initiate a proliferative progenitor region that is a precursor to both the inner ear and the neurogenic epibranchial placodes. Significantly, we find that only after FGF action is attenuated can the subsequent action of Wnt signalling allow otic differentiation to proceed. In addition, gain and loss of function of Wnt-signalling components show a role for this signalling in repressing epibranchial fate. This interplay of signalling factors ensures the correct and ordered differentiation of both inner ear and epibranchial systems.


Subject(s)
Ear, Inner/embryology , Ear, Inner/physiology , Fibroblast Growth Factors/physiology , Wnt Proteins/physiology , Animals , Chick Embryo , Ear, Inner/metabolism , Embryo, Nonmammalian , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Models, Biological , Wnt Proteins/genetics , Wnt Proteins/metabolism
12.
J Cell Biol ; 173(3): 333-9, 2006 May 08.
Article in English | MEDLINE | ID: mdl-16651378

ABSTRACT

Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Homeodomain Proteins/physiology , Melanocytes/cytology , Receptors, Notch/physiology , Signal Transduction/physiology , Stem Cells/cytology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/genetics , Cell Survival/drug effects , Cell Survival/physiology , Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Epidermal Cells , Epidermis/embryology , Epidermis/metabolism , Gene Expression/genetics , Hair Color/genetics , Homeodomain Proteins/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Intramolecular Oxidoreductases/metabolism , Jagged-2 Protein , Melanocytes/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, Transgenic , Receptor, Notch1/metabolism , Stem Cells/metabolism , Transcription Factor HES-1
13.
Dev Biol ; 291(1): 144-53, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16427619

ABSTRACT

Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2.


Subject(s)
Melanocytes/physiology , Proto-Oncogene Proteins/physiology , Skin/cytology , Stem Cells/physiology , Animals , Cell Differentiation , Cell Lineage , Cell Survival , Hair Follicle/cytology , Hair Follicle/metabolism , Melanocytes/metabolism , Mice , Mice, Knockout , Pigmentation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins c-kit/biosynthesis , Signal Transduction , Skin/metabolism , Stem Cell Factor/metabolism , Stem Cells/metabolism
14.
Development ; 132(24): 5589-99, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16314490

ABSTRACT

Emerging evidence from stem cell (SC) research has strengthened the idea that SC fate is determined by a specialized environment, known as the SC niche. However, because of the difficulty of identifying individual stem cells and their surrounding components in situ, the exact mechanisms underlying SC regulation by the niche remain elusive. To overcome this difficulty, we employed melanocyte stem cells (MSCs), which allow the identification of individual SCs in the niche, the lower permanent portion of the hair follicle (HF). Here, we present molecular makers that can distinguish MSCs from other melanocyte (MC) subsets in the HF. We also describe a simple and robust method that allows gene expression profiling in individual SCs. After isolating individual MSCs from transgenic mice in which the MCs are marked by green fluorescence protein (GFP), we performed single-cell transcript analysis to obtain the molecular signature of individual MSCs in the niche. The data suggest the existence of a mechanism that induces the downregulation of various key molecules for MC proliferation or differentiation in MSCs located in the niche. By integrating these data, we propose that the niche is an environment that insulates SCs from various activating stimuli and maintains them in a quiescent state.


Subject(s)
Cell Lineage , Hair Follicle/cytology , Melanocytes/physiology , Stem Cells/physiology , Animals , Cell Proliferation , Down-Regulation , Gene Expression Profiling , Green Fluorescent Proteins/metabolism , Melanocytes/cytology , Melanocytes/metabolism , Mice , Mice, Transgenic , Stem Cells/cytology , Stem Cells/metabolism
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